Conformation of Viral Nucleic Acids in Situ

CONFORMATION OF VIRAL NUCLEIC ACIDS IN SlTU T. 1. Tikchonenko Institute of Virology, USSR Academy of Medical Sciences, and Department of Virology, Moscow State University, Moscow, USSR

I. Introduction.. .......................................................... 11. Single-Stranded Nucleic Acids. ............................. A. Rodlike end Filamentous Viruses.. ...................... B. Spherical (Isometric) Viruses. ........................... 111. Double-Stranded Nucleic Acids. ......................................... A. Bacteriophages.. ........................................ B. Other Viruses.. . . . . . . .......................................... IV. Concluding Remarks. . . . .......................................... References. . . . . . . . . . . . . . ..........................................

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I. INTRODUCTION The question of nativity is the perennial problem facing every biochemist and biophysicist engaged in analyzing the properties of complex natural macromolecules, viral nucleic acids being a case in point. At the first stages of investigation this is rather an auxiliary problem of isolating the compound sought for without damaging it and establishing its composition and structure. Nativity is then understood as a number of rneasures taken to prevent irreversible denaturation or degradation due to drastic isolation procedures, or, as the case may be, to the action of degradation factors of the cell itself (nucleases, for example) which survived purification. But, this aspect of the problem will not be of interest to us as it is usually a matter of experimental techniques. But it is at the next stages of investigation-when the more complicated task is set of studying finer features, for example, comparison of nucleic acids in vivo and in vitro, in the object (virus) and in the solution after isolation-that the problem of nativity acquires its full meaning. As a matter of fact, it is transformed into the problem of reversible conformational changes accounted for by the different environment of the molecule prior to and after its release from the viral particle. It is natural, however, that such changes should be referred to as denaturational rather tentatively and only to such an extent where the suggestion of their reversibility cannot be proved experimentally. Unfortunately, experimental verification of the reversible character of a change often depends upon the ability (or possibility) to imitate in vitro the conditions in which the given molecule “lives” in vivo. But at present such an experimental task is condemned to failure as we know practically nothing about the “living conditions” inside viral particles. 201

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It goes without saying, that conformational change which accompanies the release of RNA or DNA from the viral particle into solution, does not, as a rule, apply to the primary structure of the molecule but is observed in a higher ordered structure, i.e., secondary, tertiary, or quaternary structures. As is clear from the title, the problems to be discussed in this review will be confined to viruses, and, to be more cxact, to the question of similarities and differences between the structure and properties of usual forms of nucleic acids in solution and that of nuclcic acids inside viral particles. It may he stated a priori that such differences should exist, as the viral particle is a rather peculiar and highly specialized object designed for storage and transport of the genetic substance of viruses. It must be understood that the properties of nucleic acids in situ ought to be affected by the environment which they have in viruses, by the special functions which they have to perform, by their close partnership with protein, and by a number of other intraviral factors of which we know very little, if anything. By way of warning, it should be stated at the outset that the experimental investigation of nucleic acid structure in virus particles has just begun and no detailed picture should be expected. The bulk of the information dealt with below is obtained by means of X-ray diffraction and electron microscopy, various combinations of optical methods, and chemical and physical modifications combined with some way of recording corresponding results. The first two methods allow one to carry out direct investigation of the structure of nucleic acids inside viral particles. Electron micrographs of separate viral particles and viral crystals or ultrathin sections as well as X-ray diffractlon patterns of viral crystals usually furnish unequivocal information and need, therefore, no additional comment. As to the optical methods, the data obtained in this way are not, unfortunately, as unambiguous as those supplied by the first two methods. There are a number of theoretical and practical difficulties to be overcome which will be discussed in detail below. It should be noted, however, that determination of true absorption values of objects which scatter light in the region where they absorb is always a matter of difficulty when conformation of intraviral nucleic acid is studied by means of absorption, anisotropic absorption, or optical rotatory dispersion (ORD) . The use of the extrapolation method is often objected to from a theoretical point of view (for the sake of convenience this question will be discussed in the section dealing with phage). The more specific problem of obscurity of UV dichroism data (form dichroism, determination of orientation angles) and some others will be considered below.

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11. SINGLE-STRANDED NUCLEIC ACIDS

This section is based on a description of the data for single-stranded RNA which distinctly falls into two divisions: (1) the rodlike viruses and ( 2 ) the spherical viruses. As to the virus research along these lines, it is not very extensive and the data are rather scanty. The first consideration will be practically confined to an analysis of the structure and properties of RNA in tobacco mosaic virus (TMV) and DNA in phage Fd, and the second consideration to the same problems using the data on turnip yellow mosaic virus (TYMV), broad bean mottle virus (BBMV), bromegrass mosaic virus (BMV), tomato bushy stunt virus (TBSV), etc. A . Rodlike and Filamentous Viruses 1. Secondary Structure

a. X - R a y Diffraction and Electron Microscope Data. From X-ray diffraction and electron microscope evidence the TMV particle is known to be a cylindrically shaped riucleoprotein of diameter 150 k and length 3000 A. The protein component of this virus consists of helically arranged subunits (pcptide chains). The pitch of this helix is 23 k , the repeat period (three turns of helix) is 60 k , yielding approximately 130 turns in the whole particle (Watson, 1954; Franklin, 1955, 1956; Franklin and Klug, 1956; Franklin et al., 1957; Caspar, 1956; Caspar and Klug, 1962, 1963; Klug and Caspar, 1960; Huxley and Zubay, 1961). The most peculiar feature of the structure of RNA in the TMV particle is that of the whole single-stranded polynucleotide chain being buricd deep inside the protein shell at a distance of 20 A from the internal channel of the virion; this centrally located RNA chain winds between turns of helically arranged protein subunits. As a matter of fact, the mode of packing of the protein subunits predetermines the character of the RNA winding. It is noteworthy that the minimum density a t a distance of 40 i%from the axis in the viral protein repolymerized without RNA has the same width and depth as the other minima in the radial density distribution of this protein, as the X-ray diffraction evidence indicates. It testifies to the fact that the RNA fits well in the symmetry of the viral protein. The structure of RNA inside the viraI particle is in agreement with the above features of the tertiary structure of the protein shell. According to X-ray diffraction evidence, the pitch of the RNA helix in the TMV particle is 23 A, its diameter is 80 k , that is, 49 nucleotides per turn of the helix or 3 nucleotides per protein subunit. Calculations of Ginoza (1958) confirmed that a single-stranded RNA chain having a molecular weight of 2 x loa with a total length of 33,000 k is capable of

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forming a 3000 A helix with a pitch of 23 A, should the distance between phosphorus atoms in the helix be 5 A. Before comparing the structure of RNA in TMV with its structure in solution it should be emphasized a t once that no single-stranded polynucleotide chain of such geometry can exist in solution and it does not. For comparison the A configuration of single-stranded RNA has a pitch of 29 A, a diameter of 17 A, and 11 nucleotides per helix turn (Zubay and Wilkins, 1960; Rich and Watson, 1954; Brown and Zubay, 1960; Klug et al., 1961; Fuller, 1961). These figures, naturally, describe the helical portions of RNA and have nothing to do with the structure of the whole molecule. The above features of RNA packing in the viral particle are responsible for very important conclusions. If secondary structure is understood as the spatial organization and periodicity existing as a result of interaction of adjacent monomers and neighboring chain links, we are bound to admit that in this sense the RNA in the TMV particle has no ordered secondary structure of its own. Ordered secondary structure is usually associated with some type of hydrogen bond spiralization of the chain. In the case of intraviral RNA, the nucleotides are localized in such a way that no hydrogen bonds or any other bonds can exist either between opposite bases within one turn or between the bases in adjacent turns. It is doubtful that between the bases of intraviral RNA there should develop interactions causing base stacking in solution (Michelson, 1963). For, this kind of interaction requires preferential perpendicular orientation of bases to the chain axis. It is only in this case that the planar bases are situated one over the other to form a stack. I n TMV, the RNA bases are oriented preferentially parallel to the long axis of the particle. One cannot preclude some kind of interaction being possible even with such geometry for the helix; but as yet we do not possess any positive evidence for it. Hence the conclusion that between the bases of RNA in TMV particles there is no interaction which can create an ordered secondary structure of its own. Nevertheless, the data described above leave absolutely no doubt that such ordered structure of RNA in TMV does exist and that the content of this formal secondary structure is practically equal to 100%. Wagner and Arav (1968) in experiments dealing with the interaction of mononucleotides with positively charged polypeptides gave theoretical confirmation for the possibility of the appearance and existence of an ordered structure of this kind a t the expense of interaction of mononucleotides with the ordered structure of their partner. In their experiments, mononucleotides adsorbed on poly-L-lysine acquired ordered structure, base stacking, and the hypochromic effect typical of base in-

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teraction in oligonucleotides. So, mononucleotides bound to alkaline polymers behaved as if they had phosphodiester bonds. b. Spectrophotometry. The most important conclusion of the previous section, that the RNA in the TMV particle has no secondary structure of its own, can be confirmed by means of spectrophotometric evidence. The first to be mentioned here are the pioneering experiments of Fraenkel-Conrat (1954) on the interaction of formaldehyde with TMV, though the later study of the mechanism of action of CH20 introduced some ambiguity into the conclusions of this author. In the experiments of Fraenkel-Conrat, 24-hour incubation of intact virus particles with 1% CHzO at 40°C gave but a small (2-3%) increase in absorption at 260 mp, while under similar conditions the hyperchromic effect of free TMV RNA amounted to 28 to 29%. There exists some vagueness in interpretation of the results obtained by investigation of the polynucleotide secondary structure by means of CHZO and similar agents. That is, that the presence or the absence of ordered structure is judged by the reactivity of base amino groups toward CHzO while the very reaction of addition of CHzO to the bases is, as a rule, followed only in an indirect way, by the disappearance of hypochromism of the molecule. In solution these two phenomena-the primary reaction of CH20 addition and the following collapse of the ordered secondary structure-may occur simultaneously but in the viral nucleoprotein the situation may prove more complex. Thus, there are grounds t o believe that the mobility of bases of intraviral nucleic acids is in some way restricted (Dobrov et al., 1967; Tikchonenko and Dobrov, 1968; Inners and Bendet, 1969; Simmons and Glazer, 1966). Hence, the addition of CHzO to amino groups does not always lead to the collapse of the ordered structure of the bases and the deveIopment of hyperchromism, even if the intraviral RNA possesses some degree of spiralization. Some light could be thrown on this problem by determination of shifts in the long wave length absorption spectrum of the bases as the primary reaction of formation of methylol derivatives of bases is accompanied by a definite hyperchrornism a t 270 to 290 mp with no major alteration in absorption at 250 to 260 mp (Haselkorn and Doty, 1961; Grossman et al., 1961; Tikchonenko and Dobrov, 1968). Unfortunately, the absolute magnitude of optical shifts in this case is insignificant and is difficult to take into account in the case of turbid virus suspensions when correction for light scattering should be made (see below). It is logical to suggest that if RNA in the virus has no secondary structure of its own and if such structure appears after release from the virus, then under conditions favorable for spiralization this process will

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be accompanied by marked changes in the optical properties of the RNA. Absorption in UV is the easiest t o record in this case as spiralization connected with base interaction should entail a noticeable hypochromic effect. According to Bonhoeffer and Schachman (1960)) TMV degradation into protein and RNA under moderate heating in media of high ionic strength was accompanied by clear-cut hypochromism of 15 to 20% at 260 mp in comparison to the initial absorption value. These are convincing enough data in favor of the fact that RNA in TMV has no (or almost no) ordered secondary structure and that it is acquired in solution. The value of hypochromism offered by these authors seems to be somewhat low when compared to the normal level of hypochromism usually found in free TMV RNA under similar conditions (25-30%). But some uncertainty in determination of the true value of RNA absorption in light-scattering objects renders this small discrepancy insignificant. After analysis of all the results described above it is possible to make the firm statement that RNA in TMV particles has no secondary structure whatsoever. As to the other viruses of this group, we do not possess any positive data, except, perhaps, for the reference of Day (1966) to the unpublished results of D. A. Marvin and Schaller on the filamentous phage Fd. According to this author, in the experiments of Marvin and Schaller singlestranded DNA turned out to have a similar hypochromism in the free state and in the viral particle, but no figures are cited. Based on the assumption of identical secondary structure for single-stranded DNA in vitro and in situ, Day tried to determine the percent of a-helix in the protein of intact virus by the difference between ORD values for the whole phage and free DNA, respectively. The estimated percent of a-helix turned out to be abnormally high-90% as compared to 25 to 35% for TMV (Simmons and Blout, 1960). It is possible that such a discrepancy is connected with the erroneous initial assumption about the identical structure of DNA in solution and in the virus. Hoffman-Berling et al. (1966), emphasized that 40% of the amino acids present in Fd protein is generally considered as non-a-helix promoting. c. Optical Rotatory Dispersion. There are two aspects of the problem of conformation of intraviral RNA. The first aspect is associated with such external factors as protective action of the protein shell; the second, that having to deal with such inherent properties as regularity and irregularity of the secondary structure per se. As to the first aspect, the very existence of the nucleic acid inside the viral particle protects it to some extent from the influence of the environment and makes it more resistant to the action of agents which readily destroy RNA helicity in solution.

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It is known that the secondary structure of RNA in solution is extremely unstable and depends largely upon environmental conditions. It is clear, therefore, that the degree of spiralization of free RNA and optical and hydrodynamic properties depending on this factor vary widely with change in temperature, pH, ionic strength, etc. (Spirin, 1963). So, if intraviral RNA had such irregular structure as it has in vitro we would be right to expect higher physical stability and higher chemical inertness. But in the previous parts of this review it was shown that the behavior and properties of intraviral RNA are utterly different from the forms of RNA we are accustomed to in solution. It turned out that it is not only the defects of secondary structure which must be accounted for, but an altogether new conformation must be elucidated. For example, the lack of hypochromism and interaction between the bases of RNA in TMV which was discussed above is usually understood in terms of a total lack of order in the arrangement of the bases. But the truth is that the position of bases in intraviral RNA has a higher degree of organization as compared to free RNA forms. Unfortunately, none of the methods described above can offer quantitative information about the percent of formal secondary structure in intraviral RNA, although the high degree of order encountered, for example, in the patterns of X-ray diffraction testify to practically a perfect RNA helix in TMV. As to the methods usually used for identification of secondary structure of polynucleotides and quantitative determination of its regularity in solution, most of them cannot be applied to intraviral RNA because of the existence of a protein shell. Thus, the drawbacks of direct spectrophotometric methods (see above) make us place our hopes in ORD and UV dichroism. The results obtained by means of the latter method are considered in detail in the section dealing with tertiary structure and are only mentioned in this section. Optical rotary dispersion in preparations of rodlike viruses was studied by a number of authors (Simmons and Blout, 1960; Marvin, 1966; Day, 1966; Simmons and Glazer, 1966) but it was only Simmons who saw as his immediate aim the study of the properties of intraviral RNA. The other authors elucidated the secondary structure of protein components and, first and foremost, the helical configuration of protein subunits and percent of a-helix. Simmons compared the ORD of whole TMV, repolymerized viral protein, and free RNA from TMV in the spectral region of 230 to 360 mp. The dispersion of native TMV was greatly different from that of repolymerized protein and clearly anomalous. As these two structures have only one difference, i.e., RNA present in the virus, it is logical to ascribe to RNA the anomalous character of the ORD. This

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suggestion was strengthened by a strongly positive Cotton effect for TMV in the base absorption region which is typical of RNA in salt solutions. But, calculated ORD patterns from free RNA and repolymerized protein spectra gave a dispersion not characteristic of the whole TMV, Similarly, if ORD patterns of repolymerized TMV protein are subtracted from those of whole TMV, the picture of dispersion of intraviral RNA will be greatly different from measured ORD patterns. It may be suggested that the lack of simple additivity in ORD of repolymerized protein and free RNA is a sign of peculiar conformation of the nucleic acid in the virus. The alternative point of view of explaining the absence of additivity by the conformation of the protein component of the virus must be ruled out because of the X-ray diffraction data cited above which proved identity of structure between the repolymerized protein and the protein component of the virion. Detailed study of ORD patterns from native and denatured RNA preparations and whole virus showed that RNA in salt solutions has a typical positive rotation with a maximum at 260 mp. On denaturation in 8 M urea it is shifted to the long wave length region of the spectrum, its value thereby decreasing. It is assumed that this shift of the maximum to the long wave length region of the spectrum is due to the rupture of intramolecular hydrogen bonds, and a decrease in the value of the positive rotation maximum is an indication of the collapse of ordered secondary structure. From this point of view it is extremely interesting that TMV has a strong positive rotation in the spectral region above 275 mp with a maximum at about 285 mp. Accordingly, the “estimated” ORD curve for intraviral RNA has its inflection point (270-275 mp) close to that of denatured RNA in urea. At the same time, the absolute value for the positive rotation maximum of intraviral RNA was shown to be even higher than that for native RNA. This evidence introduces two important conclusions which are quite consistent with the above-described conception of intraviral RNA structure. First, the long wave length shift of ORD in intraviral RNA suggests the absence of planar base-base hydrogen bonding. Second, the strong positive rotation testifies to rigid orientation of the RNA bases in TMV. Later, Simmons and Glazer (1966) showed that addition of ethylene glycol to viral suspensions causes a drastic change in ORD patterns, one of the consequences being a sharp decrease in the value of the Cotton effect which reaches a value corresponding to the contribution of the protein a-helix. These changes are not connected with the disintegration of viral particles and the release of RNA into the medium as these effects are reversible and if ethylene glycol is diluted below a definite concentration

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a strong positive Cotton effect is restored. Ordered arrangement of the bases in intraviral RNA was shown also by means of UV dichroism (see below). It is of interest that regular location of the bases revealed both by means of UV dichroism and ORD did not respond to an increase in temperature up to 50 to 60”C, a t which temperature the viral particle disintegrated. It is well known that at such temperatures free TMV RNA in solution loses its ordered structure almost completely, and no dichroism is observed a t 40°C (Dvorkin and Spirin, 1960). These data allow one to speak not only about the regular arrangement of the bases in the intraviral RNA but even about their superstabilization as compared to the physical stability of helical portions of RNA in solution. One of the most important results obtained by Simmons and Glazer is that of reversibility of ethylene glycol action, i.e., ready dissociation and reassociation of the bonds which fix and immobilize bases in relation to each other and to the particle’s axes. Hence, the conclusion that the immobilization of bases in intraviral nucleic acid is not caused by steric hindrances limiting base rotations, but is the consequence of additional stabilization of secondary and tertiary structure although the physical forces which make such a structure stabilized are different from those which are operative in solution. Simmons and Glazer interpreted their results with ethylene glycol as an indication of the nature of these forces. They think that the action of ethylene glycol which disorders the regularity of the RNA bases in TMV should be ascribed t o the specific ability of this substance to rupture hydrophobic interactions between the planar bases and the protein component. It should be noted, however, that the suggestion of these authors about the specific action of ethylene glycol requires further verification and more convincing arguments. d. Other Methods of Investigation. Some data on the conformation of intraviral nucleic acids can be also found in rather heterogeneous experimental material dealing with the action of a number of chemical and physical factors on rodlike viruses. I n this connection the results of chemical modification of TMV are most interesting. Schuster and Wilhelm (1963) as well as Singer and Fraenkel-Conrat (1967) reported greatly different reactivity of various bases of intraviral RNA from TMV toward nitrous acid and N-methyl-W-nitronitrosoguanidine (“nitrosoguanidine”) , respectively. These two substances are well-known mutagenic agents whose action on nucleic acid largely depends upon the state of reacting functional groups and, other things being equal, is dctermined by the secondary structure of the macromolecule. The results of the experiments of Schuster and Wilhelm on RNA deam-

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ination in solution and in TMV are cited in Table I. It can be seen that deamination of adenine and guanine in free RNA proceeds at approximately the same rate which is about 1.5 times higher than that for cytosine. Although deamination at pH 5.0 is, certainly, slower than a t p H 4.2, the same level of modification may be achieved by sufficiently long incubation. This means that although the degree of RNA spiralization in solution depends on the pH of the medium (4.2-5.0) it does not determine the final results of the reaction. This is, evidently, due to the denaturation of helical RNA regions during long incubation. The situation is rather different in the case of TMV which, as demonstrated by the electron microscope, remains intact throughout the incubation. The most essential difference reported for deamination of free and intraviral TMV RNA is that guanine in TMV undergoes no deamination TABLE I THEREACTION OF RNA BASES WITH HNOa I N FREESTATEAND INSIDE TMV PARTICLES' ~~~

~

Free RNA

RNA in virions

Basesb

Control TMV RNA

29 hours pH 4.2

310 hours

pH 5.0

144 hours pH 4.2

Adenine Guanine Cytosine Uracil

26.3 22.5 17.2 35.5

19.7 16.3 14.3 33.5

22.2 19.3 15.3 33.5

21.4 23.0 11.1 33.5

The reaction conditions: 1 M NaNOz, 21OC;time indicated in the table.

