Studies on the stabilizing forces of simple RNA viruses

J. Mol. Biol. (1971) 56, 259-276

Studies on the Stabilizing Forces of Simple RNA Viruses I. Selective

Interference with Protein-RNA Interactions in Turnip Yellow Mosaic Virus J. M.

Plant Agricultural

KAPER

Laboratory, Crops Research Division Research Service, United States Department of Agriculture, B, ltsville, Md. 20705, U.S.A. Virology

(Received 3 July 197’0, and in revised form 2 November 1970) To allow an experimental study of the disengagement of the RNA from its linkage with the protein in turnip yellow mosaic virus, several methods were developed which affected the capsid structure to such an extent that upon breakage of the protein-RNA linkage, the RNA was allowed enough mobility to undergo either internal rearrangements or to be released in the reaction mixture. These “capsidexpanding” methods encompassed alkali treatment, moderate heating, and the treatment with 12 M-formamide, all in the presence of high ionic strength (1.0 MKCI). Moderate alkali treatment of turnip yellow mosaic virus gave rise to an in. xitu fragmentation of the phosphodiester chain of the RNA followed by a complexing of the RNA fragments inside the intact capsid. In alkali treatment with pH above 11, a large proportion of the virus particles released their fragmented RNA into the reaction mixture. The release of RNA was an all-or-nothing process, and followed single-hit kinetics for the majority of particles in a population. The rate of RNA release increased as the pH was raised, but appeared to be independent of the degree of fragmentation and/or complexing that preceded it. Hence, the release of RNA is primarily controlled by the degree of capsid expansion. Moderate heating or treatment with 12 M-formamide provided a means of capsid perturbation which allowed a direct analysis of the breakage of protein-RNA linkages as a function of pH. With both methods it was found that at pH values under neutrality turnip yellow mosaic virus retained its stability, but that a pH of 7 or higher induced dissociation into capsids and free RNA. The same pH dependence of dissociation of turnip yellow mosaic virus was also found after high doses of ultraviolet light irradiation. Whereas the viral capsid degraded into slowly sedimenting fragments upon irradiation under slightly acid as well as under neutral conditions, the majority of the virus particles remained stable at pH 6, but not at pH 7. It was concluded that in turnip yellow mosaic virus the integrity of the proteinRNA linkages is dependent upon the pH, and that these linkages are broken between pH 6 and 7. The type of interaction compatible with such behavior would be an ionic linkage between protonated histidinyl residues and negatively charged nucleic acid phosphates, or could consist of juxtaposed protein carboxylate and nucleotide amino groups, sharing a hydrogen-bonded proton. The latter model would provide a structural role for the extraordinarily large number of cytidilic acid residues in turnip yellow mosaic virus RNA. 269

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1. Introduction Controlled degradation of biologically functional macromeculoles can be used as a specific experimental approach towards understanding the nature of the forces which determine their quaternary and/or tertiary structure. The existence of certain proteins and of DNA as highly organized structures has become an established fact; and in the last few years, varying degrees of intramolecular organization have also been recognized to exist with different forms of isolated RNA. Another dimension of complexity is added to the experimental solution of the organization of biological macromolecules, when they occur in specific combinations, as is the case with nucleic acids and proteins in the form of viruses or other nucleoproteins. Here, one does not only deal with the folding and the intra- and inter-subunit interactions of each of the components individually; but in addition there is the problem of their integration into a biologically and chemically compatible superstructure. The geometric aspects of some of the least complicated forms of nucleic acid-protein integration, the simple RNA viruses, have been worked out to a considerable extent (for a review, see Kaper, 1968). The study of the dynamic aspects of these virus structures, i.e. their inherent capability of disassembly and reassembly in solution under the proper conditions, has made much progress in the case of tobacco mosaic virus (for reviews see Lauffer, 1968; Kaper, 1968; Caspar, 1963), while the reversible dissociation of several small isometric RNA viruses is under active investigation in a number of different laboratories (see accompanying paper, Kaper t Geelen 1971; Hiebert, Bancroft & Bracker, 1968; Kaper, 1969a; Bol & Kruseman, 1969; Lebeurier, Wurtz 15 Hirth, 1969; Hung & Overby, 1969). Another small isometric virus whose physical and chemical properties have been analyzed extensively, and whose macromolecular architecture has been worked out in considerable detail, is turnip yellow mosaic virus (Klug, Longley & Leberman, 1966; Finch & Klug, 1966). This virus has actually served as a protype for the geometry of several small RNA viruses. Unfortunately, to date no successful reassembly of TYMV+ from its constituent components has been reported. For this reason, studies on the nature of its inter- and intra-component interactions have been confined to degradation reactions. Controlled degradation of a simple virus such as TYMV should, ideally, result in a differentiation of the forces responsible for the stability of the complete virion, if it is possible to interfere selectively with the following categories of interactions; (a) interactions between the protein subunits, (b) interactions within the protein subunits individually, (c) intra-chain interactions of the RNA and (d) protein-RNA interactions. In the past several years, this laboratory has been engaged in various efforts to degrade TYMV in a selective and controlled manner. The work through 1967 has recently been reviewed (Kaper, 1968). It comprised the alkaline degradation of TYMV, at high ionic strength, into intact protein capsids and RNA (Kaper, 1960, 1964; Kaper & Halperin, 1965) ; a series of studies on the interference of organic mercurials with protein-protein interactions of TYMV (Kaper & Houwing, 1962a,b; Godschalk & Veldstra, 1965,1966); and the discovery that the protein-RNA interactions in TYMV make only a minor contribution to the stability of the virus in the pH-range below neutrality (Kaper & Jenifer, 1965,1967,1968). Above neutrality, TYMV is stabilized primarily by protein-protein interactions. The extraordinary t Abbreviation

used:

TYMV,

turnip

yellow

mosaic

virus.

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strength of these interactions was recently demonst&ed once more by the fact that protein denaturrtnts such as 8 ~urea or 12 M-formamide, at high ionic strength, dissociated TVMV into its components but left the capsid structure intact (Jonard & Hirth, 1966; Jon&i, Ralijrtona & Hirth, 1967; Bouley & Hirth, 1968). The above alkali- or urea-formamide-induced dissociation of TYMV into intact capsids and RNA could possibly be explained by a common mechanism, consisting of two principal steps: (1) A selective breakage of sny existing protein-RNA interactions, and (2) an RNA release step, invoked by some alkali- or urea-form~mide-mediated capsid perturbation or expansion, allowing the RNA strand to work its way out. Recent reports concerning an aggregation or complexing phenomenon of TYMV RNA, preceding its release under alkaline conditions (Bosch, Bonnet&nits & Van Duin, 1967 ; Pley, Talens, Bosch & Mandel, 1969), can also be interpreted with this two-step mechanism of breakage of the protein-RNA association and capsid expansion, thus sllowing a superstructuring of the RNA (Kaper, 1969b). Finslly, the recent discovery of a certain degree of abnormal proton-binding by TYMV in its integrated state (Kaper & Alting Siberg, 196%) could further illuminate the specific chemical nature of some of the groups involved in the protein-RNA interaction.

