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Purification and chemical analysis of Shope papilloma virus
VIROLOGY
2’7, 273-281 (1965)
Purification
and Chemical S. J. KASS’
Analysis AND
of Shope
Papilloma
Virus
C. A. KNIGHT
Department of Biochemistry and Virus Laboratory, University of California,
Berkeley, California
Accepted July 19, 1965 Rate eonal density gradient centrifugation of preparations of Shope papilloma virus revealed three components (slow, middle, and fast) which were morphologically and serologically related. The slow sedimenting component was noninfectious, contained little or no DNA, and had a buoyant density of 1.29 in CsCI. The middle and fast sedimenting components were infectious and had the same buoyant density of 1.34 in CsCl. The middle sedimenting component, yielding a single band upon equilibrium sedimentation in CsCl, was found upon analysis to contain protein and 12% DNA, but no essential lipid and no detectable polyamines. Protein end-group analyses by treatment with hydrazine or with carboxypeptidase A showed the carboxyl terminal amino acid residue to be threonine. The amino-terminal group was unreactive t,oward fluorodinitrobenzene. The viral protein was analyzed for total amino acid content. Infectious DNA was extracted from the virus by treatment with 80% phenol, and shown to be free of contaminating virus by equilibrium density gradient centrifugation in CsCl. INTRODUCTION
The chemical composition of preparations of Shope papilloma virus (SPV), obtained by differential centrifugation of wart extracts was investigated some years ago by Taylor et al. (1942) and Knight (1950). Since such preparations have subsequently been shown to contain three or four components (Schachman, 1951; Williams et al., 1960; Breedis et al., 1962) it seemed desirable to reinvestigate the chemical and physical properties of SPV. Several groups of investigators have succeeded in fractionating SPV by density gradient centrifugation and have determined something of the morphology of the particles in various fractions by electron micros1 Taken in part from a thesis submitt,ed by S. J. Kass in partial satisfaction of the requirements for the Doctor of Philosophy degree, Department of Biochemistry, University of California, Berkeley, 1964. Present address: Section of Genetics, Weizmann Institute of Science, Rehovoth, Israel. This investigation was supported in part by Public Health Service Research Grant AI 00634, from the National Institute of Allergy and Infectious Diseases.
copy (Williams et al., 1960; Breedis et uZ., 1962; Crawford and Crawford, 1963). Some other properties of these fractions have been determined as well. From these observations, it appears that most of the material in a preparation of SPV purified by differential centrifugation is virus-related in the sense that it consists mainly of a mixture of whole virus particles, virus particles lacking DNA, of the and aggregates or mixed aggregates first two species. In the present study, some of the earlier investigations on purification of SPV have been reexamined and expanded; the results obtained, together with new findings about the physical and chemical properties of SPV preparations, are presented here. MATERIALS
AND
METHODS
Isolation and puri$cation of virus. Isolation of SPV from naturally occurring papillomas -was described earlier (Williams et al., 1960). Modification of the preparation by homogenizing the papillomas with the following buffers at pH 7.1-7.3 produced virus in essentially the same yield and with approximately the same content of aggregated 273
274
HAS
AND KNIGHT