* All data expressed as mole percent of bases.

at all, even if incubated for 6 days a t pH 4.2. At the same time both free guanine and that in double-stranded DNA and single-stranded RNA is deaminated at the greatest rate as compared with adenine and cytosine (Littman, 1961 ; Schuster, 1960; Tikchonenko e t al., 1966a, 1967). Although no rate constants are given by the authors, it may be estimated qualitatively that the reaction rate of adenine also greatly decreases while the deamination rate of cytosine in the virus decreases very little. As a result unlike free RNA, intraviral purines begin to be deaminated at a slower rate than pyrimidines. Since in the virion the deamination rate of one base changes very little, that of the second base considerably, and the third base undergoes no deamination a t all, it may be concluded that the specific character of their reaction in the virus is not connected with such factors as permeability of the protein shell. A change in permeability would have equally affected the reactivity of all the bases. Again, the complete resistance of intraviral guanine to dea-

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mination cannot be explained from the viewpoint of the traditional conception of partially spiralized RNA structure. The experimental results cited above showed that hydrogen bonding and base stacking in free RNA cannot be such a blocking agent. Hence the conclusion that here some specific blocking mechanism is operative for the guanine amino group which could be compared with, for example, blocking of some free €-amino groups of lysine in viral protein (see Tremaine and Goldsack, 1968; Perham and Richards, 1968). Chemical inertness of guanine as well as some other aspects of chemical modification of RNA in TMV should be, in all probability, ascribed to the lack of uniformity in the state and environment of the four bases along the polynucleotide chain in the virion. This is, first of all, typical of purine nucleotides which have a low reactivity in TMV. There is reason to believe that the decreased reactivity may be due to interaction between the purine bases and the protein. It will be appropriate to remember here the conclusions which Fraenkel-Conrat and Singer (1964) arrived at after experimenting with mixed and heterologous reconstructed TMV. They showed that i t is only polynucleotidcs rich in purine that can be reconstructed with TMV protein. It is quite possible that pyrimidines do not react a t all with the protein component of the virus, or if they do, the bond arising thereby is not strong enough to protect the pyrimidine amino group from chemical attack. The interaction of purines with the TMV protein is likely to be highly specific and connected with the sequence of nucleotides in the polynucleotide chain (see also Caspar, 1963). I n the virion this interaction is operative together with salt bonds between the RNA phosphate groups and the alkaline groups of protein. It is, probably, the result of either the hydrophobic interaction of the planar bases with the protein or hydrogen bonds between the amino groups of purines and the hydrogen-accepting groups of the protein, or both. Mixed reconstruction is so difficult just because of the highly specific character of these bonds and the “finest agreement” between the primary and secondary structure of RNA, on the one hand, and the corresponding arrangement of protein subunits, on the other hand. It is known that rod-shaped viruslike particles formed as a result of self-assembly of viral protein without RNA are characterized by low stability. Repolymerization of the protein in the presence of homologous RNA gives typical viral particles with usual physical stability. If homologous RNA is substituted by RNA from different sources or some TMV strains, the reconstruction either does not take place or if it does, the particles synthesized have an intermediate physical stability (Schramm et al., 1955 ; Schramm and Zillig, 1955; Hart, 1958; Fraenkel-Conrat, 1956, 1957,

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1963; Fraenkel-Conrat and Singer, 1957, 1964; Fraenkel-Conrat et al., 1959 a,b; Fraeiikel-Conrat and Williams, 1958; Holoubek, 1962; Matthews, 1966). The anomalous change in chemical reactivity of intraviral RNA resembles differences in the relationship between residual infectivity and mutagenesis level in the virus and free RNA treated with nitrous acid (Vielmeter and Schuster, 1960; Mundri and Gierer, 1968; Schuster and Schramm, 1958; Gierer and Mundri, 1958; Singer and Fraenkel-Conrat, 1966, 1967). I n all cases TMV particles had a much lower level of inactivation and a higher level of mutagenesis than free RNA. Such data taken from the recent paper of Singer and Fraenkel-Conrat (1967) are cited in Table 11. The reasonable explanation will be the fact that modification of guanine leads to inactivation only (Vielmeter and Schuster, 1960; TABLE I1 MWTAQENICITY OF TMV RNA AND TMV AFTER CHEMICAL MODIFICATIONS

RNA TMV RNA TMV

Treatment

pH

HNO 3 HNOs Nitrosogurtnidine Nitrosoguanidine

4.7 4.5

-

I

Remaining infectivity (yo) Mutagenicity" 20 15 1.0-20 0.2-20

19 14 2.4-2.7 23-37

a The ratio of numbers of local lesiom given by a similar number of infective particles (as determined on Xsnthi) aa compared to simultaneously tested control reconstituted RNA.

Singer and Fraenkel-Conrat, 1967) while deamination of other bases brings about mutation or inactivation. Guanine in TMV displays an abnormally low sensitivity to deamination, hence the unproportionally low value of inactivation and abnormally high level of mutagenesis as compared to the behavior of free RNA. A similar situation was observed in Fraenkel-Conrat's experiments with nitrosoguanidine mentioned above. In the case of free TMV RNA, nitrosoguanidine reacted mostly with guanine and, t o a much lesser extent, with cytosine. Determining infectivity and mutagenesis in preparations of RNA and the intact virus, these authors found a high level of mutagenesis and a low level of inactivation of RNA in the virus and a rather low level of mutagenesis in isolated RNA. These results can be interpreted like those in the case of nitrite, although the mechanism of action of nitrosoguanidine has not been elucidated. Analysis of the experimental results leaves almost no doubt about the higher resistance and chemical inertness of some bases in intraviral

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RNA. Probably, the same phenomenon is also responsible for the lower sensitivity of TMV, as compared to free RNA, to the inactivation by CHzO which was described by Staehelin as early as 1958. According to his data, 0.1% CHzO caused complete inactivation of free RNA within 1 hour whereas in the case of intact TMV even 24-hour incubation with 1%CHZO entailed only a partial loss of infectivity. Unfortunately, the experimental results reported by this author do not allow one to evaluate the specific contribution of the protein with a sufficient level of confidence. On the one hand, the protein shell hinders CH20 diffusion into the particle “on its way” to the RNA, especially a t the last stages of the reaction when cross-links appear in the protein (see review by Bachrach, 1966). On the other hand, the reaction of CH2O with protein also causes inactivation of virions. That is why, if the first factor will increase the resistance of TMV to the action of CH2O the second factor will neutralize it to some extent. And although the exact proportion of each of these factors in the total inactivation level is not known, the difference between the sensitivity of the native virus and free RNA is so great that it could be explained only in terms of conformational peculiarities of the RNA in TMV. Interesting information about the properties of intraviral RNA can be also obtained from the experiments with TMV exposure to UV light. Tao et al. (1966) and Goddard et al. (1966) showed that exposure of viral suspensions to UV light does not cause dimerization of neighboring pyrimidines although this very reaction was one of the main causes of inactivation of free TMV RNA. The absence of such dimers may be unequivocally explained in terms of the concept of the absence of interaction between the bases of intraviral RNA and the specific geometry of its helix which was elucidated above. It should be noted that some authors observed that after exposure to UV light the bonds between the protein and nucleic acid in the virus become much stronger. RNA preparations obtained from UV-irradiated virus contain much more protein. Tao et al. (1966) think that these phenomena are likely to be the result of cross-linking between RNA uracil and protein subunits of the virus. TMV was labeled with 36S,exposed to UV light, and then disintegrated by detergent, The label was shown to be present in the RNA and its quantity amounted to one peptide per molecule of RNA. Appearance of cross-links between RNA and protein in the UV-exposed virus could be the explanation for the old findings of Bawden and Kleczkowski (1959). They stated that it is not the intact virus but the UV-irradiated free infectious TMV RNA which is photoreactivated (photoreactivating systems are known t o remove pyrimidine dimers) . The cross-linking between the protein and nucleic acids was confirmed by ex-

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periments where various nucleoproteins and mixtures of bases and amino acids were exposed to UV light (Smith and Aplin, 1966; Smith and Meun, 1968; Smith and O’Leary, 1967; Smith et al., 1966). On the other hand, Kleczkowski and McLaren (1967) are rather skeptical about this interesting cross-link hypothesis as they observed no increase in specific infectivity after TMV deproteinization by phenol which ruptures the protein-RNA bonds induced by UV light. But one can not exclude the possibility that phenol has its effect in some, not very “delicate,” way which causes the cleavage of the RNA molecule where it has been cross-linked (see the data of Kaper and Jenifer below). The lower sensitivity of the double bond of the TMV pyrimidine ring to radiation hydration revealed by Tao et al. (1966) and Evans et al. (1966) is also likely to be connected in some way with the specific conformation of its RNA. According to the data of the first group of authors free TMV RNA is inactivated by UV light in H2O faster than in D20. At the same time the rate of inactivation of intact TMV in DzO and HrO turns out to be the same which means the absence of photolysis of pyrimidine double bonds. Evans gave direct evidence for pyrimidine photolysis being absent among primary photochemical reactions occurring in TMV after exposure to UV light. The most probable explanation for such an anomaly is that of TMV RNA being localized in the hydrophobic core of the viral protein. This point of view is in good agreement with the idea of Simmons and Glazer (1966) concerning the hydrophobic interaction between protein and RNA in TMV. Unfortunately, we still do not have a t our disposal methods of determining the hydration of intraviral nucleic acid and all of our knowledge pertains to the total hydration of virions (Lauffer and Bendet, 1954). Among the speculations on this subject the opinion of Tremaine and Goldsack (1968) and Perham and Richards (1968) should be mentioned who, on the contrary, think that the internal portions of protein which make contact with the RNA as well as the surface (the internal channel included) are hydrophilic and consist, for the most part, of polar amino acids. But these authors proceed from the theoretical concept for simple monomer proteins suggested by Kendrew (1962), Wyckoff et a$. (1967). To what extent all this is true for the complex polymer of ribonucleoprotein is not known. The problem which is being discussed is very closely related to the question concerning the nucleoprotein nature of viruses. Although the very fact that both protein and nucleic acid are present in the viral particle seems to be the real answer, the situation is, in fact, much more complicated. Watson (1953), for example, denied the nucleoprotein nature of phages; he thought them to be just ‘(a bag with liquid content.”

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From such a point of view the presence of protein and nucleic acid in the viral particle may be looked upon as the biological analogy of two political systems peacefully coexisting but having no diplomatsic relations. Hence, to solve the question of whether or’not TMV is a nucleoprotein in the true sense of the word, it is necessary to prove the presence of specific bonds and interaction between the protein and nucleic acid. Salt bonds are of primary importance as they are the main force in the interaction responsible for the very existence of tissue nucleoproteins. It is understood that the salt bond should arise between the phosphate groups of the RNA and the free amino groups of the viral protein. Naturally, such a situation is possible, only in the case of a polar hydrophilic environment for the RNA molecule in the protein “flesh” of TMV. This question was dealt with in the very old and not quite unequivocal experiments of Kaushe and Hahn (1948) who used, for this purpose, acid and basic dyes. According to their data, intact TMV bound acid dyes equivalent to 14,000 base groups per mole of viral protein and basic dyes equivalent to 45,000 acid groups per mole of viral protein. A purified preparation of TMV protein contained the same quantity of acid groups but there were 11,000 additional basic groups. Thus, the removal of RNA from the virus was accompanied by the unmasking of 11,000 basic groups which were believed by the authors to participate in the formation of salt bonds with the RNA. As TMV RNA has 6000 nucleotides, the degree of correspondence seems to be quite satisfactory, at least arithmetically. To arrive at a final answer to this question, one must have direct evidence that the appearance of additional basic groups is really due to the rupture of the salt bonds with RNA and not to conformational changes of the protein itself after removal of the RNA. The present findings of Perham and Richards (1968) and Fraenkel-Conrat and Colloms (1967) give a more definite, but still not final, answer to this question. According to the data of these authors who used selective chemical modification of viral protein, immidoester (methyl picolinimidate) reacts with the c-amino groups of the 53rd and 68th residues of lysine in isolated viral protein. In intact virus it reacts only with the 68th residue (as a matter of fact, the question about the reactivity of the 53rd lysine residue in TMV is more complicated but it does not change the picture). The c-amino group of the 53rd lysine residue is involved in an ion pair either with an RNA phosphate or with a carboxyl of the same or an adjacent peptide. The RNA bonding is more probable as Fraenkel-Conrat and Colloms (1967) found that acetylation of the viral protein a t the 53rd residue of the peptide chain renders impossible re-

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construction of infectious virus from the mixture of protein and RNA whereas acetylation of the 68th residue did not affect reconstruction. The experiments of these two authors seem quite convincing and account for the salt bond for one third of all thc RNA phosphate groups as there are three nucleotides per one peptide chain. The mode of blocking of the remaining phosphate groups is still unknown. To sum up, at least some part of the protein neighboring the phosphodiester backbone of the RNA molecule should have a polar, hydrophilic nature. Does this mean that all portions of the RNA molecule have the same environment? Evidently, the answer should be in the negative1 Because the RNA-protein salt bonds do not explain either the change in reactivity of the bases in the intraviral RNA, or the absence of photolysis of pyrimidine in TMV exposed to UV light, or the results of Simmons and Glazer (1966). It is quite possible that the bases of intraviral RNA, unlike its phosphate groups, are surrounded by hydrophobic sites of the peptide chains. Such an assumption would reconcile the seemingly contradictory experimental results. This point of view is in complete agreement with the results of numerous investigations of the interaction of nucleic acids and nucleotides with basic proteins. It has been shown, unequivocally, that the interaction between nucleic acids and protein involves not only nonspecific salt bonds between the acid phosphate groups and positively charged side chains of the polyamino acid. Besides these nonspecific bonds, there is the contribution of bases which is quite specific and is due to nonelectrostatic hydrophobic interaction of these two components. It should be added that differences in the composition of the polynucleotide bases and differences in the amino acid composition of the polypeptides has a noticeable effect on the strength of such bonds and the specific character of their interaction (Chargaff et al., 1953; Spitnik et al., 1955; Lucy and Butler, 1955; Johns and Butler, 1964; Akinrimisi et aZ., 1965; Tsuboi et al., 1966; Leng and Felsenfeld, 1966; Sober et al., 1966; Ohba, 1966; Olins et al., 1967; Wagner and Arav, 1968). 2. Tertiary Structure

Some parameters of the tertiary structure of TMV RNA in vivo are, naturally, predetermined by the entire structure and the very existence of the virion. From this rather formal point of view, intraviral RNA should be described as an open, single, rigid, rod-shaped helix with a diameter of 80 A and a length of 3000 A. All we know about the tertiary structure of single-stranded RNA in solution (see the review by Spirin, 1963) is absolutely contradictory to the above description and, therefore, does not need any comment. And it is only in a solution of low ionic

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strength and a t low temperatures that the tertiary structure of free RNA is more or less similar to that in situ, a t least formally. I n this case RNA is visualized as a rod-shaped particle with diffuse contours, 1200-2500 A long and 30-40 A wide (Rice, 1961 ; Spirin, 1963). One can see that even the parameters of this most similar form, to say nothing about some other properties, make it different from the tertiary structure of intraviral RNA described above. These problems are being intensively investigated by means of flow birefringence and flow UV dichroism. These methods allow one to obtain valuable information as to the orientation of planar bases towards the long axis of the nucleic acid molecule or viral particle. It is believed that the flow UV dichroism method can furnish quantitative information, a t least in principle and a t least under favorable conditions. However, Gray and Rubinstein (1967) doubt this optimism based on their data on the identity of dichroic spectra and dichroic ratio between A and B configurations of DNA. (The inclination angles of these configurations differ by 20".) Tobacco mosaic virus may be successfully investigated by this method if viral particles in preparation are well oriented and the degree of orientation is correctly estimated, i.e., knowledge of the angular distribution of the particles in the sample is indispensable. The second stumbling block is to separate from the total preparation spectrum the specific spectral contribution of nucleic acid bases. The ideal solution of the first task would be to have well oriented gels of the type used for X-ray diffraction examinations. But simple calculation will show that it is only by using capillaries of about 10 p wide that this aim will be achieved with such methods as UV dichroism. Such fineness has proved too much to desire (Schachter et al., 1966). That is why orientation of particles in flow is used to this end. This presents some additional difficulties as to determination of the particles' orientation. At the same time, as follows from Fraser's equation (1953), knowledge of the size of the fraction of the oriented particles (F) is essential for determination of the orientation angle of chromophores to the particle axis 8. That is:

where 8 is the angle between the molecular axis and the normal to the chromophore, and R is the value of the dichroic ratio. The value of f can be calculated for objects where R is measured and 8 is known. For example, if for the double-stranded poly A used by Gabler (see below) the planes of adenylic acid residues are oriented at an angle of about 10"

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from the perpendicular to the molecular axis, i t . , 6 = 90” - 10” = 80” (Rich e t al., 1961) and R = 0.88 (Gabler, 1967), then we shall have f = 0.09. So low a value for f will be acceptable if one takes into account that the length of its molecule in the experiments cited did not exceed 300 A. Bendet and Mayfield (1967) found themselves in still greater difficulty trying t o determine the degree of orientation of filamentous phage Fd because they used as a marker T2 DNA. The dichroic ratio ( R ) of this DNA at 260 mp was determined to be 0.4, the base plane tilt in the B form about 0”, and the value of f was calculated to be 0.5. But as the authors had to deal with phage Fd the transition is made by a statement which is too sophistical to be called anything else: “T2 is unusually stiff hydrodynamically and since Fd is probably more rigid than DNA, f = 0.5 would seem to be a conservative estimate for this parameter.” It is only too natural that the calculation based on such a foundation is rather questionable. For the above reason all the authors dealing with this problem (Schachter e t aE., 1966; Bendet and Mayfield, 1966, 1967; Gabler, 1967) speak about “more or less perpendicular (or parallel) ” position of bases toward the long axis of the particle without giving exact values for 8 and it was only Mayfield (1968) who succeeded in putting forward an approximate theory which suggests a quantitative relationship between the dichroism of partially oriented solutions of rodlike particles and the anisotropic light scattering manifested by such solutions. This theory makes use of differences between that scattered light which is perpendicular and parallel to the particle axis to estimate the degree of orientation. A plot U measure of light scattering) us. All/& (a measure of diof T ~ ~ / T (a chroism) for various values of 6 allows easy extrapolation to perfect orientation. I n its turn, knowledge of the dichroism for the perfectly oriented rods allows one to calculate an effective chromophore orientation at various wavelengths. At any rate, the theoretical and experimental curves fit well in Mayfield’s data. The second obstacle, that of separating the specific spectral contribution of intraviral nucleic acid is being overcome for TMV (but not for Fd). As early as 1955 Franklin found that oriented TMV gels have a higher positive birefringence than that of repolymerized protein (P.TMV) . If one takes into account that the only difference between the whole TMV and P.TMV is the presence of RNA in the former, the conclusion may be drawn about the preferential orientation of RNA bases parallel to the long axis of the particle. But this conclusion is somewhat tendentious as birefringence helps to determine the optical properties of the whole preparation and cannot be used to examine the orientation of the separate chemical components of the RNA (sugar, phosphate, or base).

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The technique of flow UV dichroism allows one to determine selectively the orientation of components which absorb in UV light. Such investigations were performed by a number of authors with rodlike and filamentous viruses: Butendant et al. (1942) ; Seeds and Wilkins (1950) ; Perutz e t al. (1950) ; Schachter et al. (1966) ; Schachter (unpublished results) ; Bendet and Mayfield (1967) ; Gabler (1967) ; Mayfield (1968). In the simplest case, when the aim is to investigate intact viral particles, the total dichroiem pattern obtained represents the four nucleic acid bases and three aromatic amino acids (tryptophan, tyrosine, phenylalanine) . It is not out of place to mention here that maximum absorption of asymmetric polymers containing conjugated planar chromophores occurs when the light electric vector is parallel to the planar bases. And, respectively, minimum absorption should be expected with the vector pcrpendicular to the plane of the chromophore (Beaven e t al., 1955). The experiments of the above author showed that when the electric vector was polarized parallel to the TMV molecular axis, minimum absorption was observed. The minimum absorption value was recorded with perpendicularly polarized light, the medium value, with nonoriented TMV preparations. As the absorption spectra of bases and aromatic amino acids overlap to a considerable extent, it is next to impossible to separate specific spectral contributions from the protein and RNA in the case of the intact virus. Such an attempt was made by Schachter et al. (1966) and Gabler (1968). These investigators, like Franklin (1955a,b) , measured the dichroism of intact TMV and P.TMV. Monochromatic UV light allowed them to confine the components investigated to those absorbing in the region of 230 to 300 m p . Assuming that the dichroism of the viral protein component and that of the P.TMV is the same, it was possible to obtain dichroic characteristics of intraviral RNA by substracting the dichroic value of the P.TMV from the dichroic spectrum of the whole virus. But the results obtained by means of the above methods cannot be interpreted directly because of the possible effect of so-called form dichroism. There is always a possibility of some part of the dichroism value being due not to the actual structure of the macromolecule but to its form and the refractive index difference between it and its surrounding medium. But Bendet and Mayfield (1967) and Mayfield (1968) showed that the dichroic ratio for phage Fd and TMV does not practically depend upon the concentration of monovalent cations in the medium when the refraction index under such conditions changes from 1.333 to 1.400. Some increase in positive dichroism noted by these authors on addition of glycerol to the medium was attributed to better orientation

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of the viral particles due to higher viscosity of the solvent. Thus, it may be concluded that form dichroism should not interfere with the interpretation of the results obtained, at least in the case of rodlike and filamentous viruses. Using the above technique, Schachter et al. (1966) showed the dichroism of both intact virus and protein to be positive in the region of 240 to 294 mp, and negative in the region of 295 to 301 mp. The dichroic ratio in the region of 280 t o 301 mp was almost similar for intact TMV and P.TMV whereas a t shorter wavelengths the dichroic ratio of TMV by far exceeded that of P.TMV. The dichroic spectrum and the value of the dichroic ratio for intravial RNA was obtained, as was indicated above, by subtraction of the P.TMV curve from that for TMV. The identity of the dichroic spectra of the protein component of P.TMV and TMV is of paramount importance for the method of obtaining difference spectra for the RNA. And there are grounds to believe that they are identical or, at least, nearly identical. The reason for such a statement is the great similarity of the dichroic ratio profiles of intact TMV and P.TMV in the spectral region of 280 to 300 mp, i.e., that of absorption of the aromatic amino acids. Second, similarity of X-ray diffraction pattern and birefringence values for highly humid P.TMV and TMV gels testify t o the identical structure of protein in these two objects (Franklin, 1955a,b), And third, this point of view is also confirmed by the results of physicochemical and immunobiological investigation of products of viral protein repolymerization (see Fraenkel-Conrat and Ramachandran, 1959). Viral RNA has a characteristic positive dichroism over the whole UV spectral region with a maximum a t 2540 which is evidence in favor of the preferentially parallel orientation of planar bases to the longitudinal axis of the particle, Based on the tertiary structure of RNA in TMV particles it may be concluded that the sugar-phosphate backbone is perpendicular to the long axis of the particle. Such a position of the RNA helix would agree with the geometry of the a-helix of viral protein both in intact and repolymerized viral protein (Fraser, 1952). The axis of the protein a-helix has a preferentially perpendicular orientation to the long axis of the particle where the amino acid side chains (aromatic in this case) are oriented preferentially parallel to the long axis of the particle. It should be emphasized that we may be certain only about the orientation of tryptophan and, to a lesser extent, tyrosine as it is the absorption of the former amino acid that dominates in the UV spectrum of thc protein. Mayfield (1968) made an attempt to determine the exact value of the tilt angle of the TMV chromophore groups, but, unfortunately, this author for some reasons used intact virus. Therefore, the data cited below