2. Outline

of Experimental

Approach

The major obstacles encountered in attempts to analyze the nature of intercomponent interactions in viruses by means of controlled degradation reactions, are problems of selectivity of interference and of experimental visualization of the ensuing disengagement, particularly if it concerns the disengagement of the RNA from its linkage with the protein. In the present study, where the protein-RNA linkage of TYMV WELS the principal object of investigation, two categories of reactions were used. (a) Reactions which interfere with the protein-RNA linkage, but which leave the interprotein subunit bono5 intact As was mentioned above, in this category of reactions en experimental problem was posed by the necessity to visualize the dissociation of the protein-RNA linkage. This was because, in addition to its interaction with the protein, the RNA was also physically embedded in the capsid structure. The experiments carried out in this series were therefore selected for their known (or suspected) involvement of conditions leading to a release of RNA from the virus with retention of the capsid structure. First, the alkaline degradation reaction of TYMV (Kaper, 1964; Kaper & Helperin, 1965) was further analyzed. Several newer ctspeots of this reaction, such as the in situ RNA complexing (Bosch et al., 1967; Pley et al., 1969), and its relevance to the interference with protein-RNA interactions (Kaper, 19693), were examined in detail. Subsequently the problem of RNA-release from the virus under alkaline conditions was investigated. The next set of experiments in this category of reactions concerned the RNArelease from TYMV by means of heating. Several publications on this topic have appeared in the past (Lyttleton & Matthews, 1958; Kaper & Steere, 1959; Hitchborn, 1968). However, each of these studies (with the exception of the earliest) was primarily concerned with heating as a means to obtain covdently intact, biologically competent, RNA. More recently, moderate heating to accomplish RNA-release from the viral capsid has also been applied to polio virus (Hinuma, Katagiri, Fukuda, Fukishi &

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Watanabe, 1965; Drees t Borna, 1965; Drees & Demme, 1966; Van Elsen t Boeyd, 1966; Boeye BEVan Elsen, 1967). Since there is little doubt that the essence of the heating processes for both TYMV and polio virus is that both allow the release of RNA with practically complete preservation of the capsid structure, the conditions for interference with the protein-RNA linkage could be investigated systematically. The final series of experiments utilized the formamide-induced release of RNA from TYMY quoted in the Introduction (Bouley & Hirth, 1968). Here it could be demonstrated unequivocally that, provided a means is offered for the RNA to be released from the physical constraints of the capsid, the conditions for selective interference with the protein-RNA interactions can be pinpointed accurately. (b) Reactions which interfere with the protein-RNA linkage after neutralization of the inter-protein subunit bonds Any reaction with or any effect upon the TYMY protein, resulting in degradation of the capsid structure in the absence of RNA, but not in its presence, can be used in this particular experimental approach. In the past, such a differential effect had been found in reactions with p-mercuribenzoate (Kaper & Jenifer, 1965). It led to the finding that the protein-RNA linkage in TYMV was pH-sensitive, and could be broken by exposure to a pH of 6 to 7, or higher. In the present study, a differential effect of ultraviolet irradiation of TYMV was discovered and utilized in a similar manner to examine the pH sensitivity of the structure of the ultraviolet light irradiated virus.

3. Experimental (a) Materiala Turnip yellow mosaic virus was isolated from infected Chinese cabbage plants which had grown in an artificially lighted room at 20°C for 21 days after inoculation. The virus was purified according to Steere (1956) in the earlier work (studies of RNA-release from TYMY by means of alkali treatment), and by the method of Dunn & Hitchborn (1965) for all other experimentation. For all experiments concerning in Bitu complexing of RNA oia mild alkaline treatment, the virus was purified from freshly harvested leaves. In other experiments the leaves had been stored frozen at -20°C. Naturally occurring top component was removed from purified virus preparations by repeated short-duration ultracentrifugation. TYhW protein capsids were prepared by alkaline degradation of the virus (Kaper, 1960,1964), Centrifugal homogeneity of the starting preparations of TYMY and of the capsids were comparable to those shown in Fig. 1 of a previous publication (Kaper, 1964). All chemicals used were of analytical quality. (b) Apparatus Ultraviolet spectra and optical density measurements were obtained with the Cary model 14 recording spectrophotometert, and the Beckman DU manual spectrophotometer. Preparative ultracentrifugation was performed with the Spin00 model L ultracentrifuge. Analytical ultracentrifugation was performed with the Spinco model E ultracentrifuge equipped with schlieren optics, and with the photographic ultraviolet optical system in all experiments before October 1968. The latter optical system was subsequently replaced by a high-intensity ultraviolet light-source, monochromator, and photoelectric scanning system of the same manufacturer. Densitometer tracings of sedimentation patterns obtained with the photographic ultraviolet optical system were made with a Spinco model RB analytrol with densitometer attachment. t Mention of any speoi60 equipment, trade produots, or oommeroisl companies does not oonstitute its endorsement by the U. S. Government over similar products or companies not mentioned.