material and empty (nucleic acid-free) capsids: 0.05 dP phosphate, Veronal-NaCl (0.02 A&O.2 M), and 0.1 dl et,hylenedinmine tetraacetate (EDTA). Other buffer mixtures were t,ested and found inferior t’o t’hose just listed, either because they were less efficient in the extraction of virus or because Obey extracted excessive amounts of nonviral materials along with the virus (Kass, 1964). Likewise, heating homogenates at 50”60” for 30 minutes was tried as a purification step, but was abandoned when it showed no advantage. Therefore, virus was routfinely isolated and purified by differential centrifugation in the presence of one of t’he three buffers listed above. Density gradient centrifugation of purified SPV. In rate zonal centrifugation, purified virus obtained by differential centrifugation was layered on a preformed gradient of glycerol or sucrose of density 1.17-1.05 and centrifuged in a swinging-bucket rotor as described earlier (Williams et al., 1960). In order to achieve greater efficiency of separation, materials from the visible bands of the first density gradient centrifugation were diluted with an equal volume of water and sedimented at about 62,000 g for 2 hours; the resulting pellet was resuspended and subjected to another cycle of rate zonal sedimentation. Since mechanical losses were rather great in this process, further cycles of rate zonal sedimentation were not attempted. For equilibrium density gradient centrifugation, virus preparations were mixed with CsCl (Maywood Co., optical grade) at a final density of 1.30 g/ml and centrifuged in the SW-39 Spinco swinging-bucket rot,or at 34,000 rpm for 18 hours (Meselson et al., 1957). The components appearing as a result of this procedure were located and collected as in the rate zonal method. Density of the fractions was measured by weighing aliquots in a 50-~1 pipette (H. Pederson, Carlsberg Laboratories, Copenhagen). Infectivity titrations. Infectivity measurements were made by placing test solutions on l-inch-square areas of depilated skin of domestic rabbits and spreading and injecting the material in a regular pattern with an electric tattoo needle. Owing to large varia-
t,ions in susceptibility among diff crent animals (Beard, 1956), comparisons of infectivit#y of virus fractions were performed by inoculating a series of dilutions on different areas of the same animal, and noting the number and size of the papillomas produced in a given time (usually 35-40 days). Estimation of protein. Routine protein estimation was made by the calorimetric procedure of Lowry et al. (1951), using crystalline bovine serum albumin as a standard. These result,s in turn were standardized in terms of virus by analyzing t’he latter for nitrogen and use of the relationship : virus = Nessler nitrogen X 6.1. The nitrogen content of SPV was calculated to be 16.4%, based on a composition of 12 % DNA with an N content of 15.2 % (sodium salt: Chargaff, 1955) and 88 % protein of t’he amino acid composition reported here. Extraction of DNA. SPV purified by repeated cycles of differential centrifugation was shaken for 5-10 minutes at room temperature with an equal volume of redistilled 80% phenol in the presence of 0.1 M NaCI, 0.15 M neutralized trichloroacetat,e, and 1O-2 &r EDTA (see Weil, 1961). Phenol was removed from the aqueous phase with ether, and et’her was removed with a st,ream of nitrogen gas. In some experiments, separation of DNA from other materials in the aqueous phase of the extraction mixture was accomplished by equilibrium centrifugation in CsCl of density 1.70 at 34,000 rpm in the SW-39 rotor for 40 hours at 11”. Fractions were removed by puncturing the bottom of the tube and were assayed for infectivity both undiluted and diluted 1: 10 in buffered saline. CsCl appeared to interfere with the infectivity assay, since the diluted material produced the same number as or more papillomas than t’he undiluted material. The density of the fractions was measured by weighing, as described above, and the DNA was located by the absorbancy at 260 rnp. Estimation of DNA. The DNA content of SPV fractions was estimated as phosphorus X 10.9 (Chargaff, 1955). However, when the quantities of material available were insufficient for P analysis, DNA was estimated from the ultraviolet absorption, using cor-
PURIFICATION
AND
CHEMICAL
ANALYSIS