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are the mean values of tilt angles of all chromophores responsible for dichroism, i.e., the bases and aromatic amino acids taken together. For the five wavelengths observed by Mayfield (247, 270, 284, 291, and 298 mp) the calculated values of the angles were 45 +- lo,49.5 -t. 0.5", 48.5 -I 0.5", 48 -L 0.5", and 60 t 2", respectively, which amounts to a mean angle of about 50". There is no doubt that a series of experiments with intact TMV and repolymerized viral protein will allow one in the future t o determine the orientation angles of the bases proper. While in TMV the bases are oriented parallel to the particle axis and RNA helix, in the case of helical polynucleotides in solution the situation is quite different. Preparations of double-helical DNA oriented in one way or another have strong negative dichroism, i.e., they absorb light intensively when its electric vector is perpendicular to the DNA long axis (Beaven et al., 1955; Dvorkin, 1961; Dvorkin and Krinskiy, 1961 ; Seeds, 1953 ; Thorell and Ruch, 1951). Base orientation in the RNA molecule is a more complex matter. As was shown by Frishman et al. (1963) ,the positive birefringence of highly polymeric-oriented RNA after partial degradation gave way t o negative birefringence. Such anomalous behavior is accounted for by the fact that in the first case orientation of the bases in the helical portions of the molecule in relation to the long axis of the rodlike particle was recorded (Spirin, 1963). In other words, the planar bases in the helical portions of the RNA, are normal to the axis of the helix, like in DNA. But as the axes of these helical portions are oriented perpendicular to the long axis of the whole rodlike molecule of RNA, the bases will have a parallel orientation in relation to the axis of the rod. Mild hydrolysis of the molecule in solution destroys the complex tertiary rod structure yielding helical fragments with small single-stranded ends. Now, the orientation of these fragments in flow will orient the bases toward the axis of the helices, and hence the appearance of negative dichroism, as is the case with DNA. All this experimental evidence suggests that the bases in the helix of intraviral RNA in TMV have an essentially different orientation as compared to that of RNA in the helical portions of the molecule in solution where the orientation angle of the bases is of the order of 10 to 20" (Michelson, 1963). But, if the analysis of measurements on the whole virus reveals the dichroism which represents an average for the protein and nucleic acid, the subtraction of the spectral contribution of the aromatic amino acids will yield the average for the four bases. And in spite of the fact that the above cited X-ray diffraction evidence may be interpreted in favor of the uniformity of the tertiary structure of intraviral RNA along all its length, the results of chemical modification of TMV (see above,

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Section II,A,l,d) testify to the nonuniform character and environment of the four bases inside the virion. From this point of view the attempt of Gabler (1968) would appear to be very interesting and promising. This investigator compared the dichroism not of the virus and P.TMV but that of P.TMV and protein repolymerized in the presence of poly A (A.TMV) . Hart and Smith (1956) and Fraenkel-Conrat and Singer (1964) were the first to synthesize viruslike particles of this kind containing a homopolymer of adenylic acid. Such viruslike paticles are morphologically similar to TMV but they have no infectivity, lower physical stability, lower content of phosphorus, and their average length is less than that of TMV. The last two facts are responsible for some features of the dichroic spectra of A.TMV, and particularly the lower value of positive dichroism below 2900 k.Like in the previous case, dichroic spectra of poly A were obtained by subtraction of the P.TMV dichroic spectrum from that of A.TMV. Intraviral poly A was shown to have both a maximum positive dichroism and a maximum value of dichroic ratio in the region of maximum absorption, i.e., a t 260 mp, while a t shorter and longer wavelengths the author detected, quite unexpectedly, some regions of small negative dichroism. The negative dichroism of intraviral poly A in some spectral regions compared to the positive dichroism of RNA inside TMV introduced some complexity into the interpretation of the above results. Generally speaking, such a discrepancy could be accounted for by an extrapolation error for the different wavelengths; as such a probability is greatly increased in the case of A.TMV because of its low UV absorption value due to a polynucleotide deficiency. An alternative explanation, which the author thinks hardly probable without clarifying such a point of view, is a different character of orientation of a part of the adenine residues. But, if one takes into consideration the high degree of stereospecificity typical of the interaction between the protein and nucleic acid components of TMV, such a state of things would seem probable when instead of homologous RNA the protein reacts with a synthetic homopolymer (see above discussion of the problem of mixed reconstitution of TMV) . Nevertheless, one is inclined to agree with the author that the coincidence of the maximum dichroism region and the maximum absorption region allows one to conclude that at least the greater portion of the bases in intraviral RNA is for the most part parallel to the long axis of the particle. With the dichroic ratio value R = 1.2 (that of TMV was equal to 1.6 in these experiments), the tilt angle of adenine bases to the long axis of the particle was shown to be 52". Remembering that, according to the data of Mayfield, the mean statistical orientation angle of all chromophores was about 50", the above figure for the orien-

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tation angle of adenine seems to be quite realistic if not practically identical. Besides the above differences in the dichroism patterns of intraviral RNA and poly A, the reasons for which still remain obscure, poly A and A.TMV have one more peculiarity in common. The curve describing the dichroic ratios for poly A in A.TMV has a plateau in the spectral region of 2500 to 2750 b. The presence of such a plateau should be explained in all likelihood, by the presence of just one chromophore group in poly A. Comparison of poly A structure in solution and in A.TMV will reveal the same differences as in the case of free and intraviral RNA. Poly A has a negative dichroism a t acid pH where it is helically organized at all wavelengths with a minimum of -0.4 ml/mg-mm a t 2510 A. At the longer wavelengths the dichroism gradually increases to reach zero values a t 2900 A. These data are unequivocal evidence in favor of the fact that adenine residues in free poly A are not oriented parallel to the axis of the molecule as is the case inside A.TMV, but instead, perpendicular. The perpendicular orientation of adenine in this case is consistent with the above-mentioned model (Rich et al., 1961) where the bases are oriented a t an angle of 10" toward the long axis of the molecule. A similar picture of base orientation was suggested by Bendet and Mayfield (1967) for the filamentous phage Fd, containing single-stranded ring DNA (Marvin and Hoffman-Berling, 1963a,b; Marvin and Schaller, 1966). These authors found that UV flow dichroism of phage Fd was positive for wavelengths longer than 262 mp and shorter than 239 mp. The region between these two wavelength values revealed negative dichroism. The impossibility of preparing repolymerized protein for this phage prevented Bendet and Mayfield from obtaining the difference spectrum and stimulated all kinds of speculations. They constructed an artificial dichroic spectrum based on the known values of the specific absorption of DNA and protein of the Fd phage and on the assumption of their additivity. The latter assumption is rather problematic, which is recognized by the authors, because between the experimental and theoretical spectra there is a deviation-a 5 mp shift to the long wavelength region of the spectrum. As a result of this and some other assumptions indicated above, the calculated value of 8 for DNA in phage Fd was de5". Unfortunately, no data are presented on the termined to be 25 orientation of the bases in free single-stranded DNA of Fd phage, although there are grounds to believe that it must be similar to the base orientation in RNA. Tryptophan and, possibly, tyrosine in the protein component of this phage are oriented practically parallel to the long axis of the particle, like in TMV.

*

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Thus, it may be considered as proved that inclusion of RNA, synthetic polynucleotide, or single-stranded DNA in the virion entails the radical change of its structure (change of diameter, pitch of helix, base orientation, etc.). It should be emphasized that the values for the tilt angle of the bases determined in the papers cited in this review, are not encountered in any conformational transitions observed in ribo- and deoxyribonucleotides, either single or double helical. (Langridge and Rich, 1963; Tomita and Rich, 1964; Gomatos, et al., 1964, Zubay and Wilkins, 1960; Rich and Watson, 1954; Brown and Zubay, 1960; Klug et al., 1961 ; Fuller, 1961). It is not excluded that such a position of the bases represent some universal principle of organization in a t least some nucleoproteins, as RNA bases in ribosomes are also oriented preferentially parallel to the electric axis of the ribosomal particle (Michelson, 1963; Morgan, 1963). B. Spherical (Isometric) Viruses 1. T e r t k r y Structure

a. X - R a y Diffraction and Electron Microscope Data. Until recently spherical isometric viruses were believed to consist of a central body (core) made of nucleic acid and an outer protein shell. They were thought to be different from rodlike viruses because of the minimum interaction between the protein and nucleic acid confined to surface contact: the shell-the central body. The bulk of X-ray diffraction and electron microscope data was obtained by the Cambridge group for the RNA-containing TYMV. They showed that such a point of view seemed t o be a simplification. Klug and Finch (1960) were the first to suggest the new concept of the structure of spherical viruses. They showed that the X-ray scattering curves for the native virus is no simple sum of the diffractions from the mixture of viral protein and RNA. I n accordance with their data the RNA in the virus must have a complex configuration. It was shown later that, as in rodlike viruses, the RNA in TYMV is in intimate contact with protein, the type of protein symmetry determining that for the RNA, although, maybe, not so accurately and completely as in TMV. Fortunately, though TYMV protein does not repolymerize in vitro to form protein viruslike particles containing no RNA, TYMV preparations have a so-called “top component” which constitutes empty viral protein coats having no RNA (Markham, 1951). This component crystallizes like the intact virus yielding mixed crystals (TYMV plus top component) as well. I n both cases the result is a cubic unit cell

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with a side of 700 A. X-Ray diffraction patterns from the protein component of native TYMV and from isolated protein coats appeared to be identical within the limits of resalution of this method (Klug et al., 1966a)b). The isamorphous structure of the protein in these two objects make it possible to obtain information about the organization of the RNA in the virion by subtracting the diffraction of the coat from that of the virus. Crystalline preparations of TYMV and top component have a diamondtype lattice with eight particles per unit cell (Bernal and Carlisle, 1948; Klug et al., 1966a)b). The large size of the unit cell limits the possibilities of the method to a great extent, the resolution not exceeding 20 being one of the problems. According to the above authors, TYMV and the top component are icosahedrons with a radius of 140 A to 150 A, consisting of 180 peptide chains. The electron micrographs of the intact virions reveal 32 morphological subunits (Huxley and Zubay, 1960; Nixon and Gibbs, 1960; Haselkorn, 1962; Finch and Klug, 1966) of which 12 are pentagons and 20 are hexagons. Comparison of X-ray diffraction and electron microscope evidence shows that the viral particle does have 32 scattering centers with a radius of 125 A. More intensive scattering from these centers as compared to that of the protein subunits allows one to think that they consist, most probably, of local RNA condensates. Such a suggestion is confirmed by X-ray diffraction patterns from TYMV crystals stained with uranyl acetate which is an electron microscopic stain specific for nucleic acid. Such staining enhanced specific scattering in the region occupied by RNA. Similar, but not identical, data were obtained in cases where X-ray diffraction from TYMV was measured in solutions of ammonium sulfate of various concentrations. As the electron density of protein is lower than that of nucleic acid, in a medium with an electron density of 0.407 el/A3 [3.7 M ammonium sulfate] the reflections typical of protein disappear (Perutz, 1946). As expected, the reflections from the nucleic acid did not disappear on disappearance of the protein reflections, although they were weaker due to the lower contrast. As was indicated above, the RNA in TYMV particles is not a perfect sphere with a radius of about 125 A but a complex structure without a uniform angular distribution. The bulk of the RNA is localized on 32 radial bumps. The radial portion of these bumps goes along the axis of the protein subunits, their effective radii being 145 and that of the RNA about 125 A. The transverse portion of the bumps winds around five to six protein subunits. All 32 bumps are located a t a distance of 85 A from each other and fit into

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the symmetry of the protein capsid. It is assumed that the RNA has a closed ring structure as, according to Klug and Finch (1960), a ring form of the nucleic acid in the icosahedral protein shell will provide more adequate packing than a linear form. The main advantage of such an arrangement is the uniform contact between protein and RNA along the entire polynucleotide chain. As yet there is no detailed picture for the distribution of the other portion of the RNA in the central part of the particle because the method is not sensitive enough. This tertiary structure of the intraviral RNA and its close contact with the protein subunits is responsible for the 32 morphological subunits which are readily distinguishable in the electron micrographs of native TYMV but are absent in empty protein capsids. To sum up, Xray scattering a t low and wide angles testifies to the icosahedral symmetry of the TYMV RNA in situ. RNA is buried deep within the protein and its large segments form bumps wound around groups of 5(12) and 6(20) structural subunits, respectively. Similar tertiary structure with close contact between RNA and protein subunits (32 capsomers) was reported by Finch, et al. (1967) for another small icosahedral virus, a BBMV. But this is not the only point of view concerning the tertiary structure of RNA in virions. The conclusions of Fischbach, et al. (1965) who studied low-angle X-ray diffraction patterns from suspensions of a number of plant viruses (BMV, BBMV, WCMV) are more conservative. They do not put forward any clear idea for the RNA arrangement in virions but proceed from the assumption that all viruses must have an outer shell of protein which surrounds a RNA core. Katz and Rich (1966) make similar conclusions analyzing low-angle X-ray diffraction data obtained from wet gels of MS2 and +X174 phages. Unfortunately, the brevity of their report does not allow for a more detailed discussion. This old concept of the central RNA-containing core is hardly consistent with the fact that none of the viruses show a large difference in density between the protein shell and the RNA core. The partial specific volume of RNA is much smaller than that of protein and might lead one to expect the RNA core to have a much larger density than the protein shell. It should be mentioned that in Klug’s model this problem does not arise as the RNA is distributed more or less uniformly within the virion. Fischbach thinks that this absence of an essential difference in electron density between the central portion and periphery of the virion is due to a large hydration of the nucleic acid and a lower hydration of the protein so that both the protein shell and the RNA core, except for the hollow center, have approximately the same average electron density of 1.2 times that of water. This point of view is somewhat strengthened

227

CONFORMATION OF VIRAL NUCLEIC ACIDS IN SITU

by the fact that in all cases the volume of the virus is roughly twice as large as the “dry volume” calculated from the molecular weight and partial specific volume of viral components (see Table 111). The excess volume must be due to solvent in close association with the protein and RNA. Unfortunately, we do not have a method for evaluation of the hydration of these two components in the virus (Lauffer and Bendet, 1954). An opposing idea concerning the hydration of nucleic acids in virions is supported by Pollard (1953)’Tikchonenko e t al. (1966a,b,c), and Maestre and Tinoco (1967). These authors proceed from the conception of a deficient hydration for the nucleic acids in the virions. Their argumentation, however, might be accused of speculative trends. The only experiment in this respect (Maestre and Tinoco, see below) dealt with TABLE I11 MOLECULAR PARAMETERS OF SOMEVIRUSES ~~

Virus

Protein

~~

RNA

Molecular weight ( X 10-6)

Virus

R17”

3.6 4.6 5.2 7.0

BMV* BBMVc WCMVd

2.5 3.6 4.1 4.6

1.1 1.0 1.1 2.4

Outer radius

(A)

133 130 147 140

~

Volume of sphere

(A x

10-6)

9.8 9.2 13.3 11.5

Dry volume (A8

x

10-6)

4.0

5.4

6.3 7.8

~~

Fischbach et al. (1965). Anderegg et al. (1963). c White (1962). Anderegg et a.?. (1961).

@

double-stranded DNA. It is not impossible that the case for hydration may be quite different for RNA-containing viruses. Finally, the evidence on selective degradation of RNA inside some plant viruses (see below) also favors Klug’s concept. According to Klug et al. (1966b), their experiments with X-ray diffraction in media of different density may give an indication, although not very direct, of the nature of the forces binding RNA and protein subunits in TYMV particles. It was shown that the low-angle scattering pattern from virus crystals in 1 and 4 M ammonium sulfate are very different. Reflections, 4n 2, typical for this mode of packing and visible in 1 M ammonium sulfate, disappeared on increasing the salt concentration to 4 M . A simultaneous decrease in the effective radius of the RNA from 125 to 117 A was observed. The authors offer two explanations for this fact. The first

+

225

T. I. TIKCHONENKO

one is that the distal portions of the RNA bumps have a lower density than the middle portion of the radial thread. Hence the increased electron density of the solvent, as in the case of protein, masks this portion of the structure. As a result, the scattering from the denser middle portions of the radial threads is recorded which leads to a smaller radius. The second explanation is that of dissociation of salt bonds between the RNA segments and protein in 4 M ammonium sulfate leading to a collapse of the RNA molecule “stretched” on the protein subunits. This 2 reflection and the smaller radius causes the disappearance of the 4n of the light-scattering structure. In this case the greater portion of the RNA, almost all the RNA, in fact, in TYMV particles, is a nucleoprotein with typical salt bonds. The authors are inclined to prefer the second point of view which is more interesting and promising but they offer practically no arguments for such a choice. The complete reversibility of the action of 4 M ammonium sulfate may raise some doubts as to the correctness of such a preference. I n this case we would be bound to conclude that the profound change in RNA conformation and the rupture of salt bonds do not affect in the least the intactness of the virion. But these same authors discussing the papers by Kaper (1964) and Stols and Veldstre (1965) concluded that the change of RNA conformation in situ brings about fatal consequences for the viral particle. These considerations are of rather a speculative nature as the conditions of Kaper and Stols were different from those of Klug et al. But this dilemma is easy to resolve in a purely experimental way. It is natural to suggest that the change in RNA conformation and the rupture of nucleoprotein bonds will be accompanied by a considerable change in the optical properties of the intraviral RNA. The second distinguishing feature of the tertiary structure of the nucleic acid of a number of small spherical viruses is a central cavity where nucleic acid is either absent or present in concentrations many times lower than that in the rest of the virion. The presence of a central cavity was proved for WCMV, BMV, BBMV, phage R17, and phage +X174 (Anderegg, et al., 1961, 1963; Fischbach, et al., 1965; Katz and Rich, 1966; Finch et al., 1967). Such things are usually proved by means of X-ray diffraction and electron microscope evidence which, when used siqultangouslg, preclude the possibility of artifacts. The size of the central cavity in various viruses is more or less similar and amounts to 90 A t(BMV) or 100-120 k (BBMV). This is less than 1 3 % of the total volume of the particle. Phage R17 is the only one to have an inner cavity of much less size (30 d in diameter) which is 0.1% of the total volume of the particle (Fischbach et al., 1965). If one takes into consid.eration that a central empty . - channel was proved $0 exist in TMV (CBS-

+

CONFORMATION OF VIRAL NUCLEIC ACIDS IN SITU

229

par, 1956) and some large bacteriophages were shown to have a central cavity (see below), it is logical to raise the question about the universal character of this phenomenon and its place in the general problem of structural organization of the viruses. On the one hand, the similar principle of organization revealed for such different kinds of viruses seems to be an argument in favor of the universal character of this principle, but, on the other hand, the data obtained for TYMV prevents one from jumping t o conclusions. TYMV has been studied better than any other small spherical virus but the presence of a central cavity has not been reported. However, Klug, et al. (1966) emphasize that the present-day possibilities of the X-ray method do not allow one to say that TYMV has no central cavity a t all, but only that this virus has no cavity with a radius greater than 70 A. The problem is, that due to insufficient sensitivity of the low-angle diffraction technique no hole of diameter less than 70 8 can be detected in a sphere having a diameter of 280 8. Anderegg et al. (1963) think that the existence of the central cavity is due to the electrostatic repulsion forces of the RNA segments. But such a situation may be true in situ only if the phosphate groups of RNA are poorly shielded and the RNA segments interact not with the protein but with each other. It is not difficult t o calculate that the first point may be the case for small spherical viruses with acidic protein of the TYMV type but is not the case for viruses with alkaline protein of the BBMV or BMV type. Thus, Harris and Hindley (1962) showed that the TYMV peptide chain consists of eight lysine residues and three arginine residues, i.e., 1980 alkaline amino acids in the entire protein of the virion consisting of 180 subunits. This quantity of alkaline amino acids is capable of neutralizing only about 30% of the phosphate groups of the RNA in TYMV with a molecular weight of about 2 x lo8 (6000 nucleotides). Triamine neutralizes 14% more phosphate groups which accounts for 0.7% of the total weight of TYMV (Johnson and Markham, 1962; Markham, 1963). It is clear, therefore, that the remaining phosphate groups may be shielded both by mono- and divalent cations. That some part of the TYMV phosphate groups are actually neutralized in this way was shown by Bosch et al. (1967). But the other portion of the phosphate groups is in a protonated nondissociated form, according to Kaper and Jenifer (1965, 1967, see below). These authors showed that in the course of interaction of p-chloromercuribenzoate with TYMV a t neutral pH, protons are released into the medium (about 3600 protons per virion). It is suggested that the source of these protons might be not only the protein but also the RNA phosphates. Unfortunately, the authors of this interesting hypothesis did not compare the qumber -of protons released from the

230

T. I. TIKCHONENRO

whole TYMV and its top component, although both were used in their experiments. This could have given a simple and direct answer to the question of the state of phosphate groups in situ. These data naturally have a qualitative rather than quantitative character. Nevertheless, these results confirm that some part of the RNA in TYMV, maybe not a very large part, might really be affected by repulsive interaction which, in turn, would be responsible for the existence of the central cavity. This is not the case for BBMV whose peptide chain consists of 27 residues of alkaline amino acids (15 lysines and 12 arginines) which for the whole virus (180 subunits) means 4860 amino acid residues, i.e., more than enough to neutralize the 3700 nucleotides of viral RNA (Yamazaki and Kaesberg, 1961b). It goes without saying, that this calculation represents a great approximation and should be interpreted only as a possibility for the neutralization of the phosphate groups of this virus by the alkaline amino acids present in its protein. As to the second condition, i.e., that of interaction of polynucleotide chain segments with each other, in the case of both TYMV and BBMV, the intimate interaction with protein must limit the mobility of the RNA chain due to electrostatic repulsion. Nevertheless, in spite of these discrepancies a central cavity does exist in BBMV while in TYMV its presence has not yet been proved. To summarize the above considerations we must say that Anderegg’s hypothesis for the origin of the central cavity in sphericaI viruses needs more forcible argumentation, while the main objective of investigation should be given more attention because of its possible universal character. b. Degradation of R N A in situ. The tertiary structure of RNA in TYMV, BBMV, and some other viruses described by Klug has been confirmed by experiments on the degradation of intraviral RNA by mild alkaline treatment. According to the data of Kaper (1964), Kaper and Halperin (1965), and Bosch e t at. (1967) short time treatment (up to 8 minutes) of TYMV suspensions at pH 10.6-11 at 30°C in high ionic strength medium does not cause any noticeable changes in the morphology and structure of virions but provides for the selective degradation of their RNA in situ. Deproteinization of such suspensions by phenol gives a low molecular weight but monodisperse RNA preparation with a sedimentation constant of about 5 S and with a molecular weight of 57,000 3500. If not intact virus but isolated RNA was subjected to the mild alkaline treatment, the degradation was random and resulted in a preparation with a continuous distribution of molecular weights. From these data it may be concluded that the phosphodiester bonds in intraviral RNA are ruptured at definite points or sites located more or