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pH-stat reactions were done in all-glass thermostatically controlled reaction vessels in conjunction with the TTTl automatic titrator and the SBR%-SBUl titrigraph recorder from Radiometer. The ultrrtviolet light-source in irradiation experiments of TYMV wss the Mineralight R51 lamp with Corning filter no. 9863 of Ultraviolet Products, Inc., San Gabriel, Calif. According to the manufacturers specifications, this lamp produces an intensity of 120 pw/cm2 of 254 nm ultraviolet light at a distance of 18 inches. (c) Methoda Alkaline degradation reactions were carried out in a pH-stat aa previously described (Kaper, 1964), at 3O”C, and in the presence of 1-O BX-KCl. Length of treatment and pH varied according to the purpose of the experiment. Treatments were terminated by means of neutralization with a small volume of HCl in I.0 M-KCl. In reactions where the kinetics of RNA release from the capsids was studied, small portions were removed from the same reaction mixture at preset time intervals, and added to 0.5 vol. of 0.1 M-sodium phosphate buffer of pH 7.0. Neutralized reaction mixtures were subsequently dialyzed overnight at 4°C against an excess of buffer of relative low ionic strength (usually about O-1). The composition of these buffers varied according to the purpose of the alkaline treatment. Dialyzed alkaline reaction mixtures were used for different purposes. For compositional analysis, frsctions were diluted to an 0.D.2Bo = 1 with a suitable buffer of neutral pH and ultracentrifuged using ultraviolet optics. To sepamte residual, physically intact, alkali-treated TYMV from RNA that had been released, the reaction mixtures were submitted to two 1-hr cycles of differential ultracentrifugation at 50,000 rev./m& For complete removal of empty oapsids, additional cycles of ultracentrifugation were necessary (Kaper & Halperin, 1966). The low ionic strength dialysis step before differential ultracentrifugation is essential since RNA has a tendency towards aggregation at high salt concentration in the cold, and could consequently contaminate the alkali-treated TYMV pellet (see Results section (b)). RNA was extracted by means of conventional phenol treatment (Gierer & Sohramm, 1956), or by means of a new freezing method (Kaper & Alting Siberg, 1969a). The latter method required that the preparations be in distilled water. For total RNA analysis, reaction mixtures were extracted in their entirety; isolsted alkali-treated TYMV particles were the starting product for the preparation of in situ alkali-treated RNA. Heat degradation of TYMV at different temperatures and pH was performed by preheating 9 vol. of diluent of predetermined salt strength to the required temperature, to which 1 vol. of a distilled water solution of TYMV wse subsequently added. Unless it is stated otherwise, all incubations lasted 8 min, after which the mixtures were cooled in an ice bath. Virus concentration in the reaction mixtures was 0.3% and the ionic strength was high (1.0 ~-Kc1 + 0.02 M-buffer). Reaction mixtures were dialyzed overnight in the cold against a 0.1 ionic strength buffer of pH 7.0, and analyzed with the analytical ultracentrifuge. Treatment of TYMV with 12 M-formamide waz basically performed according to Bouley & Hirth (1968). Slight changes in buffer concentration and pH were as indicated in the text (Results section (c)). Ultraviolet irradiations were carried out at 4°C with 2 ml. of sample at a concentration of 0.3% in 0.1 M-buffers spread out in Petri dishes at about 8 cm from the light source. Irradiations lasted for 3 hr, at which time the amount of energy delivered by the source wm of the order of lo6 ergs/mma. The irmdiated samples were immediately analyzed in the analytical ultracentrifuge.

4. Results (a) Alkali-induced

interference with protein-RNA interactions in turnip yell&w mosaic virw and cmnpting of the RNA (i) The RNA complex ae related to plant tissue storage Turnip yellow mosaic virus preparations were isolated from each of two portions of a freshly harvested infected leaf batch. One portion had been stored for 24 hours

J. M. Leaf rouras ,

frrsb L

froml 1 pH Il.05

pH lO;B5

b)

KAPER

pH IO.85

iI//---’ -I,;\

ly./

Fm. 1. Alkeli treatment of TYMV from fresh- and frozen-leaf sources at pH IO.86 and pH 11.06 for 8 mm under standard conditions (see Experimental). The densitometer traoings of the ultraviolet sedimentation patterns depiat the following: (a) Reaction mixtures after neutrahxation and dialysis ver8uB 0.1 n-sodium acetate, pH 6.0. The rapidly mdimenting boundary represents TYMV from which no RNA haa been released (&ah-treated virus). Sedimentation was at 20°C. Patterns were photogmphed 12 to 16 min after the centrifuge reaohed a speed of 29,600 rev./mm (b) Phenol extracts of reaction mixtures shown under (a). These p&terns represent the sedimentation distribution of all RNA species in a reaction mixture after alkali treatment. Sedimentation w&8 at about 6°C. Solvent: 0.1 M-sodium acetate, pH 6.0. Patterns were photographed 12 to 16 min after the centrifuge resched a speed of 69,780 rev./mm. (c) Phenol extra&s of isolated alkali-treated virus. These patterns represent the sedimentation distribution of the RNA species left inside the viral capsids after alkali treatment. Sedimentation was at about 6%. Solvent: 0.1 M-sodium acetate, pH 0.0. Patterns were photogmphed 12 to 16 mm after the centrifuge reached a speed of 69,780 rev&in.

at - 20°C (frozen-leaf source), the other at +4”C (fresh-leaf source). No discernible differences oould be detected in the physical (sedimentation behavior) and biological properties (speoifio infectivity) of these virus preparations, nor of their RNA’s. Both virus preparations were alkali-treated at pH lO+% for eight minutes under standard conditions; the preparation from the fresh-leaf souroe was also treated at pH 11*05. Different portions of eaoh reaction mixture were processed as follows: (a) direct ultracentrifugation for compositional analysis ; (b) direct phenol extraction and ultracentrifugal ana.lysis of total RNA; (c) isolation of alkali-treated TYMV, followed by phenol extraction of RNA and ultracentrifugal analysis. Controls were submitted

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to identical treatment with omission of alkaline conditions. The results of this experiment, which was representative for a total of four performed on different leaf batches, are given in Figure 1. Figure I(a) shows that the amounts of RNA fragments released at pH 1035, by TYMV from either leaf source, is approximately identical, but is larger at pH 11.05. Figure l(b) and (c) show however, that the physical state of the RNA inside alkali-treated virus is affeoted by the leaf-storage conditions. A large proportion of the RNA extracted from fresh-leaf alkali-treated virus consisted of a homogeneously sedimenting component (S,O,Wabout 40 s), as opposed to the frozen-leaf alkali-treated virus which contained predominantly fragmented RNA. The control experiments yielded RNA with the sedimentation properties of native TYMS’ RNA (X,,,, about 33 s) in either case. A heating test (5 mm at 70°C) of the 40 s RNA and the 33 s RNA, resulted in complete fragmentation of the former (Fig. 2(a)), and only in the exposure of a limited number of hidden breaks iu the control RNA (Fig. 2(b)), typical for native TYMIV RNA (Haselkorn, 1962). Thus,

I I

,’/

/----

-d i /I

FIQ. 2. The effect of heating (5 min at ‘70°C) on the sedimentation properties of (a) the RNA complex obtained from an 8-m& pH 10.86, elkali treatment of TYMV; (b) the RNA extracted from the virus control. The densitometer tracings represent ultraviolet sedimentation patterns at 40 and 15 min after remhing 59,780 rev./min, respectively. Temperature: 5°C. Solvent: 0.1 Msodium met&e, pH 6.0.

weak alkali treatment of TYiMV isolated from fresh leaves (as opposed to frozen leaves) led to the in situ formation of an RNA complex, composed of smaller fragments, and sedimenting at higher rates than native !NMY RNA. These experiments therefore confumed the original Snding of Bosch et al. (1967), and solved an apparent controversy as to the cause of the formation of the complex (Halperin & Kaper, 1967). (ii) The RNA complex after in vitro freezing of turnip yellow mosaic virus Turnip yellow mosaic virus is resistant to freezing and thawing in vitro in 0.1 M-sodium acetate, pH 6.0, (Kaper & Alting Siberg, 1969u,b). Upon subsequent alkalitreatment at pH 1035, the RNA also was found to be complexed, although it sedimented more slowly (35 S) than RNA complex from an alkali-treated unfrozen control (sedimentation rate about 40 s). Thus, freezing of TYMV im vitro failed to interfere severely with the alkali-induced RNA complexing. (iii) The RNA complex as related to the method of its iaolatio?a Freezing and thawing of water solutions of TYMIV is a very mild degradation method, after which the RNA can be observed directly in the mixture as a homogeneously