rections obtained with intact virus for light scattering (Bonhoeffer and Schachman, 1960) and for ultraviolet absorption by protein. Phosphorus was estimated by a micromodification of the method of Nakamura (1952) in which digestion, color development, and dilution were done in a 1.0 ml volumetric test tube. Serological tests. Semiquantitative precipitin tests were made on serial twofold dilutions of virus materials incubated in 1 ml test tubes with a constant amount of precipitating antiserum (Bryan and Beard, 1941; Knight, 1946). Incubation was for 2 hours at room temperature, and overnight in the refrigerator, after which the sizes of precipitates were noted. The qualitative relationship of antigens was tested by the Ouchterlony technique of diffusion in agar gel (Le Bouvier, 1957). Terminal amino acids of SPV protein. Attempts were made to determine the amino terminal amino acid of SPV-protein by applying the fluorodinitrobenzene method (Fraenkel-Conrat et al., 1955) to the virus using either 1% sodium bicarbonate at neutral pH or sodium carbonate-NaOH at pH 12. The latter buffer disrupts the virus particles, but does not interfere with the coupling reaction. The carboxyl terminal amino acid was investigated by hydrazinolysis and by digestion with carboxypeptidase. Hydrazinolysis was by a modification (Tsugita et al., 1960) of the method of Niu and Fraenkel-Conrat (1955). Protein which had been stripped from purified SPV with phenol and washed with methanol (Anderer, 1959) was heated in vacua with 200-500 times its weight of hydrazine (Eastman, 95+ %). After removal of the unreacted hydrazine in vacua over H2S04, the residue was analyzed without further treatment for neutral and acidic amino acids in the Spinco amino acid analyzer. Digestion of whole virus with carboxypeptidase (Worthington 2 X crystallized carboxypeptidase A-DFP, solubilized with alkali) was performed by the method of Guidotti using 1% sodium dodecyl sulfate (Duponol C recrystallized from ethanol) to disrupt the virus particles (Guidotti, 1960;
OF SHOPE
PAPILLOMA
VIRUS
275
Light, 1962). To compensate for the partial inactivation of the protease in detergent (Light, 1962), the enzyme was added in portions of ?&,o the weight of the substrate every 30 minutes for 4% hours. The inactivation of carboxypeptidase was somewhat variable, and the conditions not’ed above did not result in t’he release of as much total amino acids as did another digestion by addition of f$cc enzyme every 2 hours for 10 hours. Under all conditions t’ested, the C-terminal amino acid was released in greater quantity t’han the penultimate residue. To stop the digestion and to precipitate protein the pH was lowered to 2.0 and the amino acids in the supernatant, fluid were analyzed in the amino acid analyzer. Since basic amino acids were not detected in qualitative tests (paper chromatography) after treatment of SPV-protein with carboxypeptidase B (Worthington) alone or together with carboxypeptidase A, quantitative assays for basic amino acids were not made in subsequent carboxyl-terminal st’udies. Amino acid composition of SPV protein. Protein stripped from SPV with phenol was hydrolyzed in vacua with constant boiling HCl (5.7 N) at 108” for 24 and 72 hours. The HCI was rapidly removed on a rotary evaporator (Ikawa and Snell, 1961) and the residue, taken up in the appropriate buffer, was analyzed on the long and short columns of the Spinco amino acid analyzer. Tyrosine and tryptophan were estimated directly in a sample of protein by a spectrophotometric method (Goodwin and Morton, 1946). Tryptophan was also determined in the same sample by a calorimetric method (Spies and Chambers, 1949). Tests for polyamines in SPV. Two microliters of SPV at a concentration of 20 mg/ml were applied to the origin of Whatman No. 1 paper and chromatographed along with reference polyamines in the solvent isopropanolHCl-HZ0 (170:44:36) (Ames et al., 1958). After drying for 24 hours the paper was sprayed with 0.1% ninhydrin in 95 % ethanol-acetic acid-collidine (160:75: 10). The limit of detection of spermidine (a typical polyamine) by t’his procedure was observed to be somewhat less than 0.03 pg.
KASS AND KNIGHT
2iG
mmI
h’ther sensitivity of SPI’. Ether sensitivity of SPV was t’ested by the mebhod of Andrewes and Horstmann (1949). After incuhation with >< volume of et’hyl ether for 20 hours at 4” t,he ether was permitted to evaporate in an open dish at room temperature and dilutions of treated and control virus were tested for infectivit’y on the same rabbits.