*

CONFORMATION OF VIRAL NUCLEIC ACIDS IN SITU

23 1

less periodically along the polynucleotide chain. Of interest is the striking agreement between the size of the resulting RNA fragments and the periodicity of the tertiary structure, i.e., the 32 bumps. The molecular weight of TYMV RNA is 2 x lo6 and the molecular weight of the fragment of the polynucleotide chain of which the bump consists is approximately 60,000. Thus, it may be suggested that the fragments obtained after mild alkaline treatment of TYMV should be either the RNA bumps themselves or the portions of the chain whose dimensions correspond to those of bumps. In the first case it is the points or sites of RNA between the bumps which are attacked; in the second case, within the bump. The first assumption seems to be more probable from the point of view of general theory. It is assumed that the RNA in the bumps is in close contact with the protein and is less accessible to alkali due to a number of factors, including steric ones. That is why with short-time exposure, hydrolysis of phosphodiester bonds by alkali is most probable at the sites where free RNA is not bound to protein. Upon prolonged incubation of the TYMV in alkali, more profound degradation of the RNA was observed and the fragments were shorter and of nonuniform size. The latter phenomenon should be ascribed to rupture of the polynucleotide chain at sites where it is difficult for the alkali to attack the phosphodiester backbone. Bumps are just the place where the RNA is in someway protected. Thus, the important feature of the tertiary structure of TYMV RNA in situ is the regular periodicity of alkali-labile phosphodiester bonds along the chain which, most likely, signifies equal size and periodicity of the RNA bumps themselves. If TYMV virions are disintegrated in a certain way, these fragments, which are portions of the bumps, are released into the medium as aggregates with a high sedimentation constant. This testifies to the existence of definite bonds in situ between these fragments. The existence of these specific aggregates may be accounted for by a number of factors, including hydrogen bonds, and hydrophobic and salt interactions. After heating or in solution of dimethyl sulfoxide these aggregates irreversibly dissociate into 5 S fragments. The existence of aggregates is impossible without the presence of monoor divalent cations, while removal of either cation does not lead to dissociation of the aggregates. The possible contribution of protein in the appearance of aggregates was not investigated by the authors. According to the preliminary data of Bosch, specific absorption of aggregates does not practically differ from that of free RNA. The mechanism for the existence and appearance of these aggregates is not known but it is

232

P. I. T!IKCBONY!!NKO

clear that they can be formed under the specific environment that TYMV RNA has in situ. The role of triamines in the stability of aggregated RNA fragments obtained upon limited degradation of RNA in situ has not been elucidated. Johnson and Markham (1962) and Markham (1963) who were the first to report the presence of bis (3-aminopropyl) amine- (1,7-diamino-4-azaheptane) in TYMV, ascribed to this compound a special role in the maintenance of the RNA tertiary structure. Their results, confirmed by the evidence of Johnson and Hills (1963) and Mitra and Kaesbcrg (1963) prove that triamine is capable of condensing the RNA polynucleotide chain and that stability of the chain to RNAase and heating is thereby enhanced. Phenol deproteinization of TYMV suspensions in a low ionic strength medium in which M bis-(3-aminopropy1)amine is present, allow one to obtain RNA preparations with a sedimentation constant of 30 S and higher. (The sedimentation constant ‘of the reference RNA which was isolated without triamine was 19s.) Electron microscopic study of these preparations showed that this rapidly sedimcnting RNA consists only of spherical particles having a diameter (of 100 to 200 A. Such a form of RNA is stable in solution for a long timc rlt room temperature (if the polyamine is present in the medium in the concentration indicated above), but disappears after 2 minutes of heating at 80°C or after treatment with RNase. At the same time the sedimentation constant goes down to 19 S-20 S (in the heating experiments). The appearance of the spherical rapidly sedimenting RNA form may be achieved not only if the virus has been initially deproteinized in the presence of triamine but also by addition of this substance to a solution of deproteinized high molecular weight RNA. That is why it is not clear what the nature of the spherical material in Johnson and Markham’s preparations is : inherent intraviral structure “fixed” on isolation or the product of secondary artificial condensation. Besides, regardless of the role of triamine in the maintenance of ordered tertiary structure for TYMV RNA, this structure cannot be universal as polyamines are not indispensable for plant viruses. Matthews and Ralf (1966) who reviewed all the experimental data on TYMV wrote that selective degradation of intraviral RNA could be achieved not only by alkali treatment but also by means of ethanol denaturation of the virus, short-time heating at 45”C, and even by storage of the virus in the nonfractionated sap of infected plants. In the first two cases low molecular weight RNA fractions were released directly into the medium after disintegration of the virions; in the third case RNA fragments were obtained only after preliminary phenol deproteinization. Degradation of the RNA in TYMV particles by means

CONFORMATION OF VIRAL NUCLEIC ACIDS IN SITU

233

of agents which do not attack the phosphodiester bond itself was also described by Kaper and Jenifer (1965, 1967, 1968) who worked with pchloromercuribenzoate and by Stols and Veldstre (1965) who used quaternary ammonium compounds possessing aliphatic substituents of long chain length (C6). In the experiments of the former authors, the reaction of p-chloromercuribenzoate at pH 4.6 with the SH groups of viral protein caused rapid inactivation of the virus but no degradation of virions was involved. Degradation was observed only after the material was transferred to a solution at neutral p H and was accompanied by the release of fragments of depolymerized RNA. The particles could be prevented from degrading by removal of the p-chloromercuribenzoate from modified TYMV with the help of mercaptoethanol prior to transferring them into neutral solution, but no infectivity was restored thereby. RNA isolated from such particles also had pronounced sedimentation polydispersity. Thus, p-chloromercuribenzoate and quaternary salts destroy the physical structure of TYMV protein-RNA only when they are attacked in their combined form. Unfortunately, some of these experiments were not carried out as thoroughly as the alkaline degradation and there exists a possibility of RNA hydrolysis by nucleases a t the moment of release or in the virus with an altered protein shell. RNA degradation in modified virus can really be the result of nuclease attack (Kaper and Jenifer, 1968; Philipson, 1965; Katagiri, et al., 1967; Incordona and Kaesberg, 1964; Kaper, 1968). However, the investigations of Bancroft, et al. (1967, 1968) with cowpea chlorotic mottle virus (CCMV) allows one to suggest another mechanism of RNA degradation in situ. This phenomenon is of importance and needs a more detailed explanation. The hydrodynamic behavior of cowpea chlorotic mottle virus suggests that it exists in two forms, depending on pH. The virus has a sedimentation coefficient of 88.3 S and a diffusion coefficient of 1.50 rt= 0.04 X lo-? cm2/sec at p H 5. At p H 7 the sedimentation coefficient is 77.8 S and the diffusion coefficient 1.37 -C 0.03.10-7 cm2/sec. The two forms of virus are interconvertible in terms of gross physical behavior, but once the virus has been converted to the slowly sedimenting or swollen form, infectivity is lost irreversibly because the nucleic acid is ruptured during swelling. It was noted that during incubation of the virus a t different pH’s the infectivity remained constant from p H 3.6 to 6.0 whereas a sharp decrease in infectivity occurred above this range. This process was accompanied by a conversion of the RNA from its 23 S form to small pieces with sedimentation coefficients of about 7s. I n order to assess the extent and cause of the degradation, unlabeled virus which had been kept at p H 7 was mixed immediately before phenol extraction with labeled virus,

234

T. I. TIKCHONENKO

which was not subjected to pH 7. There was no degradation of the labeled RNA, so that RNA degradation did occur inside the slow (swollen) form of the virus. But if the origin of the 7 S RNA in the above-cited investigations is enzymic, then the enzyme must be available. It is present in crude sap extracts and may also be present in small quantities in purified preparations. Whether it has anything to do with the observed phenomenon is difficult to prove. However, it seems unlikely for several reasons: ( a ) both the degradation of RNA in situ and loss of infectivity were prevented when Mg2+ was present during incubation a t pH 7, ( b ) if nucleases are added to the swollen virus the RNA is much more extensively degraded and the virus particles fall apart, and ( c ) the amount of degradation of RNA inside virus kept in sap for a week a t 4°C was no greater than that found for purified virus heated for 1 to 2 days a t 37°C and pH 5. All these data emphasize, however, that breakage may occur by mechanical means-probably associated with shifts of structural subunits and that, in certain cases, such shifts may render the RNA open to nuclease action; but of course, this would be a consequence of conformational change in the structure of the protcin component of virus. If the action of nucleases is also excluded for the case of TYMV, then RNA degradation due to ethanol or thermal destruction of the virus or as a result of the action of p-chloromercuribenzoate and quaternary salts, should be ascribed to con formational reconstruction of the protein. In this case it can be visualized that the RNA bumps are firmly bound to the capsomers of the protein and if the latter undergoes conformational changes the distance between the capsomers changes (increases) which leads to rupture of the polynucleotide chain due to mechanical shearing. Such a more general mechanism for fragmentation of intravirus RNA could also explain alkaline degradation, the more so in that the rate of RNA attack by alkali seems to be too high for the virus with its limited diffusion (Kaper, 1960). This alternative point of view on the mechanism of rupture of the phosphodiester bond does not change anything in the above conception of tertiary structure which is based upon close contact with protein. On the other hand, RNA degradation in situ cannot be explained from the point of view of the “central body” concept without involving nucleases. 2. Secondary Structure

The tertiary structure of RNA in spherical viruses, analyzed for the case of TYMV, allows for a great variety of possible conformations for the secondary structure of the nucleic acid. On the one hand, the close

CONFORMATION OF VIRAL NUCLEIC ACIDS

IN @TU

235

contact between the protein and RNA in the bumps means that the nucleic acid in TYMV is in a position potentially similar to that in TMV. On the other hand, in TYMV there is a possibility of the RNA segments interacting with each other, which preconditions the appearance of a secondary structure of the type existing in solution. At the same time a qualitatively similar situation may also take place if the intraviral RNA is represented by a “central body.” From the traditional point of view complementary interaction of RNA segments is possible in the central zone occupied by the RNA while the periphery contacting the shell is the place for possible conformational changes due to interaction with protein. a. X-ray Diffraction Studies. In the wide-angle X-ray powder diagram of TYMV strong reflections can be seen in the 5 and 12 regions due to helical portions of intraviral RNA (Klug and Finch, 1960; Klug et al., 1961). These bands were absent from the scattering curves of the top component. Similar data were obtained for TBSV. These data are not quantitative but they may be interpreted to mean that both TYMV and TBSV should have an ordered secondary structure of the type existing in solution. The authors, however, were somewhat surprised to find no proportionality between the content of RNA in viral particles and the intensity of X-ray reflections in the bands typical of helical structure, as diffraction patterns for TYMV and TBSV appeared to be practically identical. Katz and Rich (1966) in their short communication are also doubtful about this point of view concerning the secondary structure of singlestranded RNA and DNA in situ. According to these authors small angle X-ray diffraction data obtained from wet gels of +X174 and MS2 particles do not give any evidence for a double-stranded internal structure. b. Optical Methods of Investigation. Spectrophotometric and spectropolarimetric studies of various viruses demonstrate the existence of helical portions of nucleic acid, but there is no unanimity a t all with respect to percent of spiralization. Some authors think that the secondary structure of single-stranded RNA and DNA in viruses does not differ, either quantitatively or qualitatively, from the structure of nucleic acid in solution (under conditions favorable for spiralization). This point of view is supported by Yamazaki and Kaesberg (1961a,b) ; Haselkorn (1962) ; Kaper et al. (1965) ; Shepherd et al. (1968) ; Scraba et al. (1967) ; Sinsheimer (1959) ; Crawford (1966). Usually Schlessinger (1960) and Zubay and Wilkins (1960) are also considered to be among this group of authors. But this is a misunderstanding as these authors saw their main task as the study of percent of RNA spiralization in ribosomes and used viruses only as a reference.

236

T. I. TXCHONENKO

Schlessinger and Zubay consider the complete identity of the structure of ribosomal RNA in vitro and in situ a proved fact. But they refrain from final statements as far as viruses are concerned, just pointing out the similarity of the optical properties of RNA in ribosomes and viruses. Such a careful approach is explained by the scanty and contradictory experimental results that they had with viruses. For example, Schlessinger, determining the hyperchromism resulting from the alkaline degradation of TBSV, TYMV, and southern bean mosaic virus (SBMV), had no free RNA from these viruses for reference. But while in ribosomes and ribosomal RNA of Escherichiu coli the hyperchromic effect 2%, in TYMV and TBSV it upon alkali hydrolysis did not exceed 41 amounted to 47%)and even in southern bean mosaic virus it was as high as 56%. Zubay and Wilkins, on the contrary, melting TYMV obtained a hyperchromism not exceeding 20%. Alkali hydrolysis really should give a higher value for the hyperchromic effect (see below) but the difference observed is too great. The second group of authors adheres to the opinion that the percent of RNA spiralization in viruses is lower than the content of ordered secondary structure in high ionic strength solution (Bonhoeffer and Schachman, 1960; Bachrach, 1964, 1965; Matheka et ul., 1966). Besides, Maestre and Tinoco (1967) allow for a change in the conformation of the nucleic acid in small viruses but do not attempt to characterize it quantitatively. Unfortunately, these two groups of authors worked with different viruses and employed different methods for determining the percent of helical structure. Therefore it is difficult to account for the discrepancies observed : different viruses may really have nucleic acids of dissimilar secondary structure or there may be some experimental error. The authors of the first group evaluated the hypochromic effect and specific absorption of intraviral RNA by means of the following procedure suggested by Schlessinger (1960) and Yamazaki and Kaesberg (1961a). The absorbancy at 260 mp (corrected for light-scattering) was determined a t p H 7 for a virus suspension and RNA solution; they were later degraded to nucleotides and denatured protein in 0.3 N NaOH or KOH for 24 hours a t 37°C and the absorbancy was measured again. The latter value was corrected for the decrease in absorption of uracil at high p H and also in both absorbancy determinations the small contribution to the absorption due to protein was subtracted. But not all the authors followed this procedure in every detail, which led to the lack of uniformity in the results obtained and made it impossible to compare them. For example, Kaper made no correction for neutral pH, and Zubay did not subtract protein absorption, etc.

*

237

CONFORMATION OF VIRAL NUCLEIC ACIDS I N SITU

Schlessinger, Yamazaki, and Zubay employed the above method to determine the specific absorbancy of RNA in TBSV, TYMV, SBMV, and WCMV but they made no attempt to determine the percent of spiraliaation using the data obtained. But later Kaper et al. (1965) and Shepherd et a!. (1968) used this method to compare the secondary structure of RNA in vitro and in situ and to calculate the percent of helical structure. All these calculations were based on the well-known conclusions of Doty, et al. (1959) who used an empirical value obtained from experiments with synthetic double-stranded poly AU. They found that an absorbancy equal to 0.6 for the constituent mononucleotides corresponds to complete interaction (100% formal secondary structure), and that an absorbancy equal to 0.9 for the mononucleotides is obtained in the absence TABLE I V DETERMINATION OF PERCENT OF HELICAL STRUCTUREI N RNA in Vitro A N D in Situ

1. The initial absorbancy a t 260 mp and pH 7 2. The light-scattering correction 3. The true value of absorption of virus at 260 mp and pH 7 4. The correction due t o the contribution of protein 5. The true value of absorption of RNA a t 260 mp and pH 7 6. The absorbancy after alkaline hydrolysis 7. The correction due to the contribution of the protein 8. The hypochromioity 9. Percent helical structure

OF

WCMV

Virus

Free RNA

1.000 0.1%

1.000 -

0.875

0.104 0.771 1.241 0.104 0.68 73%

-

1.000 I.457

0.69 70%

of secondary structure a t high temperature. All subsequent authors who wanted to calculate percent of formal secondary structure for any kind of RNA used these very coefficients. They assumed that a 66% hyperchromism for RNA degradation to mononucleotides or a 50% hyperchromism for melting (see below) corresponded to 100% spiralization. I n this context lower values of hyperchromism meant a lower content of ordered structure. The arbitrary character of this assumption is selfevident as the complete hyperchromism of ribopolynucleotides of different origin (base stacking plus double-helix) has, in fact, a wide range of values from 24 to 45% (Michelson, 1963). The values for virus RNA, including a 56% hyperchromism for SBMV are in complete agreement with the conclusion about different values of hyperchromisrn for RNAs of different origin. Kaper’s estimation of percent of helical structure of RNA in a strain of WCMV (Table IV) might be used as an example of such calculation.

238

T. I. TIKCHONENKO

It is seen from the table that the percent of spiralization in free and in-

traviral RNA is practically the same and is equal to 70 to 73%. Similar results were also obtained by Shepherd e t al. (1968) for pea enation mosaic virus and Haselkorn (1962) for TYMV. Kaper uses free RNA as a reference and his value of hyperchromism for intraviral RNA is above doubt, at least formally. But Yamazaki and Kaesberg (1961a)’ Shepherd et al. (1968)’ and Schlessinger and Zubay use only alkaline hydrolysis of the viral suspension relying on “the normal value of hyperchromism” like Doty e t al. (1959). Thus, estimation of the percent of formal secondary structure, which is rather doubtful even in the case of a correct experimental approach, bccomes but a formal mathematical operation. If we follow these authors’ opinion that ordered portions of secondary structure of RNA in situ do not react with protein (preserving thereby their double-stranded configuration) , we may arrive at a very interesting conclusion for TYMV. Matthews and Ralf (1966) drew attention to thc fact that if Haselkorn’s (1962) value of spiralization of TYMV RNA is correct then cytidylic acid must represent the only unpaired nucleotidc in the RNA of this virus (TYMV RNA contains 38 mole% of cytosine vs 17 mole % of guanine). If the interaction with protein is carried out at the expense of unpaired nucleotides, then cytidylic acid will be the only partner of protein in TYMV. Evidently, there is a possibility for experimental verification of this suggestion. Some information about the secondary structure of single-stranded DNA in phage @174 (Sinsheimer, 1959), and the minute virus of mice (Crawford, 1966) may be derived from the reaction with CHaO. Both authors reported a 25-26% hyperchromism a t 260 mp (37°C) as a result of incubation with CHzO but the reference experiments involving the incubation of free DNA under similar conditions were not carried out. The data concerning intactness of the virions a t the end of incubation and the contribution of protein and light scattering to the total absorbancy are also absent. All this makes it difficult to estimate quantitatively the degree of DNA spiralization in situ. The main conclusion of all these authors, that the secondary structure of single-stranded nucleic acids in spherical viruses has no particular specificity, seems to contradict Klug’s model and to agree with the concept of the Anderegg group. But one can not exclude the possibility that the contradiction in the first case and agreement in the second is nothing but a logical abstraction. In the first place, in all the small viruses mentioned above the RNA tertiary structure may be different from what was observed in TYMV. For example, their nucleic acids may prove to be connected with the protein component to a much lesser degree as compared with TYMV and the small interaction taking place between nu-

CONFORMATION OF VIRAL NUCLEIC ACIDS IN SITU

239

cleic acid and protein in this case may affect very little the optical and other properties of intraviral RNA. Second, it may be assumed that the interaction of nucleic acid and protein in some spherical viruses is qualitatively different from that in rodlike viruses and may not entail disordering of inherent ordered secondary structure. Both possibilities are supported by the experiments with the combined (hybrid) reconstruction of BBMV, CCMV and BMV (Hiebert et al., 1968). In their experiments reconstruction of spherical viruses was carried out in the presence of the protein from this same virus and practically any form of single-stranded nucleic acid, including ribosomal and soluble RNA and DNA from phage S13. In other words, the role of the nucleic acid in reconstruction of spherical viruses was rather nonspecific. The absence of specificity in this case contradicts what is known about TMV (Fraenkel-Conrat and Singer, 1959, 1964; Holoubek, 1962; Caspar, 1963). These differences may be accounted for only by the fact that in these spherical viruses the association of RNA with protein is not restrictive. According to Hiebert this means that the bulk of the RNA of these viruses may be “functionless in a structural sense.” The approach to this problem suggested by the second group of authors, i.e. that of partial despiralization of the RNA in situ seems to be simpler, both from the point of view of method and theory. They use only the first of the corrections for the optical density value which was introduced by Haselkorn and others (light scattering, absorption of protein, and decrease of base extinction in alkali). Besides, the virus and its components do not undergo alkaline hydrolysis that is a drastic and not very well-studied procedure. This is in principle, a matter of comparing the true absorption value for the nucleic acid in the virion and after its thermal destruction. In this way there is no danger for the investigator t o enter the shaky ground of the relationship between various kinds of hyperchromism in polynucleotides. If the true absorption of the RNA decreases after destruction of the virus, it may be suggested that additional spiralization takes place in solution. If the absorption does not change or, otherwise, increase, then the content of formal secondary structure is equal to or higher than that for the RNA structure in solution. By varying the ionic strength of the medium and/or temperature of disintegration it could be possible to control this process of conformational transformation. It has turned out that thermal degradation of TBSV (Bonhoeffer and Schachman, 1960) and foot and mouth disease virus (Bachrach, 1964, 1965; Matheka et al., 1966) causing the release of RNA into the medium give a hypochromic effect in high ionic strength media. In low ionic strength media the value of true absorption, as a rule, increased. The hypochromic effect observed in the medium with a high content