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sedimenting boundary (35 s) without the customary tailing of RNA fragments due to a partial exposure of hidden breaks (Kaper & Alting Siberg, 1969aJ). When this method was applied to fresh-leaf alkali-treated virus, it yielded an RNA complex P4Xl.Wabout 42 s) virtually without any accompanying fragmented RNA (Pig. 3(a)), although it caused some RNA aggregation visible as a skewness of the boundary’s leading edge. Phenol extraction of the same alkali-treated virus yielded a slower sedimenting RNA complex (about 36 s) as well as a significant proportion of RNA

(b)

FIU. 3. The stats of the RNA oomplex inside alkali-treated virus derived from both fresh-leaf and frozen-leaf soumes, as revealed by freeze disruption and by phenol extra&ion of the virus. The densitometer traaings represent ultraviolet sedimentation patterns of the following. (a) The RNA released by freezing of alkali-treated virus from a fresh-leaf source. (b) The RNA released by phenol extra&ion of alkali-treated virus from a fresh-leaf souroe. (c) The RNA released by freezing of alkali-treated virus from a frozen-leaf soume. (d) The RNA released by phenol extraction of alkali-treated virus from a frozen leaf source. All sedimentation experiments at about 6°C. Solvents: 0.1 M-sodium acetate, pH 6.0. Patterns were photographed 12 to 16 min after the aentrifuge had reached a speed of 59,780 rev./min.

fragments (Pig. 3(b)). Both methods were then applied to frozen-leaf alkali-treated TYMV. Freezing yielded a larger amount of rapidly sedimenting (though severely aggregated), RNA complex (S,,,, about 39 s) than phenol extraction (Fig. 3(c) and (d)). Thus, the above experiments showed that regardless of leaf-source, alkali treatment of TYMV leads to in situ RNA complexing. However, the degree of rigidity and the strength of the complex (as judged from sedimentation rate and proportion of fragmented RNA before heating) can be different. (iv) The RNA wmplsx a.s related to the pH of alkali treatment Table 1 shows the effects of a limited variation of the pH of alkali treatment (pH 10.5 to 11.05) on a number of parameters which characterize the RNA complexing process. The following trends were identified: (1) The proportion of virus particles releasing their RNA increased with this pH increment. (2) The proportion of alkalitreated particles containing complexed RNA decreased in the same order. (3) The

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1

EfSect of the pH of all&i treatment on some parameters churacterizing the in situ TYkfV-RNA compkxing process Proportion TYMV particles which released RNA (%)

Proportion alkalitreated particles with complexed RNA (%)

S a0.w of complexed RNA (s)

10.5

20

90

43

10.85

30

82

39

11.06

60

60

35.5

PH

Sedimentation homogeneity of fragments constituting the RNA complex polydisperse less polydisperse 7.6 8) G%o.a about monodisperse (Sa0.r = 5 5)

sedimentation rates of the isolated RNA complexes decreased. (4) The heated RNA complexes exhibited decreased sedimentation polydispersity and average sedimentation rates, indicating a more complete fragmentation to the limiting 5 s value (Kaper & Halperin, 1965). Thus, it appeared that optimal in situ complexing of the RNA could already be achieved at moderately higher pH values, and that fragmentation to 5 s RNA was no essential prerequisite for the complexing process. (b) Alkali-induced release of RNA from turnip yellow mosaic virus with retention of the cupsid structure Previous investigations have shown that alkaline treatment of TYMV at high ionic strength leads, in its extreme form, to an all-or-nothing release of RNA fragments, while the capsid structure is retained. Schlieren sedimentation diagrams of degradation mixtures exhibited three boundaries in various proportions at any time during the reaction. These corresponded with alkali-treated TYMV, protein capsids, and RNA fragments, in order of decreasing sedimentation rates (Kaper, 1964). Analytical ultracentrifugation with ultraviolet optics showed, for all practical purposes, only two major optical density increments (Kaper & Halperin, 1965). These were identified with the alkali-treated TYkIV and RNA boundaries, because protein capsids contribute insignificantly to the total ultraviolet light absorption at 254 nm. Such absorption increments can therefore be taken as representative for the amounts of viral RNA still encapsidated, and of RNA released, respectively. In the present study it was determined by this method that TYMY preparations, which differed significantly in their capacity to form rigid and well-defined RNA complexes (see Fig. 1 (b) and (c)), released similar amounts of RNA upon alkali treatment at pH IO.85 (fresh-leaf TYMY, 30% ; frozen-leaf TYMY, 25%), and at pH 11.05 (fresh-leaf TYMY, 50% ; frozen-leaf TYMV, 51%). A systematic analysis of the RNA release from three different virus preparations, at pH values varying from 10.4 to 11.7, was performed at an early stage of the present study. In Figure 4, this RNA release (= 100% -o/O encapsidated RNA) is plotted as a function of the pH of treatment. In comparison with the newer data of Table 1, it indicated a somewhat slower release of RNA from virus preparations isolated by the butanol-chloroform method (Steere, 1956).

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II5

110

IX

PH

FIG. 4. The RNA All alkali treatments mental). The three

release (= lOO%-y0 enoapsidsted RNA) from TYMV as a fun&ion of pH. lasted 8 min and were performed under standard conditions (see Experiourve8 represent three different virus preparations using the same oonditions.

The above differences are minor when the RNA release figures are compared with recent data published by Pley et al. (1969). These investigators observed a much slower release of RNA, upon alkali treatment of TYMY, than is suggested by the data of Table 1 and/or Figure 4. In addition, they claimed considerable differences in RNA release between identically treated TYMV preparations from fresh- or frozenleaf sources. A typical example of the slower RNA release, taken from their data, is that less than 25% of the RNA from either type of virus preparation was released after eight minutes at pH 11.5, or 60 and 85%, respectively, after eight minutes at pH 12.5. Even more striking is an experiment in which TYMY was treated at pH 11.5 for 155 hr, apparently without complete release of the RNA. These discrepancies probably originated in their method of estimating the RNA release from TYlW, which is based on measuring the ultraviolet light absorbance in the 2.5 hour, 100,000 g, supernatants of neutralized reaction mixtures. These mixtures have an ionic strength of about 045; their ultracentrifugation in this laboratory resulted in the pelleting of a sign&ant amount of released RNA, due to its aggregation tendency at high ionic strength. This RNA is subsequently discarded with the alkali-treated virus and/or capsid pellet. It could be the source of erroneous results, and cause underestimation of the extent of RNA release. In Figure 5 the time-course of RNA release from TYMY at different pH values, determined for a single virus preparation, is given. For each of the treatments (except the one at pH 10.85) a more rapid release was noticed in the beginning, after which a linear relationship between the logarithm of the amount of RNA still enclosed by the capsid and the time of treatment was assumed. Extrapolation of the pH 11.23, pH 1140, and pH 11.66 curves to zero time, indicated that at least 55 to 70% of the population of virus particles released its RNA with single-hit kinetics. In order to

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I XI

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I 30

I 40

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Time hid

FIG. treated

5. The under

time-course standard

of RNA conditions

release (= 100~&-~o encapsidated (see Experimental) at four different

RNA) from pH values.