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of monovalent cations was not as pronounced as in the case of TMV but, nevertheless, it was definitely present. Assuming as a first approximation that hypochromism is proportional to percent spiralization it is possible to calculate roughly the content of helical structure. Bachrach reported that the percent of formal secondary structure in foot and mouth disease virus amounts to 43% whereas in a state of maximum spiralization in vitro the percent of ordered sites rises to 64%. Similar data were obtained for TBSV. Both Bonhoeffer and Schachman and Bachrach account for their results in a rather simple way: 50% of the RNA in the virus has a typical secondary structure while the other half of the RNA has no secondary structure of its own but acquires the lacking percent of spiralization if there are favorable conditions in the medium. The estimation of the absolute value of percent spiralixation in this case suffers from the same fault that was described above but relative values have a much higher degree of confidence. The lower content of ordered secondary structure of intraviral RNA resulting from the above experiments should not be looked upon as a means of discriminating between Klug’s model and the point of view of Anderegg, as both groups provide for interaction with protein and the quantitative aspect has not been evaluated so far. But both groups have one point of agreement. Regardless of the possible quantitative difference of RNA spiralization in situ and in vitro, the nucleic acid in the virus is under stabilized conditions. Both in the experiments of Zubay and Wilkins (1960) and in the experiments of Bachrach (1964,1965) and Matheka et al. (1966) heating did not affectthe optical properties of the viral suspension up to the moment of degradation, which to some extent resembles the behavior of TMV in ORD experiments. As we are dealing here with the level of hyperchromism which does not change with temperature, this anomaly must be associated with the helical portions of the molecule. Consequently, bases in the spiralized portions of the polynucleotide chain should have some additional stabilization. In the case of TMV, base immobilization was assumed to be due to interaction with the protein, leading to the complete loss of inherent secondary structure. I n the case of the spherical viruses described above such an explanation is inappropriate a t least, partially. For, either the mechanism of stabilization of RNA secondary structure in spherical viruses is essentially different from that for TMV RNA, or the secondary structure of intraviral RNA is not identical to that of RNA in solution. The first of these alternative points of view is born out by the fact that the secondary structure of DNA and RNA can be stabilized by alkaline protein and polyamines without damaging it (Michelson, 1963). The second possibility is based upon the scantiness of knowledge

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pertaining to intraviral RNA: there is just the value for specific absorption, and the X-ray diffraction curves of dry TYMV powder contradicting the data of Anderegg and Katz and Rich. c. Action of Mutagenic Substances. The results of the study of induced mutagenesis on spherical viruses with single-stranded nucleic acids confirm the absence of any important qualitative difference between the secondary structures of DNA and RNA in vitro and in situ. But here there is no complete agreement either. Some authors (Van der Ent et al., 1965; Belych et al., 1968) believe that there is some difference in sensitivity to X-ray and UV irradiation with h = 2537 k between intact phages +X174 and MS2 and their isolated nucleic acids. Similar data indicating a higher resistance of infectious RNA from arboviruses to UV exposure, heating and HNOz were reported by Mika et al. (1963). A high radioresistance for nucleic acid in solution as compared to whole phage may be due either to conformational differences in structure, or to specific conditions and environment of the nucleic acid molecule in situ and in vitro. Zavilgelskiy and Tovarnizkiy (1966) and Dityatkin et ul. (1968) reported equal sensitivity to UV light with A = 2537 A for phage +L-7 and its free single-stranded DNA. And only exposure to UV light with A = 2650 A and above revealed some differences due to UV absorption by the protein component. Similar data were obtained by Gendon (1966) for poliovirus and its infectious RNA treated with hydroxylamine. As to UV-induced mutagenesis, all the authors report a higher level for phage particles than for isolated nucleic acid. For example, Belych et al. (1968) reported that the number of mutants formed as a result of UV irradiation of intact phage +X174 exceeded by 2 to 2.5 times that formed by irradiation of free DNA. But according to Krivisskiy, mutagenesis induced in phage 4x174 and its DNA by HN02 did not reveal any pronounced differences in the level of mutagenesis in DNA in situ and in vitro (Belych and Krivisskiy, 1966; Tchernik and Krivisskiy, 1965). The possible reason for different levels of mutagenesis induced by HNOz and UV light might be the comparatively drastic condition of deamination. One can not exclude the possibility that the acid pH used in HNOz-induced mutagenesis masks rather small differences present in the secondary structure of single-stranded DNA in the virus and solution (see also Freese, 1965).

111. DOUBLE-STRANDED NUCLEIC ACIDS But for some exceptions the entire section dealing with double-stranded nucleic acids is based on the data for bacteriophages and, to be more

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precise, on analysis of the problem of the secondary and tertiary structure of DNA in the big complex phages such as T-even or Sd. A. Bacteriophages 1. Tertiary Structure of D N A

Even a superficial review of the experimental data will reveal a number of fundamental facts which constitute the essence of the problem. If phage T2 has but one DNA molecule (Thomas and Pinkerton, 1962; Rubinstein et al., 1961; Burgi and Hershey, 1961) with a molecular weight of 120 to 130 X lo6 and a length of about 5 to 6 x lo5 A (Cairns, 1961; Thomas, 1963), there arises the question of how it is situated inside a phage particle which is an icosahedron with a maximum diameter (Cummings and Kozloff, 1960). And that T 2 is no exception is of 1190 i% confirmed by the data for phage Sd. The molecular weight of this DNA is of the order of 70 X lo6 (Tikchonenko and Zak, 1966) ; its length is 3.6 X lo5 (electron microscopic data of Kisselev et al., 1963), and the diameter of the head, which is an octahedron, averages 600 A. I n other words, the ratio of the length of the DNA molecule to the diameter of the phage particle is of the order of 400:l to 500:l. Such a ratio for the length of the DNA molecule to head diameter, which roughly represents the number of segments in which the DNA should be arranged in the phage head, is also observed in other viruses. Hence, there arises the problem of the organization of these segments both in relation to each other and to the longitudinal axis of the particle. There are several experimental approaches to this problem : electron microscopy, bircfringence, and X-ray diffraction (Bendet, 1963 and Thomas, 1963, were the first to review this problem). a. Birefringence. As bacteriophage preparations, unlike simple viruses, have not been crystallized, an investigator faces the problem of obtaining oriented preparations, i.e., samples with viral particles situated more or less similarly in relation to any axis of symmetry. The considerable degree of asymmetry typical of many phages due mainly to their long tails allows one t o overcome this difficulty more or less successfully. As a rule, satisfactory orientation of particles in preparations can be obtained by means of centrifugation in narrow capillaries, by drawing fibers from concentrated suspensions, by the action of surface tension on drying, and in some other ways. Some of these orientation procedures are also used in X-ray diffraction studies of the DNA arrangement in situ. Bendet et al. (1960) were the first to obtain partially oriented preparations of phage T2 by drying a drop of purified suspension of the phage under a cover slide. Some idea of the degree of orientation of phage par-

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ticles may be obtained from electron micrographs of replicas shadowed with platinum. Electron micrographs of such replicas show an arrangement of closely packed particles which looks like crystalline structure, but poor resolution of the phage tails prevents one from evaluating the orientation. If the latter drawback were to be overcome electron microscopy could be successfully used for the purpose of orientation evaluation. Unfortunately, no reliable criterion for orientation has been suggested as yet; the test crossed Nicol prisms in the polarizing microswpe is by no means absolute and often gives artifacts. Nevertheless, the sign of the birefringence of such preparations in Bendet’s experiment was negative while the birefringence of empty protein shells turned out to be positive. As oriented DNA preparations also displayed negative birefringence, disappearing on treatment with DNase, Bendet concluded that the negative sign signifies that DNA segments are preferentially localized along the longitudinal axis of the particle. A quantitative approach to the problem of the degree of orientation of DNA segments in the head of phage T2 was first made in the experiments on birefringence in flow and electric field (Bendet, 1963; Gellert, 1961; Gellert and Davies, 1964). Gellert tried to exclude the possibility of his results being ascribed to free DNA by doing all the operations with phage in the presence of DNase and purifying his material under extremely mild conditions. The rotary diffusion constant, and, consequently, the fraction of oriented particles, proved to be the same for all gradients both for the intact phage and for empty protein shells (‘Lghosts”).This fact supports the belief that free DNA had nothing to do with the degree of orientation of the particles. It should also be noted that the sign of flow birefringence was negative for the whole phage and weakly positive for phage “ghosts,” like in Bendet’s data. But quantitative analysis of this data showed that the degree of orientation of segments does not exceed 10-15% (Gellert, 1961). In a later report of these authors a still lower value is given-9%. In his recent report Bendet does not give the exact figure for orientation of the DNA segments along the longitudinal axis of the particle but still insists on “preferential orientation.” Both groups of authors proceed from a model of parallel arrangement of DNA segments, of a side-to-side type of aggregation. In this case, the question of orientation of these segments toward the long axis of the particle is really essential for making a model of the packing of DNA in the phage head. But our electron microscopic observation (see below) showed that the bulk of the DNA is evidently arranged as an ellipsoid formed by turns of the segments, From this point of view the question of the orientation of the bulk of the DNA in relation to the long axis of the particle is not so acute. I n

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this case it is confined to solution of the particular problem of orientation of a small fraction of intraphage DNA. As to the possible reasons for the discrepancy of Bendet’s and Gellert’s figures, it must be, evidently, due to differences in particle orientation. b. X-Ray Diffraction. The first X-ray diffraction study of oriented whole T2 and T7 particles was carried out by Nort and Rich (1961). Later, Kilkson and Maestre (1962) carried out similar experiments with phages T2 and MS. Dembo et al. (1965) investigated quite a number of phages by this method. The data from the first two groups of authors were rather similar while our data to a great extent were different. In all cases, orientation was achieved by drawing fibers from fresh centrifuge pellets. The X-ray diffraction evidence of Nort and Rich and Kilkson and Maestre testified to the high degree of orientation of the nucleic acid in oriented gels as compared to nonoriented gels. Besides reflections at 3.4 A, typical of the B configuration of DNA a clear-cut reflection at 24 A was recorded (so-called equatorial spacing) which was ascribed by the authors to regular parallel packing of DNA segments. As oriented strands of free DNA also give such an equatorial reflection the authors thought it necessary to prove that the reflection does not disappear even at 100% humidity. Naturally, if free DNA were the source of this reflection, the dissolving and disordering of oriented fibers would lead to disappearance of the 24 A diffraction. Unfortunately, this test is rather more qualitative than quantitative and does not eliminate the problem of extraparticulate DNA. It should be noted that such behavior is typical of free DNA. Nucleoprotein complexes, including artificial DNA-protamine complexes, are characterized by strong linkage between aggregated DNA strands (Thomas, 1963). As a result, the behavior of the 24 A equatorial spacing as a function of relative humidity may be quite different. Manipulating phage suspensions in an attempt to orient them may lead to squeezing the DNA out of the particles in the form of a complex with the internal protein, such as alkaline protein firmly bound to DNA (Levine et al., 1958; Minogawa, 1961; Chaproniere-Rickenberg, 1964; Bachrach and Friedman, 1967; Kokurina and Tikchonenko, 1969; Kokurina et al., 1969). Therefore, the change in the equatorial spacing of complex aggregates to DNA-internal protein may be similar not to the behavior of free DNA but to that of deoxyribonucleoproteins. As to phage T2, it is not the best object for X-ray diffraction study because of its comparatively high physical lability. Besides T2, we (Dembo et al., 1965) used for this purpose bacteriophage DDVII Bacillus dysenteric flexneri which has a much higher physical stability as compared to T2 (Tikchonenko et al., 1966). This phage has a tail 3.5

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times as long as its head diameter, which considerably increases the particle’s asymmetry and improves the orientation of the particles in the fiber. It was assumed that if the DNA in the phage head is preferentially oriented along some axis of the particle, its reflections will be stronger than those from nonoriented particles. But the X-ray patterns from fibers of phage DDVII had just slightly more intense reflections 3.4 A to the fiber axis which testifies to the absence of orientation along the axis of the phage. For T2, the increase in the 3.4 A reflection intensity was rather small too, although it was somewhat higher than in the case of phage DDVII. Summing up all the data we come to the conclusion that there is an absence of any significant orientation of the DNA along the longitudinal axis of the investigated phages. This absence of orientation does not, certainly, signify the absence of ordered tertiary structure. Its existence is indicated by the “packing” reflection in oriented preparations described above as well as small-angle X-ray diffraction patterns (Dembo et al., 1965; Katz and Rich, 1966). In this case the mean sizes of regular packing regions inside the heads of phages T2, Sd and DDVII were determined by Debay-Scherrer’s method, from the diameter of the diffraction ring corresponding to Bragg’s reflection a t 24 A (h/2 sin 8 = 24 A). Theoretically, there may be two interpretations for diffuse maxima in the X-ray diffraction curves from aggregates of chain molecules (Vainshtein, 1963). First, it may be regarded as an indication of the real size of the ordered region in accordance with Debay-Sherrer’s formula. Second, in accordance with Vainshtein’s theory, it may be considered a measure of the disorder of the system. It is difficult to stick t o either of these alternative points of view based only on the above experimental evidence as both the size of the domains and the radius of interaction turned out to have similar values although of different physical meaning. But electron npicroscope evidence (see below) is in favor of the first point of view. Table V shows the data for DNA packing in the phage head (Dembo et al., 1965). As can be seen from this table the size of the regular region in the head, i.e. the size of the DNA crystalline domains, are similar in all three phages investigated in spite of the fact that the phages themselves are greatly different as far as their head size and molecular weight are concerned. Katz and Rich (1966) also report rather similar dimensions for such regular domains determined by the same method. According to their data, for phages T2, T4, T6, T7, and P22 the size of the domain is about 110-140 A. Though, phage particles do not have a preferential orientation of their DNA segments along the long axis of the particle, these data testify to the fact that the tertiary structure of the DNA in

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different phages has some common principle of organization (longitudinal aggregation) which accounts for the presence of similar regular domains. Some time ago when the tertiary structure of intraphage DNA was but a speculation, the high ratio of the length of the DNA molecule to the head diameter prompted the conclusion that the DNA molecule should be folded many times to be placed in the phage head. I n the models of Nort and Rich and Kilkson and Maestre the segments are arranged in the form of antiparallel side aggregates which are bound to give a more or less pronounced degree of orientation along the longitudinal axis of thc phage. This, naturally, gave rise to the difficult problem of bendings (kinks) in the rigid DNA molecule. There were some more speculative attempts to solve this problem by allowing for a loss of secondary structure a t the place of bending (Stent, 1965). According to TABLE V THESIZEOF REGIONS OF REGULAR PACKING I N SOMEPHAGES Molecular weight of Phage

DNA

(X 10-6) ~

T2

Sd DII-VII

130

70 30

Type of head symmetry

Diameter of head (A)

Size of domains

(4

~

Icosahedron with elongated middle part Octahedron Icosahedron

1190 X 800

100 f 10

GOO 500

96 f 10 96 f 10

the hypothesis of Dunn and Smith (1958) who found in phage T2 about 1 mole of methylaminopurine per every 200 moles of adenine, this anomalous base which has just one hydrogen bond with thymine provides a “hinge” which allows the DNA to bend. Thomas (1963) thinks that at the bending sites the DNA may not have an ordered secondary structure at the expense of interaction with internal protein. As in the case of 6-methylaminopurine, the number of molecules of internal protein roughly corresponds to the number of these hypothetical bends (about 300 molecules based on a 5% content of internal protein with a molecular weight of 15,000 (Minogawa, 1961). The interaction of DNA and internal protein in solution reported by Chaproniere-Rickenberg et al. (1964) confirms such a possibility to some extent. c. Electron Microscopy. The available electron microscope data on the arrangement of DNA in the phage head sdggest two problems to be discussed. First is the existence of a central cavity of the type encountered in small spherical plant and bacterial viruses (see above). The

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second question concerns the general character of the DNA segments’ arrangement in relation to each other and to the axis of the particle. i. The central cavity. Kilkson and Maestre (1962) and Maestre and Kilkson (1962) were the first to suggest the existence of a central cavity in T2 particles. Later, this suggestion was to some extent confirmed by Cole and Langley ( 1963) whose experiments involved the inactivation of phages by slow electrons. Although both the experimental approach and analysis of the data obtained seem to be rather doubtful today, the very. idea of a central cavity has won recognition because it has been supported by direct experimental evidence. I n uitrathin sections of the phage head one may see a central cavity. This was the case in T 2 (Cummings and Wanko, 1963; Klimenko et al., 1967), in h (Cummings et al., 1965), and in DDVI (Klimenko et al., 1968). As to the dimensions and the shape of the cavity, opinions differ. Cummings and Wanko think it to be an ellipsoid with 150 X 70 axes. In Klimenko’s micrographs (Klimenko et al., 1968) it is usually a sphere with an 80 A diameter. The micrographs of other investigators who used the same technique, however, do not show even a minute hole (Moll, 1963; Margaretten et al., 1966; Simon and Anderson, 1967). Thus, it has not been clear what conditions govern the visibility of a cavity in sections, and whether a cavity exists within the head of intact phage. However, all of the above authors applied various methods of fixation and embedding which could have affected the results. For this reason Klimenko et al. (1968) have studied how various conditions of fixation affect the visibility of a cavity in the head of the DDVI bacteriophage in thin sections [this phage is closely related to T2 but, unlike it, its head is more stable to various treatments (Maaarelli et al., 1967, 1969a; Tikchonenko et al., 1969a)l. This investigation examined sections obtained from samples fixed with different fixatives and under different conditions (see Plate I). The results are presented in Plate I and partially in Plate 11. They show that in sections a cavity with a diameter of about 80 A exists near the center of fixed, dehydrated, and embedded phage. This cavity has been seen with all of the mild fixation methods tested. If, however, the DNA denatured significantly as a result of fixation (Dobrov and Tikchonenko, 1968) the cavity disappeared while the periphery of the DNA condensate vacuolated. But even under mild fixation a cavity was by no means seen in every sectioned phage head, This fact does not preclude the existence of a cavity in each embedded phage particle. There is always the probability that a given phage head is cut so that its cavity is not included in the section. Apparently, this probability decreases when the thickness of the section increases, but. in too thick sections the cavity

PLATE I. The phage DDVI in thin sections. Phage particles were fixed: (A) with osmium tetroxide for 2 hours a t 0°C (Caulfield, 1957); (B)with 3% glutaraldehyde for 2 hours at 0°C (Sabatini et al., 1963); ( C ) with 3% glutaraldehyde for 2 hours at 0°C and postfixed with osmium tctroxide as in ( A ) ; (D) with 1.6% formaldehyde for 2 hours at 37°C; (E) with 1.6% formaldehyde for 2 hours at 37°C and postfixed with osmium tetroxide as in ( A ) ; (F) with 3% glutaraldehyde for 20 hours at 0°C; ( G ) with 1.6% formaldehyde for 20 hours at 0 ° C ; (H)with 1.6% formaldehyde for 20 hours at 37°C and postfixed with osmium tetroxide as in (A); (J) with 1.6% formaldehyde for 7 days at 0°C; (K) with 1.6% formaldehyde for 7 days at 37°C.

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would not be visible due to a lack of contrast. The optimum thickness of the section is about 300400 A. Thus, the absence of a central cavity in micrographs of the above-mentioned authors might have been the consequence of either drastic conditions of fixation or improper thickness of the sections. Neither, however, do these described results prove the existence of a cavity in the head of intact phage. One can not exclude, absolutely, the possibility that the cavity appears as a result of an uneven contraction during dehydration or embedding of the specimen. Nevertheless, such a possibility seems to be highly improbable in view of some other features of the tertiary structure of intraphage DNA described below. ii. The condensed form of D N A in vitro. It was reported by Klimenko et al. (1967) that treatment of T2 phage, adsorbed on a positively charged carbon film from an alkaline medium with acid solutions of phosphotungstic acid (PTA) resulted in the lengthwise rupture of the phage head envelope and the release into the medium of condensed DNA (this phenomenon may be called pH-PTA shock). Such condensed DNA had a ring-shaped toroidal structure often with two or more fibers coming from it (see Plates I1 and 111). The successive treatment with uranyl acetate destroyed the preexisting densely packed structure of the DNA which corresponds to the observations of Schlote and Kellenberger (1962). Similar results with pH-PTA shock were obtained recently by Kellenberger (1968) with T4 phage. In most cases these rings of condensed DNA had an elliptical form with a long axis ranging between 900 and 1900 A. The thickness of the rings ranged from 180 up to 450 A, the mean value being about 280 A. The question arises whether such a ring structure is the primary one, i.e., represents the native intraphage tertiary structure preserved under conditions of pH-PTA shock. If it were ring-shaped inside the intact head the micrographs of thin sections through the phage pellet obtained by these authors (Klimenko et al., 1967, 1968) also should show figures representing different sections of that ring. Nothing of the kind was observed. It may be assumed that inside the phage the DNA is organized with a different symmetry. One may assume, due to the essential similarity of all sections through separate phage particles, that the DNA is packed with the symmetry of a rotating body, having the form of an ellipsoid with a small central cavity, and that upon rupture of the coat this Structure is readily transformed into a ring-shaped one. The form of the primary product of decondensation of the intraphage DNA after its release from the particle strongly supports the existence of a central cavity in intact phage. On micrographs provided by Klimenko et al. (1967) these rings are

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often seen to break down into a system of thinner connected rings. Hence, one may suggest that the initial ring consists of a number of rings formed by comparatively thick fibers. It is necessary to emphasize the complete absence of any secondary DNA helices with a diameter of 75 b or less, as postulated by Cole and Langley (1963) and Kilkson and Maestre (1962). The conclusion can be drawn that the main elements of DNA packing, a t least in the rings and thick fibers, as seen in Plates I1 and 111, are regions composed of a number of parts of two-stranded DNA molecules which are aggregated side-by-side forming a multistrand cable or bundle. The distribution of fiber thicknesses showed a number of maxima. The first of these occurred in the region of 60 to 80 A. This coincides with the value reported by Rubinstein (1960), who observed DNA fibers formed upon destruction of the phage by hydrodynamic forces. The second maximum was situated in the range of 110-150 b,and the majority of fibers fall in this interval. It is appropriate to remember now that the mean size of the regular domain in T2, T4, T6, T7, DDVII, Sd, and P22 phages was estimated to be 100-140 b, as shown by small-angle X-ray diffraction data (Nort and Rich, 1961; Dembo et d., 1965; Kate and Rich, 1966). This coincidence suggests the existence of DNA bundles of the indicated thickness also within the intact phage heads, their thickness corresponding t o the size of the ordered domain. The uniform size of the DNA fibers from different phages suggests that they seem to represent the main structural subunit of DNA tertiary structure in situ. Unfortunately, w r y little is known about the principles governing the organization and the very existence of these fibers. But one thing seems to be quite definite: it is not a superhelix and consists, as stated above, of a lateral aggregation of DNA segments with the distance between rows being about 24 b (recall the 24 b spacing on X-ray diagrams in Section III,A,l,B) . This supposition is strongly supported by the easy decondensation of the fibers by uranyl acetate. In addition, DNA instead of a ring produced a tangled coil of filaments about 20 b in diameter, corresponding t o individual double-stranded helices. If these fibers or bundles really exist in the phage head then the DNA must make a number of kinks to form such a structural subunit, bePLATE 11. Particles of T2 phage treated with 2% PTA. ( a ) The disruption of the phage head; the DNA is in the form of a compact mass; (b) one of the rings has two fibers coming from it; (c) a ring has one fiber coming from i t ; (d) one of the rings has three fibers coming froin i t ; aggregation of DNA fibers is observed; (e) a ring without fibers; ( f ) a ring with different thickness of walls and with two fibers coming from it; (g) the protein envelope of the phage particle broken along the long axis into two symmetrical parts; (h) the contents of the phage head are released in the form of rings linked with each other.