TYMV

alkali

see whether any of the preceding events (i.e. breakage of the protein-RNA interactions ; in situ fragmentation and complexing of the RNA) influenced the rate of RNA release, a direct comparison of the kinetics of this release at pH 11.27 was made between TYMV and the alkali-treated preparation derived from the same virus batch. Practically no differences were found. Apparently, the rate of RNA release is controlled entirely by the viral capsid. (c) Interference with protein-RNA interactions in turnip yellow mosaic virus, and the use of other methods to retie RNA from the capsids In this section of Results, experiments are described where two other methods, to effect RNA release (see Outline of Experimental Approach section (a)) were applied in an effort to differentiate the pH-controlled process of protein-RNA disengagement (Keper & Jenifer, 1967) and the capsid-controlled RNA release.

known

(i) pH-Dependence of RNA release by means of heating The experimental objective was to determine which temperature would be sufficient to effect immediate release of the RNA after its disengagement from the protein, and then to vary the pH to find out at what value breakage of the protein-RNA linkage would take place. The obvious problem with heating as a means of capsid perturbation is that it may be expected to work in the same direction as increasing the pH, not only wit.h respect to RNA release, but also in interfering with the protein-RNA linkage. Therefore, a systematic analysis was performed of the effects of elevated temperatures, and different pH values close to neutrality, on TYMY buffered in solutions of high ionic strength. Although Plate I gives only the schlieren sedimentation diagrams of this analysis, each reaction mixture was also ultmcentrifuged with ultraviolet optics to identify the chemical nature (nucleoprotein or protein) of the dissociation products, and to estimate the amounts of RNA released (see percentage

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figure under each sedimentation pattern). In each case, where the schlieren sedimentation diagrams indicated signi6oant dissociation of TYMV, the absorption inorements of the ultraviolet sedimentation diagrams were associated primarily with a very slow boundary at the meniscus, and a fast-sedimenting boundary with the sedimentation coefficient of TYMV. Thus, in these experiments, the RNA was released as small, slowly sedimenting fragments. (It has to be noted that unlike the alkaline degradation where fragmentation of the RNA is inherent to the high pH, the fragmentation of the RNA upon heating is probably due to the fact that in this study no particular effort was made to use a freshly isolated TYMV preparation containing a maximal proportion of covalently intact RNA, or to take the necessary precautions against ribonuclease attack (Hitchborn, 1968).) The components with sedimentation rates between that of the virus and the released RNA were proteinaceous, judging from their lack of absorption in the ultraviolet sedimentation diagrams. The sedimentation coefficient of the principal boundary of the proteinaceous dissociation products centered around 50 s in the different experiments. In all likelihood, therefore, this component can be identified with the empty protein capsids. The shoulder trailing the capsid boundary in many of the experiments was also proteinaceous, and probably represents capsids damaged sufficiently, because of loss of protein subunits and/or deformation, to cause a reduction in sedimentation rate. Similarly damaged protein capsids were found in the early heating experiments of Lyttleton & Matthews (1958). Prom Plate I it is clear that complete dissociation of TYMV, under the conditions of these experiments, is favored by moderately elevated temperatures, and by pH levels at, or slightly above, neutrality. When the sedimentation diagrams in each temperature series are considered separately, the limiting pH above which RNA release starts to take place is pH 7 at 45”C, pH 6 at 55”C, and pH 5 at 65°C. A further increase in temperature to 75°C led to precipitates in the pH 6 reaction mixtures, and more severely at pH 5. This precipitate was proteinaceous, which could be deduced from the fact that no absorption was lost in the ultraviolet sedimentation diagrams. This precipitation precluded any conclusions regarding the dissociation at 75°C. No experiments were performed at temperatures below 45°C because at 30°C no significant RNA release takes place until pH values of the order of 10.5 are reached. At this alkalinity the RNA release is probably mainly pH-induced, which makes it impossible to interpret TYMV dissociation at 30°C in terms of breakage of the protein-RNA linkage (see section (b) above). A reasonable conclusion from the experiments of Plate I is therefore that at temperatures ranging from 45 to 65°C the observed dissociation of TYMV is primarily controlled by the process of disengagement of RNA from its interaction with the protein. This process is pH-dependent and is triggered at pH values slightly under or at neutrality. (ii) pH-Dependence

of RNA

release with 12 af-fomamide

The experiments described in this section used 12 M-formamide as the capsid perturbing agent effecting RNA release (Bouley & Hirth, 1968), whereupon the pH was varied to bring about dissociation of the protein-RNA linkage. Two reaction mixtures were made up with the following compositions: 0.4% TYMV, 12 M-formamide, O-9 M-KCl, and 0.1 M-sodium acetate or 0.1 i+r-Tris-HCl buffers. The final pH values were 6.0 and 7.5, respectively. These mixtures were incubated for ten minutes at 30°C and subsequently dialyzed at 4°C against 0.05 M-

PHI

pH5

75%

PLATE I. pH dependence of RNA release from TYMV by means of moderate heating. Experimental conditions are described in the Experimental section. Buffers used in the 1.0 M-KC1 solutions were the following. Tris-HCl, pH 8.0; potassium phosphate, pH 7.0; potassiumcacodylate, pH 6.0; and potassium acetate, pH 50. The schlieren sedimentation diagrams represent the heating mixtures after overnight dialysis at 4°C against a pH 7 buffer of 0.1 ionic strength. The figures under raoh schlieren diagram show the percentage of RNA released, as estimated by ultraoentrifugation with ultraviolet optics. Ultracsntrifugation was performed at 20°C. Photographs were taken 16 to 20 min after centrifuge reached a speed of 29,500 rev./min.