PLATE 111. Particles of T2 phage treated with 1% uranyl acetate at pH 5 and ultrathin sections of T2 phage. (a) Particles treated for 15 seconds; (b) particles treated first with 0.5 M acetate buffer (pH 5 ) and then with 1% uranyl acetate; tangled coils of DNA are seen; (c) particles treated first with 2% PTA (pH 5 ) and then with 1% uranyl acetate for 15 seconds; disruption of phage heads along the long axis are seen instead of rings and tangled coils of DNA; (d) ultrathin sections of phage stained with 1% uranyl acetate and lead; note cavity; ( e ) the same, but the only stain is lead; (I) the same, but the only stain is 1% uranyl acetate; the cavity seems to be larger than that seen in (d) and (e).

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cause there is only one DNA molecule in phage particles. This idea is supported by the stepwise decrease of the fiber diameter observed by Rubinstein (1960) and Klimenko et al. (1967). It may be explained by assuming that the number of individual DNA helices forming the fiber does not remain constant, kinks being located at different points of the fiber. For example, Rubinstein (1960) obtained a series of diameters: 75, 57, 41, and 27 A. The existence of this series may be explained as follows. Initially, there is a bundle consisting of three loops and one free end of a DNA molecule (in total there are seven helices in section) ; if all these loops were of different length, the diameter of the bundle would decrease stepwise toward the end of the bundle, resulting in a series of four diameters close to those measured. iii. The ring structure of DNA in the half-empty head. Velikodvorskaya e t al. (1968) and Klimenko et al. (1968) obtained interesting information about intermediate forms of the DNA condensate remaining in the head of T2 and DDVI phage in the course of its ejection through the phage tail. DNA ejection in these experiments was induced by adsorbtion of the phages to cell wall fragments of E . coli B or to intact bacterial cells (Plate IV). I n their observations most of the adsorbed phage particles revealed either filled or empty heads. Only a small proportion of them appeared partially filled. I n the latter case, a gap between the protein coat of the head and its inner content (DNA) was found when a small part of the DNA molecule had passed out of the head. If a considerable portion of the DNA had been ejected, the remaining material often appeared in the form of a ring, resembling the torus seen upon rupture of the T2 head in case of pH-PTA shock. The election micrographs give the impression that this ring is bound to the protein at the equatorial part of the head coat, which corresponds to the site where the “sickle” of ghosts is located. The authors suggest two models to explain the observed phenomenon. The first model treats the intraphage DNA as a “rigid” condensate, where different parts of the whole tertiary structure have fixed relative positions which do not change during the DNA ejection. Let us consider two variants of the “rigid” model. In the first variant the torus has preexisted in the head of the intact phage and has been covered with a DNA layer which is removed during ejection. Ejection would simply demask the torus. Then, the hole of the torus would be apparent in sections of intact phage. However, the size of the hole that is seen in sections is much less than the size of the hole of the torus that is seen in the head in the course of ejection. In the second variant the hole of the torus does not preexist in the intact phage. The torus and its hole originate during ejection as a result of the loss of a part of the DNA. This variant

PLATE IV. Interaction of DDVI phage with the cell wall fragments of E . coli B. Negative staining with 2% PTA (pH 7); (a) adsorbed phages with contracted tail sheaths 5 to 10 minutes after mixing; (b) indentations of the cell wall produced by the phage needles; (c) and ( d ) early phosc of DNA ejection: gaps between the protein coat and the DNA condensate; (e), ( f ) and ( h ) advanced phases of DNA ejection: DNA rings within the heads; (g) advanced phases of DNA ejection: DNA adhered to one side of the protein coat; no rings are seen.

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can not as yet be ruled out, but it seems to be rather improbable for reasons given below. The second model treats the DNA tertiary structure in situ as a mobile one. During ejection, the parts of the DNA molecule which remain in the head change their relative positions. At first, outer layers of the DNA come out creating a space in the head. Then, internal motions of the remaining condensate result in its expansion and transformation into a torus. If a small hole in the center of the intact phage head exists, it may play the role of an initiator in the formation of the hole of the torus. The main argument favoring this model is the fact that the DNA condensate after pH-PTA shock (Klimenko et al., 1967; Kellenberger, 1968) is readily transformed into a torus without any intermediate forms. So one may arrive a t the conclusion that the condensed DNA within the phage hcad tends t o transform into a torus even prior to the start of ejection. This would be accomplished when sufficient space developed within the head. It is reasonable to assume that such a transformation would involve some sort of relative sliding of adjacent parts of the helical DNA molecule within the condensate. Accepting the hypothesis of sliding DNA helices for the transformation of the primary condensate to a torus, it is reasonable to assume that this same procedure facilitates the ejection process itself: when the DNA somehow slips out of the tightly filled head and passes through the narrow channel in the phage needle. The rapidity of torus formation and DNA ejection through the needle suggests that such a postulated sliding proceeds without having to overcome considerable potential energy barriers in spite of the adhesive forccs between adjacent DNA windings. 2. Secondary Structure

a. Electron Microscopy. Although there are no direct electron microscopic data on the secondary structure of DNA in situ, this technique furnishes important indirect evidence pertinent to this problem. This information consists of the internal volume of the phage particle ( Vi), the volume that may or should be occupied by the DNA molecule (Vd), and, finally, the volume really occupied by DNA in the phage particle Vfd). These values and their sequence are indicative of an anomaly in the way of packing of intraphage DNA. Bendich and Rosenkranz (1963) were the first to mention briefly this phenomenon, and Kisselev et al. (1963), Tikchonenko (1966, 1967), and Klimenko et al. (1967) described this anomaly in greater detail. The anomaly is, in fact, the extremely dense packing of the nucleic acid in the phage head. The ratio of the internal cavity of the particle

256

T. I. TIKCHONENKO

occupied by DNA to the volume of the DNA molecule appeared t o be equal to or even less than unity. But this alluring simplicity should not conceal the whole picture which is far from being simple. I n the first place, such a simple mathemetical operation requires precise data for the size of the particle and its geometry. Information of this kind is usually supplied by electron microscopic and, sometimes, by X-ray diffraction investigations. Unfortunately, different investigators using different methods rarely obtain similar results. There is no unanimity on the question of DNA molecular weight. But, while in this instance the difficulty in estimation is due to experimental error and may, in principle, be overcome, the internal volume of the particle is really hard to assess. I n the case of spherical viruses the size of the internal volume is determined from the internal diameter of the virus. The value of the internal diameter is obtained by subtracting the thickness of the protein shell from the total (“outer”) diameter of the particle. The capsomere diameter is assumed to be the thickness of the protein shell and the nucleic acid is tacitly agreed to be evenly distributed throughout the entire internal volume. The data cited in Table VI show that both assumptions are not always right. The real volume occupied by the DNA in a virus could be estimated exactly from the size of the region stained by uranyl acetate or lead or their combination in ultrathin sections of the virus (Karnovsky, 1961). But such data have been obtained only for two or three viruses (only for T2 phage in this table) ; that is why one has to be content with a less exact and not equivalent valuethe size of the region occupied by PTA in viral particles devoid of their nucleic acid. And yet this value is a definitely better characteristic of the internal volume than the result obtained by subtraction of two diameters. But even this simple procedure has proven difficult to carry out: the size of the DNA region varies and depends on the way of making a preparation for electron microscopy. The nucleocapsid diameter in sections through the virus fixed and imbedded in polymerizing media is shorter by 20% than that of the particles in preparations stained with PTA or uranyl acetate and in metal-shadowed preparations (Watrach et al., 1963; Granboulhan et al., 1963; Wildy and Horne, 1963; Williams et al., 1961). None of the authors seem to know the reason for such a discrepancy; it is either the uniform shrinkage of the material in the course of preparation of sections or the flattening of unfixed phage particles on the film and subsequent increase in diameter in the course of staining. That is why only comparable data have been included in Table VI, i.e., those where the parameters of the phage particle have been determined by one method or comparable ones. In some cases (see columns

257

CONFORMATION OF VIRAL NUCLEIC ACIDS IN SITU

3 and 4 in Table VI) where no complete data were available, the diameters of the shells or nucleocapsids, respectively, were calculated assuming uniform contraction or expansion of the particle (see below). Comparison of the two last columns in Table VI leads to the conclusion that the size of the internal volume of the particle is markedly different from the size of the zone occupied by DNA. The latter value is, as a rule, much smaller. And here, there are two possibilities: first, that the region occupied by nucleic acid in the section is distant from the edge of the sectioned phage head by a value exceeding the capsomer TABLE VI SOME PARAMETERS OF VIRUS PARTICLES"

Virus Phage T2 Virus of wound tumor of plantsb Virus of laryngotracheitis infections Reovirus type 3b Wart virus Polyoma virus

Thickness of protein coat or diameter of ca somer

Measured diameter of region occupied by nucleic acid (A)

ti)

Calculated diameter of inner volume of particle (A)

35

1120 X 730

620

70

480

750 X 650 850 X 650 350

1075

106 X 95

865 X 885

800

595 550

50 80 50

495 390 380

325 306 260-220"

Diameter of particle (A)

1190

x

480

800

a The table is based on the data of Bradley (1965a), Klimenko et al. (1967), Margaretten el a2. (1966), Vasquez and Tournier (1962), Williams et a2. (1961), Watrach et al. (1963), Crawford and Crawford (1963). Virus with double-stranded RNA. 0 See comments below.

diameter or that of the protein shell determined as indicated below. Second, the region occupied by DNA in the particle may not reach its geometric center. For example, in polyoma virus the external and internal diameters of the zone occupied by DNA are 260 and 220 8, respectively. In other words, all the DNA of the particle, having a molecular weight of 2.5 to 3.5 x los is localized in the 20 h layer which begins at a distance of 110 A from the particle's periphery and ends at a distance of 110 A from its center (Mattern et al., 1967). Such an anomaly calls for a more detailed analysis of this problem, which will be done below using the thoroughly studied phage T2. For discussion of the problem of DNA packing in the T2 head, it

258

T. I. TIKCHONENKO

would be useful to estimate first the precise dimensions of the head. Electron microscopic observations made on shadowed, unfixed, lyophilyzed phage led to the dimensions 950 x 650 A (Williams and Fraser, 1953). However, measurements on formalin-fixed phage after negative staining a t neutral p H give head dimensions as high as 1190 X 800 A (Cummings, 1963 ; Cummings and Kozloff, 1960). What dimensions should be assigned to the wet unfixed phage head? Comparison of the electron microscopic observations with X-ray diffraction data may suggest the answer. As it was stated earlier there exists a reflection corresponding to a spacing of 24 A, which may be interpreted as the distance between the paeked rows of DNA helices (period of packing). This spacing is reduced to 19 A when the phage fibers are dried (Nort and Rich, 1961). With the simple assumption that drying results essentially only in uniform contraction of the head without significant rearrangement of its internal structure, the ratio of the outer dimensions of the dry and wet phage head must be equal to the ratio of corresponding periods of packing. Indeed, the ratio of periods is equal to the ratio of respective head dimensions of fixed and dried phages. 19 950 R 650 0.8 2 4 R = W% = SoOK , Hence, it may be assumed that the dimensions of the head measured on the fixed phage are fairly close to the dimensions of the head in the wet state. The maximum value of the head width may be taken as 900 A, which is obtained from X-ray scattering from such areas of phage gel whcre there exists an ordered packing of phage particles (Katz and Rich, 1966). For these estimations of the head size, one may calculate the irincr volume of the head. These calculations are summarized in Table VII. A series of values is given, for negative contrast and thin sections give different thicknesses of the head coat ranging from 35 to 75 A (see e.g. Bradley, 1965a). All calculations are based upon an icosahedron with an elongated middle part, as the most probable model (Moody, 1965; Boy de la Tour and Kellenberger, 1965). The volume of the central cavity in all cases was not taken into account as it occupies less than 1% of the total volumc of the particle. As the values of internal volume calculated for different widths of protein shell turned out to be different, i t is desirable to choose a criterion for the evaluation of the values obtained. Such additional information might be derived from a comparison of calculated values for the internal volume and experimentally obtained parameters of the DNA zone in sections. But as has been indicated above, the latter method is bound

CONFORMATION OF VIRAL NUCLEIC ACIDS I N SITU

259

to give values which are on the average lower by 20%. For example, if the size of the negatively stained phage particle is 1190 x 900 A, the respective values obtained from sectioned material are 900 X 800 A according to Klimenko et al. (1967) and 1000 X 800 A according to Cummings and Wanko (1963). Klimenko’s value is closer to the size of T 2 particles determined by Katz and Rich by small angle diffraction (the longest diameter-900 A ) . The region occupied by DNA in the sections and stained with uranyl acetate or lead is, in this case, an ellipsoid which, according to the first group of authors, has dimensions of 750 x 650 A and according to the second group of aulhors, 850 x 650 A. Thus, the volume of the zone TABLE V I I ESTIMATION OF INNER VOLIJMEINSIDE T 2 HEAD

Geometrical model of the head Icosahedron with elongated middle part ;* of: 1190 x EOO I; 1190 X 900 A

dimension^

Head volume

(X

106A3)

380 480

Inner volume (X 106 A 3 ) for coat thicknessb

35

A

280 360

soh;

75 A

240 310

180 220

a For the calculation of volumes of the icosahedron we derived the following for1967): Ti, = 0.718b2 L - 0.322b3where L is the length of the mula (Klimenko el d., head, and b is the width. 6 Condit iorial coat thickness (for explanatlion see the text).

occupied by DNA will be, respectively, 170 x loe and 190 x lo6 A‘, i.e., consistently lower than the figures cited in Table VII. As the longest diameter determined by X-ray diffraction appears to be equal to the longest diameter of the section (900 A ) , the latter value may be assumed t o be the true value for the particle. Now, the problem of phage contraction during sectioning, fixation, and embedding loses its significance. As was mentioned above, electron microscopic stain (uranyl acetate, lead) in the sections is localized at a distance of 75 A from the edge of the sectioned particle. This value is not necessarily the real thickness of the protein shell and also it should not be looked upon as contradicting the figure of Bradley (1965a) for the head capsomere diameter. It may be assumed that for some unknown reason the zone occupied by DNA in T2 phage begins at a distance of 75 A from the edge of the head. The alternative point of view, i.e., that of the 75 .& being an artifact would be based on a hardly probable assumption of

260

T. I. TIKCHOImhXO

selective contraction of the DNA without contraction of the entire particle. There is no less controversy as to the principles of calculating the volume of the DNA molecule. In the simplest case, which is nothing but a supersimplification, the DNA is assumed to be in the form of a cylinder, and knowing the height and diameter of this figure, its volume is calculated. The value obtained thereby, naturally, bears very little relation to the volume which DNA really occupies in the viral particle. At the same time the correct estimation of such volume requires knowledge of the mode of DNA packing, its secondary structure in situ, etc. As was already mentioned, all the authors who studied X-ray diffraction reported a 3.4 A reflection which signifies the presence of the B configuration of DNA, and a 24 A packing reflection testifying to regular arrangement of the double-helical segments or windings of DNA. Both facts mean that a t least some portion of the DNA inside the phage has the secondary structure characteristic of the native molecule as described by the Watson-Crick model. But, these data should by no means be interpreted as testifying to the fact that all intraphage DNA or at least its major part has such a secondary structure. X-ray diffraction patterns of intact phage preparations are rather diffuse which means that only some portion of the intraphage DNA has the native configuration to which we are accustomed. Let us calculate the volume ( V ) which would be occupied by T2 DNA if it were organized in such a way that it consists entirely of helical sections packed hexagonally, the spacing (D)between rows being 24 A, considering it to be completely in the native state. Assuming the weight of the DNA to be 130 x los daltons and the corresponding length ( L )to 62 x lo4 A (see, e.g., Thomas, 1963), we obtain 2

VnNA= -D2L = 413 X lo6 Aa

fi

In reality, the volume which should be occupied by native DNA may be still greater, as the electron microscope value for the length of the molecule is smaller than the length calculated for its molecular weight based on the known DNA ratio of mass and length (Frank et al., 1963). Thus, for a molecular weight of phage T2 of 130 x lo6 and an internucleotide distance of 3.4 A the calculated volume, of DNA must be 450 x lo6 As.Both this figure and the one cited above exceed by far the dimensions of the internal cavity of the phage particle where this DNA is located. And phage T2 is no exception. The same discrepancy between the

261

CONFORMATION OF VIRAL NUCLEI0 ACIDS IN SITU

volume occupied by DNA and that of the internal cavity is also encountered in other phages, for example, in Sd. The electron microscope value for the length of the DNA molecule in this phage is 3.6 x 106 A and the volume it occupies is 144 X lo6 A3 (Kisselev et al., 1963). Phage Sd is an octahedron with a diameter of about 600 A which, with a minimum width for the protein shell of about 25 A, amounts to a volume for the internal cavity of about 66-85 X 10“A3. Even if one does not take into consideration the X-ray diffraction evidence of the 24 A packing reflection and calculates not the real volume for the DNA but TABLE VIII DENSITY OF NUCLEIC ACID PACKING FOR

Virus type

Molecular weight of nucleic acid (X 106)

SOME

VIRUSES“ Density of packing

Inner diameter

Inner volume (X 108A8)

cleotide

192 210 130b 175b 204

3.7 4.8 1.1 2.8

1100 660 220 340

4105 190d

1100 590

(A)

&/nu~~

R17 WCMV +X174 +-X174

+R T2 T2

1.1 2

1.6 1.6 1.5 130 130

-

c

-

-

gm of nucleic acid/cma ~~

0.50 0.8 2.1 1.5 0.69 0.62 1.05

0 It has been assumed for simplification that the internal cavity is a sphere. The necessary values were taken from the data of Fishbach et al. (1965), Anderegg et al. (1961), Tromans and Horne (1961), Sinsheimer (1959), Burton and Ledbetter (1968). Brenner and Horne (1959). b 130 A diameter is from Sinsheimer; 175 A-diameter is from Tromans and Horne (Scliaffer and Schwerdt, 1959). c For the native DNA packed in a hexagonal lattice with a 24 d spacing between rows. d The volume occupied by DNA computed on the basis of thin sections.

that of a cylinder with a diameter of 20 A and length of 3.6 X lo5 A, we shall have a volume of 113 x lo6 A*. In other words, in this case, too, the native double-helical DNA has too great a volume to be placed in the bacteriophage particle. The results calculated for these phages and similar data for other groups of viruses are cited in Table YIII. The density of packing is seen to be unreal for all of the cases, if one bases one’s calculation on the molecular volume occupied by the native double-helical structure. To sum up, double-helical nucleic acids in their native form are incapable of being placed inside viral particles where they are usually “accommodated.” Hence, we are forced to assume that, first, intraviral

262

T. I. TIKCHONENKO

DNA may have other ways of packing and, second, there exists a different secondary structure for DNA occupying much less volume than in the Watson-Crick model. A denser mode of nucleic acid packing is also suggested by the data for small viruses containing single-stranded DNA and RNA, cited in this table. To analyze the data of Table VIII it may be assumed, with some degree of approximation, that the density of packing does not exceed the buoyant density of denatured DNA in solution. (It is necessary to remember that the absolute density values obtained by the method of equilibrium centrifugation (1.70-1.74 gm/crn5) arc, certainly, different from the true density values.) Bearing this in mind, it may be stated that the density of packing of the single-stranded DNA in phage 4x174, based on Sinsheimer’s determination is too high. But this packing density appears to be acceptable as calculated from the data of Tromans and the recent data of Burton and Ledbetter (1968) obtained for phage +R. The rest of the values cited in Table VIII do not exceed this limit. For quite a number of viruses the packing density is equal to or exceeds that of double-stranded DNA in phage T2. Consequently, the nucleic acid in viral particles can be packed more densely, provided that it is devoid of any ordered secondary structure. Based on this data it is natural to make an assumption-which will not contradict the X-ray diffraction evidence-that the loss of ordered secondary structure by some “excess portion” of the DNA is the only way to fit all the DNA inside such particles as T2, Sd, and others. A double effect is thereby achieved. On the one hand, the partial specific volume is decreased and on the other, denatured portions of DNA may be packed more economically, as compared with the 24 A distance between parallel rows. 6. Spectrophotometry. The possibility of partial denaturation of DNA was considered by a number of authors in connection with the problem of numerous bends of the rigid DNA molecule inside phage particles (Dunn and Smith, 1958; Thomas, 1963; Bendich and Rosenkranz, 1963). But the first attempt to prove this suggestion experimentally gave negative results (Bonhoeffer and Schachman, 1960). Tikchonenko et al. (1966a,b, 1967) were successful in solving this problem. Experimentally the approach of these authors did not differ from that used with plant viruses. The experiments were based on the determination of true adsorption values for the DNA in the virus and its behavior on release into the medium. It was suggested that if the DNA conformation in situ differs considerably from that in solution, then the release of the DNA into the medium should be accompanied by noticeable changes in its optical properties. The starting point of the experiments was the sug-

CONFORMATION OF VIRAL NUCLEIC ACIDS IN SITU

263

gestion of partial denaturation in situ with some drop in absorption (hypochromic effect) expected to appear after its isolation in solution. The first task was to determine the true absorption value for DNA inside the virus which is a light-scattering particle. Numerous investigations of this problem (McLaren and Shugar, 1964; Higashi et al., 1963; Basu and Dasgupte, 1967; Amesz et al., 1961; Rosenheck and Doty, 1962; Fisher and Gross, 1965; Gross and Fisher, 1965; Leach and Scheraga, 1960; Englander and Epstein, 1957; Schanenstein and Bayzer, 1955; Mayfield, 1968; Van de Hulst, 1957; Duysens, 1956) showed that the measured absorption of a solution of small particles and/or macromolecules is usually greater than the true, that is electronic, absorption (A,) as a result of Rayleigh scattering (a). In this case, the measured absorbance ( A ) will be

A = At

+u

The extinction due to scattering according to Rayleigh-Mie theory is equal to

where K is dependent on the solute concentration, on the refractive index of the solvent, and on the molecular weight of the solute. There exist several ways for determining A,: the extrapolation method, the use of a special Cary model 1462 scattered transmission attachment, and the method of turbid filters (Shibata et al., 1954; Amesz et al., 1961). The last method was not applied by the above authors to analysis of viral suspensions. I n accordance with our experience the Shibata method did not give good results with T phages. The Cary 1462 attachment allows one to record only the light scattered forward within an angle smaller than 90". The light scattered with an angle more than 90" or scattered in other directions will not be recorded. Although Amesz et al. (1961) supposed that transmitted light emerges from a particle within an angle ,smaller than go", this is not a general rule and needs special proof. I n accordance with the data of Maestre and Tinoco (1965, 1967) who used this attachment for the determination of A, in T2 and T 4 phages the estimated value of light scattering a t 260 mp proved to be about 10%. On the other hand, Englander and Epstein (1957) and Tikchonenko et al. (1966a,b) who applied the extrapolation method found that the value of u amounted to about 25%. A very similar value for u was estimated by determining At directly in a specially constructed UV-integrating photometer (see below).