(a)

(b)

PLATE II. pH dependence of RNA release from TYMV in 12 M-formamide at high ionic strength. The experimental conditions are given in Experimental and in the text. Schlieren sedimentation diagrams represent the following reaction mixtures. (a) Formamide treatment at pH 6.0, followed by dialysis OW~US 0.05 M-sodium acetate, pH 6.0 (upper pattern). The same treatment at pH 7.5, followed by dialysis ZIWSW 0.05 M Tris-HCI, pH 7.5 (lower pattern). Sedimentation was at 20°C. Photograph was taken 16 min after centrifuge reached a speed of 29,500 rev./min. (b) Formamide treatment at pH 6 without dialysis (upper pattern). The same treatment at pH 6 followed by a pH change in the presence of 12 M-formamide via dialysis against a mixture of 12 M-formamide, 0.9 M-KC& and 0.1 M-Tris-HCl (pH 7.5) (lower pattern). Sedimentation was at 20°C. Photograph was taken 16 min after centrifuge reached a speed of 42,040 rev./min. (c) Formamide treatment at pH 6.0, followed by dialysis versus 0.05 x-sodium acetate, pH 6.0 (upper pattern). The same treatment followed by a first dialysis WTSUB 0.05 M-sodium acetate, pH 6.0, and a second dialysis against 0.05 x-Tris-HCl, pH 7.5, to change the pH in the absence of 12 M-formamide (lower pattern). Sedimentation was at 20°C. Photograph was taken 12 min after centrifuge reached a speed of 29,500 rev./min.

PH’

pH.7.35

pH5

PH 5

(a)

(b)

PLATE III. pH-dependence of ultraviolet light-induced degradation of TYMV and virus capsids. The experimental conditions are given in the Experimental section. Schlieren sedimentation diagrams represent irradiation in the following buffers. (a) 0.1 M-Tris-HCl, pH 8.0; 0.1 M-Tris-HCl, pH 7.0; 0.1 M-sodium acetate, pH 6.0; 0.1 Msodium acetate, pH 5.0. Sedimentation was at 20°C. Photographs were taken 12 to 16 min after centrifuge had reached a speed of 29,500 rev./min. (b) TYMV (upper pattern) and virus capsids (lower pattern) in 0.1 M-Tris-HCl, pH 7.35. TYMV (upper pattern) and virus capsids (lower pattern) in 0.1 M-sodium acetate, pH 5.0. Sedimentation was at 20°C. Photograph for pH 7.35 experiment was taken 4 min after centrifuge reached a speed of 42,040 rev./min; for pH 5 experiment, 12 min after reaching 29,500 rev./min.

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sodium acetate and 0.05 M-Tris-HCI of the same respective pH. The dialyzed solutions were examined in the analytical ultracentrifuge. Plate II(a) shows that at pH 7.5 practically all the virus had dissociated into protein capsids and RNA, confirming the observations of Bouley & Hirth (1968). I n contrast, very little dissociation was observed at pH 6aOt. This suggested that interference with the protein-RNA interaction took place between pH 6.0 and 7.5. Direct evidence for this interference was then obtained by treating TYMV with 12 nr-formamide at pH 6, and subsequently changing the pH of part of the reaction mixture by dialysis vers~.~12 i+r-formamide, 0.9 M-KCl, and O-1 x-buffer of pH 7.5. It can be seen from Plate II(b), in which the pH 6 reaction mixture and the pH 7.5 dialysis mixtures without prior removal of the formamide are displayed, that the mixture at pH 6 contains primarily undissociated TYiWV; but that upon dialysis to pH 7.5 empty capsids constituted the principal boundary and the RNA was disengaged and released. That such a pH-dependent release of RNA is only possible in the presence of the protein-perturbing agent 12 Mformamide is evident from a control experiment displayed in Plate II(c), in which first the formamide was removed by dialysis against O-05 x-sodium acetate, pH 6, after which the pH was changed to 7.5 by means of dialysis against 0.05 M-Tris-HCl. In this case the RNA was not released because the structurally restored capsid apparently did not allow it to pass. (d) pH-Dependence of the degradation of ultraviolet light-irradiuted mosaic virus

turnip yellow

It was recently established in this laboratory that large doses of ultraviolet light irradiation could bring about extensive degradation of TYMY and of its empty protein capsids at pH values around neutrality or above. When similar doses of radiation were given at acid pH, the protein capsids also degraded, but not the virion. Thus, here it was possible to analyze the interference with the protein-RNA interactions directly in a virion which derived its stability solely from such interaction (cf. section (b) of Outline of Experimental Approach). In Plate III(a) a series of sedimentation diagrams is given, which represent TYiUV irradiated in buffers of different pH values. It can be noted that at neutrality, or above, practically complete degradation had taken place into primarily two, slower, polydispersely sedimenting products. In contrast to the result at pH 7 and 8, ultraviolet irradiation at pH 6, or lower pH, caused very little degradation which was detectable in the ultracentrifuge. To facilitate preliminary identification of the ultraviolet light-induced conversion products of TYIW, the pH 7 and pH 5 irradiation mixtures were also ultracentrifuged using ultraviolet optics. These sedimentation patterns are shown in Figure 6(a) and (b). From the optical density distribution in these sedimentation diagrams, it was concluded that both irradiation products contained nucleic acid; and since the principal irradiation product sedimented at approximately 50 S, it had to be a nucleoprotein. Thus, the ultraviolet light-induced degradation of TYMY was more complicated than a direct dissociation of the virus into free nucleic acid and probein. Plate III(b) represents the results of irradiation experiments in which TYMV (top patterns) and TYbW capsids (bottom patterns) received equal doses of ultraviolet light at pH 7.35 and pH 5.0. It can be seen that t It was learned recently that & similar pH-dependence was also found (Jonard, personal communication).

of the 8 M urea-induced

dissociation

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--II/’

t

(0)

(b)

(c)

FIG. 0. pH-dependence of ultraviolet light-induced degradation of TYMV. Experimental conditions are given in the Experimental section. The photoelectric scanner tracings (268 nm) represent sedimentation diagrams of: (a) TYMV irradiated at pH 7; (b) TYMV irradiated at pH 6; (c) TYMV irradiated at pH 5, and subsequently dialyzed against 0.06 M-Tris-HCl, pH 7.2. All ultraoentrifuge nms were performed at 2OW. Cells were scanned 12 to 16 min after the centrifuge reached a speed of 29,600 rev./mm

at pH 7.35 the TYMV protein capsids were converted into slowly sedimenting fragments, whereas at pH 5.0 protein fragments were also formed, but are not visible in the sedimentation diagram because they tended to aggregate and sediment to the bottom of the cell at low speeds. The TYMV samples showed the results expected from the experiments of Plate III(a). Hence, the undegraded TYMV, in irradiations of pH 6 or lower, probably represented virus particles which had their proteinprotein linkages destroyed, and which were therefore stabilized primarily by proteinRNA interactions. A pH-dependent dissociation of the protein-RNA linkage in irradiated TYMV was subsequently confirmed by dialyzing the pH 5 irradiated mixtures shown in Plate III(b) against O-1 M-Tris-HCl of pH 7.2. Figure 6(c) shows that such a neutralization was capable of converting the pH 5 irradiated virus particles into degradation products with the same sedimentation behavior as those resulting from a direct pH 7 irradiation.