264

T. I. TIKCHONENKO

The extrapolation method widely used by many investigators is based on determining the light-scattering correction by extrapolating a plot of log OD us. log h from a nonabsorbing to an absorbing region of the spectrum. For small particles with a (polarizability) constant, such a plot should give a straight line with a slope of -4. I n accordance with Van de Hulst (1957) for a very long thin particles the plot should also be straight with a slope of -3 while the slope for large particles should be -2. It is of great importance to the extrapolation method that the index of refraction changes drastically in an absorption region, with a large increase followed by a smaller decrease as the absorption is traversed from lower to higher frequencies. The magnitude of this effect is determined by the strength of the absorption. Mayfield (1968) and Van de Hulst (1957) are rather optimistic on this matter. They think that for biological materials the 260 mp absorption region of nucleic acids and proteins is weakly absorbing in comparison with the higher frequency transitions, and to a first approximation it is judged that the difference between the actual amount of light scattered and the amount calculated from extrapolation is small. Unfortunately, the whole consideration has a rather approximative semiquantitative character and leaves unknown the precise value for the difference between the true and calculated values of A t . Thus, the extrapolation method, widely used in many studies on viruses and other light-scattering biological systems, needs experimental or theoretical support. Schanenstein and Bayzer (1955) demonstrated the absence of anomalous light-scattering in the true absorption region of globular proteins and, consequently, the possibility of applying the extrapolation method. But this is true only for globular proteins. As for nucleic acids and viruses Eisinger (1966) and Olins et al. (1967) suggested the existence of such anomalies in the case of different deoxyribonucleoproteins as would render the extrapolation method incorrect. On the other hand, Englander and Epstein (1957), Leach and Scheraga (1960),Tikchonenko et al. (1966a,b), and Dobrov and Tikchonenko (1969) have offered evidence in favor of the feasibility of the extrapolation method for this kind of measurement. The most direct evidence was presented in the latter paper cited. To verify the applicability of the extrapolation method to the determination of intraphage DNA absorption some direct measurements of At on a number of phages were carried out by means of the above-mentioned special instrument. This is the integrating UV photometer with full white sphere which allows direct measurement of true absorption values. The theoretical background, design of the instrument, procedure for measurements, and the equations pertinent to the determination of At

CONFORMATION O F VIRAL NUCLEIC ACIDS I N SITU

265

are described in papers by Rvachev, Sachovsky, Tikchonenko and Dobrov (1968) and Dobrov and Tikchonenko (1969). I n this apparatus light-scattering suspensions are placed in spectrophotometer cells located in the center of the integrating sphere, the inner surface of which is covered with a 1 mm layer of magnesium oxide. The instrument contains a monochromator which is connected to the integrating sphere (diameter 16 cm) , a photomultiplier powered by a stabilized high voltage source, and a registering device (registering full scale deflection for 1 pamp with scale division values of 0.001 pamp). Figure 1 shows a diagram of the instrument. The cell-holder is designed as a skeletal frame, leaving most of the cell open. The cells are inclined to the

Ib

FIG.1. Diagram of the integrating sphere, (la, b ) Two steel hemispheres; (2) light-proof plug for cell-holder fixing; (3) rod connected to the handle going outward (4); (5a, b ) cell-holders for the sample and reference cells; (6) carriage for photomultiplier; (7) plate for connection with monochromator (from SF-4 spectrophotometer) ; (8) connecting plates of hemispheres bolted by six screw bolts (9); (10) inlet hole; (11) outlet hole. The handle (4) helps either to place the cells (5a or b) before the inlet hole (lo), or to direct the light beam to the wall of the spherical container (between holders 5a and b). vertical by a small angle (3-5") to prevent that light reflected from the front wall from passing back into the source. The cells are covered with aluminium tops to prevent the upward scattered light from falling directly onto the photomultiplier. Thus, the photomultiplier Iocated in the upper portion of the sphere is allowed to measure the total light intensity inside the sphere. The At determination formulas were corrected for reflection from the cell walls. It should be noted that making measurements with the integrating photometer is rather complex and that subsequent calculations are rather time consuming. This fact plus the impossibility of taking measurements at high temperatures makes the instrument inconvenient for routine use. The true At of suspensions of intact and disrupted phages with

266

T. I. TIKCHONENKO

diameters up to 600 to 700 A as measured with the integrating photometer coincided completely with the results of extrapolation-corrected spectrophotometric measurements. But the phages having a diameter more than 1000 A gave a more complicated picture (Table IX). For suspensions of these disintegrated phages the results, as expected, were identical with those obtained by the extrapolation method procedure. But with intact phages there were some deviations. Numerous measurements revealed two types of T 2 preparations. One type gave good agreement between these two methods of determination ; in preparations of the other type the At value determined by extrapolation was always lower. TABLE IX TRUE

ABSORPTION( A t ) V A L U E

OF SUSPENSIONS O F D I F F E R E N T PH.4GES A S

DETERMINED I N THE INTEGRATING RY

uv PHOTOMETER

AND

EXTRAPOLATION

At determined in sphere as percent of that determined by extrapolation0

Phage

260

1. Sd 2. DD-VII 3. T2 preparation A preparation B 4. DD-VI

101 100

103 111 112

,

Wavelength (mp) 270 280

290

99 99

100 102

101 102

100 107 105

99 106 104

100 106 106

Mean values for 10 different preparations.

We are, as yet, unable to account for the discrepancy between the optical properties in the case of the second type of T 2 and the DDVI preparation, as well as the very existence of two types of T 2 preparations. It might be possible to account for an error in the extrapolation determinations by contamination absorbing in the spectral region 320350 mp. But, since for disrupted phages the results obtained by both methods were similar in all cases, this hypothetical contamination would have to lose its absorbing ability a t the same time that the phage par(icles were dkrupted. This is highly improbable. Conrequently, it might be suggested, that the anomalous light scattering ob-erved for some preparations of T2 and DDVI phages is conn.clcd wiLh the optical properties of the virus particle itself. It is possible that the error in the extrapolation determination is somehow related to the size of the phage particles (maybe unusual aggregation).

267

CONFORMATION OF VIRAL- NUCLEIC ACIDS IN SITU

But one must keep in mind that the discrepancy between the direct and indirect method of At estimation takes place only with one type of Preparation of large phages. What conditions govern the appearance of such type of phages and their abnormal scattering we still do not know. Hence, the conclusion that there is now a direct method for the A, estimation of virus particles which gives identical results with the extrapolation method for small and middle-sized phages. Still, there exist some unknown properties of suspensions of large phages which sometimes cause errors in the extrapolated value of U . TABLE X DROP I N A VALUEO N DISRUPTION OF SDAND T2 PHAGE SUSPENSIONS AS DETERMINE D I N T H E INTEGRATI NG uv PHOTOMETER AN D BY EXTRAPOLATION^ At value of disrupted phage as percent of that of intact phage Phage and method Sd Integrating photometer Extrapolation T2 preparation Ab Integrating photometer Extrapolation T2 preparation Bh Integrating photometer Extrapolat ion

260

Wavelength (mp) 270 280

290

87 89

84 82

79 78

74 72

88 91

86 89

81 85

73 81

88 102

86 94

81 88

73 85 ~~

Sd phage was disrupted by heating at 55OC and T2 by heating at 65°C. The solvent was 0.1 M NaC1. For A- and B-types see Table IX. a

As was reported earlier (Tikchonenko e t al., 1966, 1967) the disruption of medium- and large-size phages brings about a considerable drop in the true value of absorbancy at 260 mp (on the average 1213% of the initial value). This hypochromism does not depend on the method applied for disruption of the phage particles provided that disruption conditions do not interfere with the secondary structure of the DNA. The hypochromic shift took place when phages were disrupted by either heating, detergent, osmotic shock, or even by release of DNA in the course of interaction with bacterial cell wall fragments (Velikodvorskaya, e t al., 1968; see also Plate I V ) . Typical results using different phages and different methods of evaluation of A , are presented in Table X (Tikchonenko et al., 1966a,b; Dobrov and

268

T. I. TIKCHONENKO

Tikchonenko, 1969). Our data were confirmed also by the results of Inners and Bendet (1969) and Gabrilovich et al., (1969) who observed the typical hypochromic shift on disruption of T2 phage using the extrapolation method. The use of the integrating sphere also allows one to register the typical hypochromic shift associated with disruption of T2 phage preparations of the B-type. Now it is clear that the drop in absorbancy for a T2 suspension is the same as that for Sd. Previously (Tikchonenko et al., 1966, 1967) the mean value of the drop in absorbancy for T2 phage as determined by extrapolation from a great number of experiments was reported to be somewhat lower than that for Sd phage. This small difference reported previously may be accounted for by the error due to extrapolation for some of the T2 suspensions (the admixture of B-type particles). TABLE XI HYPERCHROMICITY OF DNA INSIDE SD PHAGE PARTICLES At values

Wavelength (mp) 270 280

DNA type

260

1. Intact Sd aa percent of that of disrupted phage 2. Thermally denatured Sd DNA as percent of initial values 3. The same as (2) but in presence of 1.6y0 CH 2 0

115

118

127

134

139

143

140

140

140

156

164

168

290

The possible contribution of protein in these spectral changes is easily excluded by simple controls (mixture of DNA and hosts, or hosts alone). Spectral characteristics of intraphage DNA obtained by Tikchonenko et al. (1966a,b) and Dobrov and Tikchonenko (1969) indicated that intact phages have a higher absorbancy in the region of 250 to 295 mp than disintegrated particles, and a different wavelength of maximum absorption (262 mp for intact phage and 259 mp for disintegrated particles or deproteinieed DNA). It is interesting to calculate the spectral change occurring after release of the DNA from the particle in another way, that is, as hyperchromism of the intraphage DNA relative to the excreted DNA (Table XI). Comparison of the hyperchromism for intraphage DNA with that of free denatured DNA of the same phage revealed considerable differences in the spectral characteristics

CONFORMATION OF VIRAL NTJCLEIC ACIDS IN SITU

269

of the hyperchromism in these two cases, For intraphage DNA the maximum hyperchromism is shifted noticeably toward longer wavelengths. Such a tendency resembles, to some extent, the spectral changes brought about by reaction of DNA with CH20 (first and third lines of Table XI). The reaction of DNA with CH20 is accompanied by a considerable increase in absorbancy at longer wavelengths, cf. hyperchromism during heating in the absence of CH20. This spectral shift is characteristic of the primary reaction of CH2O with the NH2 groups of the bases leading to the appearance of their corresponding methylol derivatives (for reference see Section I,A,l,b) . Of course, such resemblance is a mere analogy but it is rather meaningful, indicating a possible reason for the abnormal optical properties of intraphage DNA. Some of the amino groups of bases inside phage particles seem to be involved in interactions similar in this respect to the reversible binding of CH2O (methylol derivatives of the bases). This interaction disappears, naturally, during release of the DNA from the phage and brings about the described optical changes. All these observations as well as other data obtained (see below) allow one to speak about the peculiar conformation of intraphage DNA, or of least a part of it. c. Chemical Modifications. On the other hand, some of the features of this conformation resemble to some extent the properties of partially denatured DNA (Tikchonenko et al., 1966a,b). This discrepancy as well as some other facts not mentioned above make it desirable to investigate properties of the secondary structure of DNA inside phage by more direct methods. Chemical modifications which permit the study of the state of functional base groups would seem one of the most direct and informative ways of establishing the secondary structure of polynucleotides. With respect to problems of our review the amino groups of the bases are of special importance. For this reason Tikchonenko’s group used several different reagents whose reactivity toward the functional groups of bases, mainly the amino groups, “measures” secondary structure and, principally, depends on their blocking by hydrogen bonds (Tikchonenko et al., 1966; 1969b,c; Kisseleva et al., 1966; Tikchonenko and Kisseleva, 1969). In spite of some differences, experiments with all sorts of modifications yield comparative reactivities for the amino groups of the bases in free and intraphage DNA under conditions maintaining the integrity of phage particles. i. Deaminatwn studies. If the DNA inside the phage particles is really partially denatured, one may expect to find the rate of deamination of this DNA higher than that for free DNA. The results pre-

270

T. I. TIKCHONENKO

sented in Table XI1 confirm such an expectation. One may see that the initial rates of deamination of guanine, adenine, and cytosine inside phage particlcs are very much higher than those for free DNA. Such a situation is possible if free amino groups of these bases are present in intraphage DNA which readily undergoes deamination. Tikchonenko and Kisseleva (1969); and Kisseleva et al. (1966) supplied electron microscopic proof for the integrity of the phage particles during the reaction. At later stages the behavior of these three bases in intraphage DNA seems to be different. While for free DNA the reaction maintains its linear character for guanine, adenine, and cytosine, in the case of intraphage DNA it remains constant only for guanine. For adenine TABLE XI1 MEANVALUES OF DEAMINATION ~ B L O C I T I E(v,) S OF B.4SES I N F R E E AND INTRAPHAGE DNA AT pH 4.10" Guanine (pmoles) DNA type

Adenine (Mrnoles) Cytosine (pmoles)

At300

Atso

Ahto

At180

DNA in solution DNA in phage

4200 8500

2260 2930

330 3470

417

375

750 1830

1000 611

Vm phage/V, DNAb

2.02

1.3

10.5

0.90

2.4

0.61

Data for Sd phage. V,,, = AC/Al where AC is the number of transformed bases in pmoles during At in minutes.

and cytosine exhaustion of bases with free amino groups leads to a sharp decrease in the rate of reaction. Consequently, the rate of reaction becomes very much closer to the rate of deamination of these two bases in free DNA. Theoretically, a higher rate of deamination for intraphage DNA as compared to free nucleic acid can be explained from two points of view: there is either a preexistence of free amino groups from the bases or a greater labilization of the DNA secondary structure in the virion due to acid pH a t higher temperature. As to the latter argument, under the conditions of deamination used 0.1, M Mg2+) free DNA by the authors (pH 4.1, 37"C, 1 M Na+ gives no hyperchromism and maintains its ordered structure (Cavalieri et al., 1956; Michelson, 1963; Zimmer and Venner, 1963). At the same time, physical stability may decrease under these conditions due to ionization of the guanine-cytosine pair. According to rather approximate calculations of Zimmer and Venner, no more than 10% of the guanine-

+

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cytosine pair and no more than 4% of the adenine-thymine pair may be ionized under these conditions. Protonization here practically does not occur in the amino groups of bases whose pK are in the more acid p H range (Dawson et al., 1962; Cavalieri and Rosenberg, 1956; Zubay, 1958; Michelson, 1963). That is why base ionization in this case will affect the results of deamination only so far as it disorders the secondary structure of DNA, And, according to Michelson, the dissociation of the hydrogen bond blocking the N-1 atom in cytosine and adenine in complementary pairs does not necessarily entail rupture of the hydrogen bond blocking the amino group. Although this point of view is not excluded by the deamination results it, probably, could be ruled out by the results obtained by means of other methods (see below). Based on the fact that the kinetic curves for the deamination of adenine and cytosine in the experiments of Tikchonenko and Kisseleva (1969) had sharp breaks after the first hour of reaction, the authors made an attempt to estimate approximately the quantity of bases with free amino groups. This calculation proceeds from the self-evident fact that the retardation of deamination of intraphage DNA is connected with the exhaustion of preexisting free amino groups. Thus, the slower rate of the second stage of the reaction should be due to deamination of helical portions of the DNA. It has been calculated that sites with irregular structure contain about 21% adenine and about 12% cytosine. Unfortunately, no such information is available for guanine due to the abnormal properties of the third hydrogen bond in the guanine-cytosine pair in this p H region (Zimmer and Venner, 1963; Tikchonenko and Kisseleva, 1969). ii. Oxymethylhydroxylamine ( O M H A ) . In order to check the conclusions concerning deamination Tikchonenko et al. (1969~)used another modifier-oxymethylhydroxylamine (OMHA) , which represents a derivative of the well-known hydroxylamine. The reaction of the latter with RNA and DNA depends upon their secondary structure (Brown and Phillips, 1965; Kotshetkov et al., 1966, 1967; Morozova and Salganik, 1964). Oxymethylhydroxylamine has a similar specificity with respect to secondary structure but a higher specificity toward the primary structure, at the expense of reacting chiefly with cytosine (Budovsky et al., 1968a,b). The wide pH optimum of the OMHA reaction allowed one to carry out the modification under almost neutral conditions. Thus, the labilizing action of acid pH on the DNA secondary structure is avoided. In addition, as under these conditions OMHA reacts preferentially with cytosine, the possible influence of other modified bases on the one studied may be excluded. In the above cited investigation the reaction of cytosine with OMHA was studied in two ways: first, by the decrease

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in absorption at 276 mp, as the products formed either do not absorb or have a low absorption in the W, and second, directly by measuring the loss of cytosine after hydrolysis with perchloric acid and subsequent paper chromatography. These authors showed that cytosine in free DNA practically does not react while at the same time a drop in absorbancy at 276 mp indicates an appreciable degree of reaction in intraphage DNA. Modification of the cytosine was completed after 12 to 16 hours of incubation at 32°C with 1 M OMHA. The direct measurements indicated that in intraphage DNA about 13% of all of the cytosine reacts with OMHA. This value practically coincides with the 12% of free amino groups of cytosine found in deamination experiments. According to the result, 12-13% of the cytosine of intraphage DNA is situated in sites having an irregular secondary structure. iii. Water-soluble carbodiimide. This is a very specific reagent for denatured regions of DNA (Augusti-Tocco and Brown, 1965; Drevitch et al., 1966; Salganik et al., 1967). It reacts specifically with guanine and thymine. Tikchonenko et al. (1969b) showed that the DNA inside Sd phage bound 24-25 moles carbodiimide per 100 moles nucleotide. What percent of reagent is bound to thymine and to guanine, respectively, is not yet known. The reaction of free DNA and intact phage particles, in this case, was carried out at 30°C and pH 8.0 in 0.01 M tris-HC1 buffer plus 0.1 M NaCI. A plateau was reached after 24 hours of incubation. These conditions are quite different from those present in experiments with HNOz or OMHA, and allow one practically t o exclude the greater sensitivity of intraphage DNA to denaturation as a possible reason for the results obtained. Summing up the above data on the chemical modification of the DNA in phage Sd it may be concluded that 30% of all the bases are localized in zones having a disordered secondary structure. And, naturally, there arises the question: to what extent does the DNA despiralization detected by means of chemical modifications correlate with the defects of secondary structure as manifested by anomalous optical properties? Certainly, chemical modification data should not be interpreted to mean that the reacting amino groups of bases necessarily preexisted in the free form. They may be in their free form, but, on the other hand, they might be engaged in some kind of interaction with protein [hydrogen or salt bonding, for example). The only important point is that this interaction should be much weaker than that for the amino groups of bases in the double helix (ie., hydrogen bonding in regular base stacking under conditions of cooperative interaction) . A good analogy for this phenomenon would be the difference in reactivity between the base amino groups from helical portions of RNA and double-helical

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DNA. And under conditions of chemical modification where the base reactivity of double-helical DNA serves as a normal level of reactivity, RNA may react much faster not at the expense of preexisting free amino groups but due to weaker blocking of these amino groups. A close comparison of the kinetics for these two processes in phage, free DNA, and a mixture of bases might prove helpful in choosing between these two possibilities. But, unfortunately, the kinetic approach to this problem can hardly be applied because of unsolved questions concerning the phage coat and densely packed DNA core permeability. At the same time, the spectrophotometric results cited above show that the partial hyperchromism of intraphage DNA is not characteristic of trivial denaturation, for which free base amino groups are indispensable. That is why it is possible to conclude that it is the same sites of DNA that produce the hypochromic effect on the DNA’s release into the medium and participate in chemical modification. Assuming the usual 40% hypochromism for free DNA from phage Sd, we may ascribe the 12-13% deficiency in hypochromism to the approximate 30% disordered secondary structure. This figure is equal to that obtained in the chemical modification experiments. This identity might, certainly, be a mere coincidence-then one third of the intraphage DNA should have anomalous optical properties while the second third has free amino groups and the spectral characteristics usual for denaturated DNA. The experiments with the fourth modifier-CHzO, evidently, makes the first point of view more probable. iv. Formaldehyde. Dobrov et al. (1967) and Tikchonenko and Dobrov (1969) tried to investigate the action of CH20 on the optical properties of DNA during its escape from the phage. Since CHzO stabilizes the phage protein coat, direct experiments on disintegration of the bacteriophage in the presence of CH2O could not be performed. They have succeeded in eliminating the interfering stabilization of the virion protein coat by preliminary addition of 0.05% sodium dodecyl sulfate (SDS) to suspensions of the Sd phage (it is stable to the SDS a t normal temperature and neutral pH). If such an intricate complex, phage-SDS-CHnO, was then either heated a t 56°C or its p H was changed to 5, phage disintegration and hypochromism took place. This means that the normal DNA conformation was restored despite the presence of CH2O in the medium. However, unlike the typical hypochromic effect apparent without CH20, in the presence of CH20 it turned out to be less, the absolute values being insignificantly different a t 260 mp but essentially decreasing with an increase in wavelength. This effect is completely comprehensible if one takes into account its total and additive character. First, regions of DNA inside the phage particle,

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T. I. TIKCHONENKO

which have reacted with CH20 do not contribute to the hypochromic effect, and, second, the DNA reaction with CHzO brings about spectral changes bearing opposite sign. I n other words, due to the first factor some part of the DNA inside the phage particle lacks a drop in absorbancy, the effect being more pronounced at longer wavelengths; on the other hand, due to the second factor, the longer the wavelength, the greater the rise in absorbancy in a part of the DNA. As a result a small change of (E200)absorbancy at 260 mp for the DNA after phage disintegration in the presence of CHBO is accompanied by a considerable change of the relative value of absorbancy at longer wavelengths. The authors conclude that a small but definite fraction of the amino groups of DNA bases inside the phage still reacts with CH20 during phage disintegration in the presence of this reagent. Thus, unlike about 30% of all amino groups-the result obtained in the experiments with carbodiimide, OMHA, and HNOr-in the case of CH2O the authors speak about a small fraction of amino groups without denoting its value, for some reason. As was indicated above, this fact, apparently, confirms the point of view that the bulk of modified reactive base groups is blocked by comparatively weak bonds and only a small fraction of amino groups is in the free form. I n this form the bases readily react with CH20 in the course of comparatively short incubations (20-100 minutes) at room temperature. By the way, it is the latter fact that is the weak point of this otherwise rather believable hypothesis. That is, the incubation time may be too short for the reaction to come to an end. It should be remembered that in the experiments with OMHA and carbodiimide the reaction reached a plateau after 16 to 24 hours. The high deamination rate is, evidently, due to the reaction of the protein coat which affects permcability to a considerable extent and a number of other factors of the intraviral medium. If this is the case, the inability of CHzO to prevent DNA from “renaturation” on its release from the virus could not be explained by the presence of but a small number of bases with free amino groups in intraphage DNA. In this case one is bound to accept that about one third of the base amino groups is not included in spiralized parts and is not blocked by any bonds. This question may be solved by further experiments along this line. But whatever the final answer to this question may be, it will not change the heart of the matter. In any case, we deal with disordering of the regular secondary structure of intraphage DNA. In the second set of experiments conducted by Tikchonenko and Dobrov, the melting of DNA inside the phages, in the presence of various concentrations of CH20, was investigated under conditions preventing

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275

disruption of the phage particle. Judging by a sufficiently formal criterion-the temperature a t which one half the complete hyperchromism develops-the melting temperature (T,) for DNA inside the phage, in the presence of 1.6% CHzO, proved to be 82.5"C; that means that it practically does not differ from the melting point for free DNA in the absence of CHzO (Inners et al., 1965). Only a concentration of CHzO as high as 5 % , brought about a slight decrease in the T , of the DNA inside the phage from 82 to 77"C, whereas the melting point of free DNA fell to 63°C under the same conditions. All this evidence suggests some superstabilization of the secondary structure of DNA inside the phage particle. However, strange as it may seem, this superstabilization is associated, or a t least coexists, with a disturbance of the cooperativeness of the interaction between bases, which is so typical of the double-stranded structure described by Watson and Crick. The melting profiles revealed for intraphage DNA in the presence of CHzO by the above-mentioned authors differ from those for free DNA by manifesting curves with reduced slopes and wider zones of thermal transition. With an increase in CH20 concentration from 1.6 to 4.8% the transition width increases from 20 to 47", being seven to eight times wider than the thermal transition characteristic of free DNA. Tikchonenko and Dobrov are inclined to suppose, that the loss of cooperativeness seen during the melting of DNA in the presence of CH20 can be easily explained by a separation of the DNA ordered secondary structure into numerous segments possessing independent behavior with respect to heating and CH20. One may easily understand that separation of the helical segments may be achieved by regions of denatured or altered DNA. As to the mechanism of superstabilization, there are three possibilities. The first may be the low permeability of the phage particle to CH20. This is excluded by the observation that the hyperchromism of intraphage DNA in the presence of CH2O is irreversible and has spectral characteristics typical of DNA reacted with CH2O. Second, there may be steric factors which hinder disordering of the stacked bases (see Michelson, 1963), whereas the amino groups of the bases react normally with CH20. This supposition is ruled out as the reaction with CHzO (methyl01 derivatives) is easy to demonstrate by noting absorption changes a t longer wavelengths. The third may be that the high stability of the DNA inside the phage particles to CH20 is due not (or not only) to stabilization of stacked bases but to an additional specific blocking of the amino groups of these bases. The last possibility seems t o the authors the most probable, although the mechanism of such "superstabilization" is not yet clear. The first (but not the

276

T. I. TIKCHONENKO

only) candidate for such a superstabilizing partner is, of course, the protein. d. Interaction with Dyes. As far as we know there are only two papers dealing with investigation of the secondary structure of intraphage DNA by means of dyes. One of the papers (Gabrilovich et al., 1968) reported the results of a study on Klebsiella phages by means of acridine orange. I n our investigation (Permagorov et al., 1969) pinacyano1 (PNC) was used with phages Sd and T2. Two things should be borne in mind when dealing with dyes of the PNC type for identification of secondary structure. First, by varying the ratio of the amount of DNA to that of the dye (P/D) and observing the changes in certain absorption maxima, it is possible to obtain information about the monomeric and dimeric form of the bound dye. Second, PNC and similar dyes on binding with DNA rotate the polarized light causing the appearance of anomalous ORD spectra in the dye absorption region. It may be considered as established that the induced ORD of the PNC-DNA complex is due to dimeriaation of the dye on native DNA (Permagorov et aZ., 1966). Neither phage Sd, nor phage T2 loses its infectivity or undergoes physical destruction upon interaction with PNC. The pattern of the change in absorption spectra on interaction with free and intraphage DNA proved to be generally the same. But these changes were observed for phages Sd and T 2 with a much greater P/D ratio as compared to this value for the PNC-free DNA complex. These differences allow one to calculate that for the native Sd and T2 phage, only 50 and 2076, respectively, of the entire DNA content come into contact with the dye. Binding of the dye by both free and intraphage DNA causes the appearance of an induced optical activity. But the measurement of "560 showed that the ORD value for the complex PNC-intraphage DNA is unproportionally small in comparison with the quantity of dye dimers present in this complex. For example, for phage T2 and Sd only 60 and 30% of the dimers, respectively, displayed optical activity. This means that PNC dimerization on intraphage DNA differs from that on free nucleic acid. This difference can be explained in the following way : 1. Two kinds of dimers are formed on the DNA inside the phage: optically active and optically inactive ones, their respective formation being due to the DNA conformation. If this is the case the quantity of optically active dimer in the phage is indicative of the quantity of intraphage DNA which reacts with the dye, just like native DNA. The content of native DNA inside phage Sd amounts to about 30%, and

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60% in phage T2. It should be kcpt in mind, however, t h a t these figures should be attributed to the portion of the intraphagc D N A which is accessible to thc dye, i.e., 50% of the D N A in phage Sd and 2076 of the D N A in phage T2. A corresponding calculation for Sd phage will show t h a t 15% of its total D N A has a nativc double-hclical configuration and 35% of thc total D N A content has a specific conformation. The character of the secondary structure of the remaining 50% of the D N h in Sd which does not react with the dye, remains unknown. For phagc T2 the quantity of native D N A was established to be 12%, with 85% of the total D N A content having a different conformation. The secondary structure of the remaining SO% of the intraphagc D N A in T2 particles cannot be tested by this method. As to thc D N A having a specific conformation, it is, most probabIy, a portion of the D N A with disordcred secondary structure. 2 . On the D N A inside phage there form PiYC dimcrs of one kind with the optical activity lower than that for P N C diniers on free DNA. I n this casc the entire intrapliage D N A will have a diffcrcnt conformation as coniparcd to that of free D N A in solution. 3. Dimeriaation of the dye on D N A inside the phage is spatially hindered due to some unknown factors. I n this case the quantity of optirally active dimers will be the lower limit of thc amount of native D N A in phage. Of thcsc probable reasons for different dimeriaation of P N C inside phagc as compared to that on free D N A the second case hardly wcms possible as ORD patterns for the complex PNC-DNA and for thc complex PNC-phage arc identical. And it is impossible t o choose b e t w c n the first and the third case using only the results obtained in the cxperiments with this dye. But if thesc data are addcd to the evidence obtained by other techniques one will be bound t o stick t o the first point of view. Of particular interest, in this respect, is the inrcatigation of Gabrilovich et al. (1968) (see below). \T’hcm dyes are used for the investigation of the structure of polyiiicrs there is always the problem of the influence of the dye itself on the structure of thc object studied. I n the papcr of Permagorov et nl. (1969) the quantity of native DNA inside the phages was estimated procccding from the assumption that P N C does not strongly affcct the structurc of D N A and the phage itsclf. This assumption was based on the high values of P/D used along with the small relative concentration of the dye. Under these conditions the contribution of PNC-incluccd perturbation on the structure of intraphage D N A appears insignificant. Besitlcs, the coincidence of the estimated quantities of the dye adsorption sites obtained bv means of smctrouhotometrv and ORD. shows

278

T. I. TIKCHONENKO

that formation of optically active dimers in phage occurs to the same degree as the general dimerization of the dye. This fact leads to the conclusion that the structure of the sites of intraphage DNA where optically active dye dimers are formed is similar to the structure of free DNA. In the experiments of Gabrilovich et al. (1968) the DNA structure in Klebsiella phages was judged by the luminescence spectrum of complexes of DNA-acridine orange. The capsid of these phages, contrary to that of T-even phages, was shown to be permeable to acridine dye. According to Tumerman (1967) luminescence spectra of the complex free DNA-acridine orange had a maximum at 530 mp in the native state and two maxima at 640 and 530 mp in the denatured state. The author reported the luminescence spectra for the two Klebsiella phages L1 and No. 380, which contain double-helical DNA, to have two maxima at 530 and 640 mp. The presence of the maximum a t 640 mp is interpreted to indicate that some part of the DNA inside the phages is in the denatured state. Disintegration of the phages with release of their DNA into solution caused disappearance of the 640 mp maximum. At the same time the authors observed a hypochromic shift which denotes “renaturation” of the DNA. Unfortunately, the paper does not contain some experimental details which are of importance for interpreting the results. It should also be noted that the absence of a relationship between the luminescence spectra and the P/D values does not allow one to quantitatively evaluate the results. But the quantitative side of the investigation carried out by Gabrilovich et al. provides a valuable supplement to the study described above (Permagorov et al., 1969). That is, the luminescence maximum at 530 mp of the complex DNA-acridine orange is due to the monomeric, and the maximum at 640 mp to the dimeric, form of the dye. Thus, the red fluorescence in the second case makes less probable the third proposition (limitation of PNC dimerization on native DNA regions in situ). The dimerization of acridine orange on nonhelical sites of intraphage DNA reported by Gabrilovich is a direct confirmation of our first explanation, i.e., that of two types of PNC dimers. I n this case optically nonactive dimers correspond to the sites of intraphage DNA with disordered secondary structure and optically active ones to the typical duplex. e. Optical Rotatory Dispersion. The results of application of this optical method to the analysis of the conformation of nucleic acids in various viruses were reported by Maestre and Tinoco (1965, 1967) and by us in the form of a preliminary note (Gorin et al., 1967).

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Maestre and Tinoco measurcd the ORD of 16 different viruses containing various types of DNA and RNA; yet their data on the structure of intraphage DNA and RNA concern practically only phages T 2 and T7. For these two viruses the authors offer complete data: ORD values for the native virus, its free DNA, and protein. Gorin et al. (1967) studied only the phage Sd. These authors found a marked change in ORD between the intact phage and osmotically shocked T 2 particles or temperature-disrupted Sd. The ORD curves are completely different, with the shocked or disrupted phage showing a positive rotation above 280 m p instead of the negative rotation shown by the intact phage. The rotation of the T2 shockate or temperature-disrupted Sd is the sum of the rotations for purified phage protein and free DNA. These authors suppose that the protein coat is not changed appreciably by osmotic shock or under mild conditions of isolation; and as the main change in ORD is above 250 mp, the difference in ORD between intact and shocked phage (Aw) must be a result of the peculiar conformation of the DNA in the phage head. There was a great difference between the ORD spectra of native DNA and that calculated for intraphage DNA. It was interesting to compare (Aa) for different phages. The ( A a ) for T2 and T7 phages were obtained by subtracting the rotation of the osmotically shocked T2 or LiC1-disrupted T 7 from that of the intact virus. Although the rotations of the intact phages are very different, the (ha) curves are similar. Both show a negative single Cotton effect near 265 mp with a trough a t around 285 mp. Trying to determine the possible reasons for the anomalous ORD of intraphage DNA, Maestre and Tinoco (1967) analyzed five factors: (1) localized melting of the secondary structure, (2) changes in the local pH of the molecule, (3) interaction of the protein with nucleic acid, (4) changes in the chemical state of the molecule due to reactions with internal polyamines and protein, and (5) changes in secondary structure of the molecule due to the decrease of the water content inside the virus. The first four of these factors are considered by these authors as improbable, for not being well-grounded reasons. For example, they did not believe partial denaturation of intraphage DNA to be the cause of the ORD anomaly, as in the case of the melting of DNA or RNA in solution the difference ORD shows a weak double Cotton effect. Of course, i t is quite different from the single Cotton effect of intraphage DNA difference spectra. But in the papers of Samejma and Yang (1964, 1965), cited by Maestre and Tinoco, ORD was measured a t temperatures exceeding that of the T , when the entire structure of the molecule collapses. Only partial denaturation is dealt with in the case of phages. It is obvious that in this case the difference ORD should be

280

T. I. TIKCHONENKO

obtained by subtracting the curve for native DNA from that of the partially denatured DNA at room temperatures. Besides, different denaturation procedures give somewhat different ORD spectra. Why, then, should thermal denaturation be preferred? Also, the conclusion about the absence of an effect of the protein capsid and internal protein upon the ORD of intraphage DNA, seems premature. Maestre and Tinoco stick to the concept of a “central body” which suggests a minimum contact between the protein and nucleic acid. These authors try to support this rather hypothetical point of view by using Kellenberger’s concept of a condensating factor (Kellenberger, 1962). They (but not Kellenberger!) seem to believe that the intraphage DNA at the last stages of virion formation is packed in the same way as in the head of the mature virion though the protein capsid is not present. In this statement everything is disputable and, hence, cannot be used to support another hypothesis. The example of 4x174 is, also, hardly convincing. The authors believe that since this phage is comparatively small there should be a more intimate contact between the protein coat and the DNA. As this phage shows a smaller magnitude in the difference curves as compared t o T2 difference spectra of ORD the statement about the minimum contact of DNA with protein may be considered correct. It will be appropriate t o remember here Klug’s model for TYMV where the small parameters of the virus do not prevent its RNA and protein from having intimate contact. As to the small discrepancy between the DNA difference spectra in phage 4x174 and in solution, it may be due to the peculiar conformation of a DNA in situ and not to the number of DNAprotein contacts. Finally, the possible role of polyamines (Ames and Dubin, 1960) in the modification of DNA ORD in vitro being ruled out experimentally, the authors did not perform similar experiments with internal protein. And interaction of DNA with alkaline proteins (internal protein is just such a one) has a pronounced effect on its ORD spectra (Inoe and Ando, 1968). So, taking into consideration DNA interaction both with coat protein and internal protein, the conclusion may be drawn that the contribution of this interaction in the change of ORD for intraphage DNA may be greater than that believed by the authors. In their very interesting and promising experiments with concentrated solutions of LiCl Maestre and Tinoco found out that difference spectra between normal DNA and DNA in LiCl proved to have a single negative Cotton effect at about 276 mp, and very similar in magnitude to the (act) for T2 and T7 phages. The change in ORD in concentrated LiCl solutions is ascribed by the authors entirely to dehydration. As the

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ORD difference spectra for DNA in the phage and in LiCl proved to be similar, the authors draw the very simple conclusion that dehydration is the only cause for conformational changes of the DNA in the phage head, the greater part of the DNA molecule being affected. As a matter of fact, it may be that partial dehydration of DNA in phages is one of the reasons for its conformational changes. And although the experiment with LiCl is a mere analogy it confirms to some extent the speculations of some other authors (Pollard, 1953; Tikchonenko et al., 1966a). At the same time, it must be noted that the Maestre and Tinoco hypothesis might be accused of some one-sidedness. I n the first place, it is very difficult to differentiate between dehydration and some denaturation changes in the secondary structure of DNA. The denaturing action of high concentrations of salt on DNA have been investigated by many authors (for references see Tikchonenko, 1965) which testifies to the great complexity of this phenomenon. Second, ORD difference spectra for DNA in the phage and in LiCl were similar but not identical, and the similarity might have been accidental. But even if this is not the case, the discrepancy between these difference spectra might be to a certain extent due to some of the above factors. Therefore, it is precarious, in our opinion, to exclude from the number of possible factors affecting the properties of intraphage DNA, either partial denaturation or interaction with protein or some other conditions of intraphage environment of which we now know nothing. All of them may be operative together with partial dehydration which is, certainly, an important feature of the DNA environment in situ. f . Experiments with Superhelical Circular D N A . The study of the structure of the DNA superhelix of A phage, phage P22, and that of polyoma and SV40 viruses allows one to make a very interesting suggestion which may have important consequences for our views on DNA conformation in situ. This is the idea of superspiralization of doublestranded circular DNA occurring only in witro, after the release of the DNA into solution, and not taking place in situ (Bode and MacHattie, 1968; Vinograd et at., 1965, 1968; Rhodes and Thomas, personal communication). The argument that these authors have to offer is that the number of supertwists per unit of molecular weight turned out to be similar for different circular DNA’s and changes regularly on being affected by various factors. Among such factors are ionic strength, intercalation of dyes, partial denaturation, and some others which should be mentioned (Bode and MacHattie, 1968; Vinograd et al., 1965, 1968; Rhodes and Thomas, personal communication). The data obtained by these authors are interpreted to mean that the configuration of circular DNA in the virus is essentially different and has among other things a

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different pitch and a different number of base-pairs per turn. On escape from the viral particle the DNA molecule “finds itself” in a different environment and tends to acquire a new configuration which is inherent to the new conditions, the B configuration being just one. Polynucleotide chains being closed by covalent bonds, such a configurational transition leads to the appearance of a secondary superhelix. Bode and MacHattie emphasize that the high ionic strength mimics, at least partially, the effect of intracellular environment on the pitch of the DNA double helix (in their experiments the number of supertwists in DNA decreases from 117 to 12 in 2 M NaC1). It is evident that such profound differences in DNA configuration, as pitch of the helix and the interbase distance, can be easily revealed by X-ray analysis. In this case it is natural to ask whether what is true of the circular superhelix is also encountered in the linear duplex? If the above hypothesis is correct the conformational change is observed only because the tertiary structure is bound to reflect the consequences of these changes due to the ring form (supertwisting). In the case of the linear duplex such alteration of the secondary structure will not entail supertwisting and, as a result, will not be registered at all. If the intraviral environments are similar for such “distant relatives” as A phage and polyoma virus, this phenomenon may be claimed to have a universal character. But, according to the X-ray diffraction data, oriented phage preparations give a 3.4 A reflection testifying to the presence of the B configuration. But it should be remembered, although, that in the section where these data were discussed (Section III,A,2,a) it wss emphasized that a different DNA configuration in situ is not exluded.

B. Other Viruses The only paper a t our disposal which bears some relation to the topic of this review, reports that heating of reoviruses in 2 M MgC12 for 5 to 15 minutes, a t neutral pH causes a specific increase in the preparation’s infectivity (Wallis et al., 1964). The infectivity increases four- to eight-fold as a result of the increased number of infective particles in the preparation. The ratio of the number of physical particles to the number of infected particles decreased from 15:l to 2: 1. This infectivity increase was reversible and disappeared upon lowering the Mg2+ concentration. The conditions of this experiment do not allow one to explain the obtained result by an aggregation-deaggregation phenomenon. The authors believe that specific activation of reoviruses at increased concentration of Mg2+ during heating may be due to RNA conformational changes. No real proof in favor of such a point of view is offered.

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Some speculative arguments for the possible conformation of DNA in the polyoma and SV40 viruses are given in the preceding section (Section 111,A,2,f). IV. CONCLUDING REMARKS The study of the conformation of nucleic acids in situ, ie., in viral particles, has just begun and no complete picture can yet be expected. Nevertheless, the analysis of various experimental data obtained by different techniques and methods allows one to draw a number of conclusions. 1. There is no doubt that the viral particle is a highly organized complex where nucleic acid and protein are involved in intimate interaction. The strength of this interaction may vary widely depending on the size, complexity, and type of symmetry of the particle and also on the type and content of the nucleic acid. 2. All types of nucleic acids in viral particles have a specific conformation of their own which is different from the one they have in solution. This is true both of their secondary and tertiary structures. For single-stranded RNA and DNA in the rodlike and filamentous viruses the highly ordered regular structure is conditioned by the interaction with the protein helix. In small spherical (isometric) viruses, besides nucleic acid-protein interaction, there appears the secondary structure similar to that of single-stranded nucleic acids in solution. The points of view of different authors on the geometry of single-stranded RNA and DNA in spherical viruses as well as on the percent of helical structure differ greatly. Double-stranded DNA in phage heads have hollow, ellipsoid symmetry, very dense packing, less spiralization, and a number of anomalous optical properties. 3. Intraviral nucleic acids have a complex environment both hydrophilic and hydrophobic. Therefore, nucleic acids are bound to enter into various and complex interactions with the other components of the virion, both ionized polar and nonpolar groups being involved. 4. Such factors as the degree of hydration, shielding of charged groups, local changes in concentration of anions and cations, and some others may have a considerable effect on the structure of nucleic acids in the virion. Due to these forces operative in the virion heterogeneity of the environment is increased manyfold. As a result, different parts of the nucleic acid molecule may exist under different conditions which is especially true of big viruses with a high content of nucleic acid. 5. Regardless of the degree of despiralization and even the complete loss of the inherent secondary and tertiary structure, the physical and chemical stability of the nucleic acid in the virion is usually higher

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than the stability of nucleic acids in solution. This phenomenon is conducive to the major function of the virus: the storage and transport of genetic information.

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