5. Discussion The stability of simple RNA viruses is based on the assumption of a minimum free energy conformation by their constituent components when they occur in their integrated state. This state of minimum free energy is maintained by a balance of forces within each of the constituent RNA and protein components, as well as by the interactions that exist between the protein and the RNA. In TYMV the proteinprotein interactions provide the greatest contribution to particle stability. This is already apparent from the natural existence of empty protein capsids in the diseased plant (Markham 6 Smith, 1949), but is seen more directly demonstrated by the

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stability of these protein oapsids under very adverse conditions of alkalinity, heat, and in the presence of protein denaturing agents such as formamide or urea (see sections (b) and (c) of Results; Kaper, 1964; Jonard & Hirth, 1966; Bouley & Hirth, 1968). There is a large amount of circumstantial evidence which suggests that the protein-protein interactions in TYIW are primarily hydrophobic. The experimentally observed stabilizing influence of high salt concentrations on the capsid structure in alkaline degradation as well as in 8 M-urea or 12 M-formamide agrees with the strengthening influence of high salt on hydrophobic bonds, while at the same time causing a weakening of electrostatic interactions. The extreme sensitivity of TYMTi with respect to phenol (Diener & Schneider, 1968) also points to a predominance of hydrophobic interactions. However, the most suggestive evidence comes from the reaction of organic mercurials possessing apolar residues with TYMV protein capsids (and also with the virus under the proper conditions), which causes a profound interference with the protein-protein interactions (Kaper & Houwing, 1962a,b; Godschalk & Veldstra, 1965,1966). Mercurial studies also provided the first direct indication that in TYMV, protein-RNA interactions make an additional contribution to the stability of the virion, and that these interactions could be broken at pH values around neutrality (Kaper & Jenifer, 1965,1967,1968). In addition, potentiometric titrations of mercurial-substituted TYMV indicated that there was a considerable number of abnormally bound protons hidden in the structure of TYMV. In a far less complicated experimental situation (Kaper & Alting Siberg, 1969a), it has recently been possible to compare the titration properties of TYMV with those of a mixture of its constitutent protein and RNA components (obtained by freeze-disruption of the virus) directly. The difference in titration curves thus obtained revealed a pH region (pH 3.8 to 5.0) of increased titration activit,y in the component mixture. Thus, t,his dissociation activity had to be attribut’ed to groups which became available for t’itration after disruption of the intact virion. They could be shown to be, in all likelihood, groups of protein and/or RNA origin, unavailable for titration due either to their direct participation in a linkage between the protein and RNA, or because of being buried during the process of integration of the protein and RKA into the virion. However, to test such a hypothetical participation of protonated groups in the union of TYMV protein and RNA, it would also have to be shown experimentally that the processes of dissociation and/or reassociation of TYMV component,s coincided with the characteristic pH region of prototropic activity. Structural studies aiming at the experimental analysis of breaking and remaking of the protein-RNA linkages in TYMV are fraught with difficulties however. In the first place, there is the problem of selectivity of interference brought up in the Introduction. It was outlined in Experimental Approach how this problem was dealt with. The first general approach (breakage of protein-RNA int,eractions while maint’aining protein-protein interactions) introduced the least chemical complications, but posed an additional difficulty with the experimental visualization of the disengagement of the RNA from its linkage with the protein, because of its physical enclosure by the latter. In Results it was shown that this problem could be solved because it is apparently possible to induce perturbations of the capsid structure by a variety of methods, each causing sufficient conformational change or expansion to allow the RNA some freedom of movement within the capsid, and eventually to work its way out. One such method is alkali treatment resulting in in situ complexing of TYMV 18

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RNA. The potential use of this process to analyze the interference with proteinRNA interactions in TYMV has been discussed previously (Kaper, 19693). However, the dependence of the physical quality of the RNA complex upon a number of rather subtle and sometimes difficult to control factors such as the history of the storage of the infected leaves (freezing), the method of RNA isolation, etc. (obviously reflecting equally subtle differences in the degree to which the different factors governing the RNA complexing can be influenced by these experimental variables) made it impractical to use RNA complexing for this purpose. The alkaline degradation studies were useful, however, in that they demonstrated that the ultimate release of the RNA from its protein package is essentially controlled by the degree of capsid perturbation, presumably brought about by the electrostatic repulsions within the protein, and by the tendency of the RNA to expand and work its way out. Using this principle of capsid perturbation to enable RNA release, a direct experimental analysis of the actual dissociation of the protein-RNA linkage was subsequently made (Results, section (c)(i) and (c)(ii)). Using moderate heating or 12 Mformamide, at high ionic strength, to expand the capsids, it could be shown that the breakage of the protein-RNA linkage was a pH-dependent process which could be triggered at pH values around neutrality. In the second general approach for selective interference with the protein-RNA interactions (breakage of protein-protein interactions followed by protein-RNA linkages) high doses of ultraviolet light irradiation were utilized to neutralize the protein-protein interactions. A pH dependence for the ultraviolet transformation of of the virus particles was found. Although the precise nature of this conversion is not understood, it is significant to note that it happened in the same pH region of neutrality or above, where TYMV is dissociated in the heat or the formamide treatments. In summary it can be stated that there is a considerable body of structural evidence which shows that breakage of the protein-RNA linkages of TYMV leading to disengagement of these components, takes place between pH 6 and 7. Since it is very likely that some form of proton release has to be associated with such an evidently pa-dependent process, one can do some speculation as to the chemical origin of such protons. The only source of protons eligible for dissociation at approximately pH 6 to 7, and which could be instrumental in some form of interaction with the RNA, would be histidinyl proton8 engaged in an ionic linkage with the nucleic acid phosphates. However, there is also the possibility that TYIW’s abnormally bound protons, discussed above, are part of a protein-RNA linkage. Their participation could also explain a disengagement of the RNA from its linkage with the protein at about neutrality. Recently, a type of protein-RNA interaction compatible with these properties (juxtaposed protein carboxylate and cytidylate amino groups, sharing one proton abnormally bound in some type of hydrogen-bonded proton transfer complex) has been proposed (Kaper, 19693). However, the experimental evidence was of a rather indirect nature. Therefore, until it can be shown directly that TYMY protein and RNA require a pH of about 4-O or below to become reintegrated, both types of linkages proposed will have to be given equal consideration. The model of a carboxyl-amino hydrogen-bonded protein-RNA linkage is attractive because it would rationalize one of TYMV’s most unusual chemical properties; the very high content of @dine phosphate residues (38 mole %) in its nucleic acid. The fact that one of every two and one-half base residues in TYMV RNA is cytidine

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phosphate invites speculation as to why such extraordinary amounts of this particular base would be present. The underlying hypothesis of involvement of ammosubstituted base residues in the linkage of the RNA to the protein capsid would provide a structural role for the cytidine phosphate residues in addition to their function as part of the genetic message carried by the RNA, Only one other virus is known to possess such a high proportion of this particular base. This virus is wild cucumber mosaic virus with 41 mole O/Oof cytidine-phosphate. Wild cucumber mosaic virus, although quite unrelated to TYMV biologically (host range, etc.), has been implicated to be distantly related serologically (McLeod & Markham, 1963). The relationship in certain structural properties, however, is very obvious. Like TYMV, wild cucumber mosaic virus capsids can be isolated in relative abundance from the diseased plant (Sinclair, Geil & Kaesberg, 1957). They also seem to be quite resistant to alkali treatment; but once degraded, they possess a pronounced tendency toward aggregation (Yamazaki & Kaesberg, 1961). The type of protein-RNA interaction proposed might therefore be quite unique in that it would only occur in those viruses which are structurally characterized by very resistant, hydrophobic bond stabilized, protein capsids. As such, this type of linkage might prevail only at one extreme of a spectrum of simple isometric RNA viruses characterized as having basically similar geometry (icosahedral surface lattice, T = 3 or 4, and structure units arranged in 20 or 30 hexagons and 12 pentagons) (Kaper, 1968), but in which the contribution of the RNA to structural stability would become gradually greater as one goes to the other extreme. The following paper (Kaper & Geelen, 1971), will deal with the proteinRNA interactions of a virus at the opposite end of this structural spectrum, the cucumber mosaic virus.

At different stages of this investigation Nanette

Smith,

Eursilla

Barnes

and R. Alting

assistance Siberg.

was given

by Jane E. Halperin,

REFERENCES BoeyB, Bol, J. Bosch, Bouley, Caspar,

H. t Van Elssn, A. (1967). Fi’irology, 33, 336. F. & Krusemsn, 5. (1969). Virology, 37, 485. L., Bonnet-Smits, E. M. t Van Duin, 5. (1967). Virology, 31, 453. P. & H&h, L. (1968). C. R. Acad. Sci. Paris, 266D, 430. D. L. D. (1963). Advanc. Protein Chem. 18, 37. Diener, T. 0. & Schneider, I. R. (1968). Arch. Biochem. Biophya. 124, 401. Drees, 0. & Borna, Ch. (1965). 2. Naturf. ZOb, 870. Drees, 0. & Demme, K. D. (1966). 2. Naturf. 21b, 357. Dunn, D. B. t Hitchborn, J. H. (1965). Virology, 25, 171. Finch, J. T. & Klug, A. (1966). J. Mol. Biol. 15, 344. Gierer, A. BE Schramm, G. (1956). 2. Naturf. llb, 138. Godschalk, W. & Veldstra, H. (1965). Arch. Biochem. Biophys. 111, 161. Godschalk, W. & Veldstra, H. (1966). Arch. Biochem. Biophys. 113, 347. Halperin, 5. E. & Kaper, J. M. (1967). Virology, 33, 760. Haselkorn, R. (1962). J. Mol. Biol. 4, 357. Hiebert, E., Bencroft, J. B. & Bracker, C. E. (1968). l’+oEogy, 34, 492. Hinuma, Y., Katagiri, S., Fukude, M., Fukushi, K. & Watanabe, Y. (1965). B&en 143. Hitchborn, J. H. (1968). J. Gen. ViroZ. 3, 137. Hung, P. P. & Overby, L. R. (1969). Biochemistry, 8, 820. Jonard, G. & Hi&h, L. (1966). C. R. Acud. Sci. Pa&, 263D, 1909.

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Jonard, G., Ralijaona, D. & H&h, L. (1967). C. R. Acud. &%. Paris, 264D, 2694. Kaper, J. M. (1960). J. Mol. Biol. 2, 425. Kaper, J. M. (1964). Biochemi.stry, 3, 486. Kaper, 5. M. (1968). In MoZecuZar Bagis of Virology, ed. by H. Fraenkel-Conrat, New York: Reinhold. Kaper, J. M. (1969a). Virology, 37, 134. Kaper, 5. M. (19695). Science, 166, 248. Kaper, 5. M. & Alting Siberg, R. (1969a). ViroZogy, 38, 407. Kaper, J. M. & Alting Siberg, R. (19693). Cryobiblogy, 5, 366. Kaper, J. M. & Geelen, 5. L. M. C. (1971). J. Mol. BioZ. 56, 277. Kaper, J. M. & Halperin, J. E. (1965). Biochemistry, 4, 2434. Kaper, J’. M. C Houwing, C. (1962a). Arch. Biochem. Biophyz~. 96, 125. Kaper, J’. M. & Houwing, C. (1962b). Arch. Biochem. Biophys. 97, 449. Kaper, 5. M. & Jenifer, F. G. (1965). Arch. Biochem. Biophys. 112, 331. Kaper, 5. M. & Jenifer, F. G. (1967). Biochemistry, 6, 440. Kaper, 5. M. & Jenifer, F. G. (1968). Virology, 35, 71. Kaper, J. M. & Steere, R. L. (1959). l’irology, 8, 527. Klug, A., Longley, W. & Leberman, R. (1966). J. Mol. BioZ. 15, 315. Lauffer, M. A. (1968). Adwanc. Virus Rw. 13, 1. Lebeurier, G., Wurtz, M. 8: Hirth, L. (1969). C. R. Acad. Sci. Paris, 26813, 1897. Lyttleton, J. W. & Matthews, R. E. F. (1958). Virology, 6, 460. Markham, R. & Smith, K. M. (1949). Parasitology, 39, 330. McLeod, R. & Markham, R. (1963). Virology, 19, 190. Pley, C. W. A., Talens, J., Bosch, L. & Mandel, M. (1969). v&oZogy, 38, 371. Sinclair, J. B., Geil, P. H. & Kaesberg, P. (1957). Phytopathology, 47, 372. Steere, R. L. (1956). Phytopathology, 46, 60. Van Elsen, A. & Boeye, A. (1966). Virology, 28, 431. Yamazaki, H. & Kaesberg, P. (1961). Biochim. biophys. Acta, 53, 173.

1). I.

Note added in proof: In retrospect it is of considerable interest to re-examine the results of the heating experiments of Lyttleton & Matthews (1958) on turnip yellow mosaic virus. In Figure 1 of their publication they give experimental evidence (inevitably with cruder techniques) for one of the main contentions of the present publication, i.e. for the disengagement of TYMV-RNA from its linkage with the protein at neutrality. Although their conditions might have caused more capsid damage, and at that period in time did not allow for an unequivocal interpretation of the experiment in terms of selectivity of interference, the following quotation from their paper attests to their foresight: “On this basis it would be reasonable to assume that rupture of the TYMV particle is caused by thermal agitation of RNA freed by pH change from its normal binding to the inner surface of the protein shell.”