T
t--l
I SUPERNATANT
RESULTS
Rate Zonal Centrifugation When an SPV preparation which had been purified by several cycles of differential centrifugation was sedimented for 2 hours
on a glycerol or sucrose density gradient, the material was resolved into three visible bands (Fig. 1) and a small pellet. Since the pellet material separated into the characteristic three bands when resuspended and subjected to another gradient sedimentation, it was assumed to be a mixed aggregate or an artifact of sedimentation in cylindrical tubes. The three banding components, called slow, middle, and fast with reference to their
FIG. 1. A sketch of the appearance of a centrifuge tube after rate zonal density gradient sedimentation of SPV. A 0.8 cm (3.5 ml) layer of SPV purified by two cycles of differential centrifugation was placed carefully on the top of the gradient, and centrifuged for 2.5 hours at 24,000 rpm in the SW-25 swinging-bucket rotor. The density range of the glycerol gradient is indicated at the left, along with a distance scale.
sedimentation
tivity present in the preparation. The fast sedimenting component of the rate zonal centrifugation banded in CsCl chiefly at a density of 1.34 although two small bands were observed near density 1.31. The buoyant densities of the three components are consistent with the chemical observat’ions that the slow sedimenting component is essentially protein, the middle sedimenting component contains the purest SPV (highest content of DNA), while the fast sedimenting component consists largely of SPV with a minor protein contaminant.
rates on the density
gradient,
were found to be stable to a second cycle of zonal sedimentation thus providing highly purified discrete fractions for further characterization. The absorbancies in the ultraviolet of the rate zonal fractions and the appearance of the particles of these fractions in the electron microscope were about the same as reported earlier (Williams et al., 1960). Equilibrium
Density Gradient Centrifugation
The material from each of the three zones of the rate zonal centrifugation was centrifuged to equilibrium in CsCl. The middle sedimenting, highly infectious component of the rate zonal separation gave rise in CsCl almost exclusively to a narrow, intense band at density 1.34. Traces of material in the middle component were seen at densities of 1.29 and 1.31. The material comprising the slowest sedimenting zone was nearly all found in a broad, diffuse band in a region of density centering around I.29 g/ml. A trace of material was seen at density 1.34, and t,his nrobablv ” accounts for the slight, infec-_
Analytical
Ultracentrifugation
ultracentrifuge Analytical runs with schlieren optics were made on the slow and middle sedimenting components. The middle component purified by one cycle of rate zonal centrifugation was markedly enriched in the 280 S component relative to the 180 S and 390 S components (H. K. Schachman, personal communication). The slow sedimenting component purified by 2 cycles of equilibrium sedimentation contained a sharp peak
with
a sedimentation
rate
of 192 S.
PURIFICATION
AND CHEMICAL
ANALYSIS
No material could be detected sedimenting faster than the peak material, but there was some inhomogeneous slower sedimenting material with a broad peak near 115 S. The isolated fast sedimenting zone was not examined in the analytical ultracentrifuge. From its behavior in gradient sedimentation and its chemical and biological properties it is quite likely that it is a dimer of the virus and corresponds to the minor fast component (390 S) observed in the analytical ultracentrifugation (Schachman, 1951). Markham (1962) has found that dimers of spherical viruses have a sedimentation rate 1.4 times that of the monomer, which is the relationship between 280 S and 390 S components. Infectivity
of the Density Gradient Fractions
End-point dilution tests for infectivity showed that about 5 X 1O-g g of the two faster sedimenting components (rate zonal centrifugation) produced papillomas, while more than 250 times this amount of the slow sedimenting component was required to produce the same response. The specific infectivities of the two faster sedimenting components appeared to be identical, but the infectivity tests are not accurate enough to reflect the small differences in purity which were detected in buoyant density and in DNA content. DNA Content of SPV The reported values for the DNA content of SPV range from 6.6 to 8.7% (Taylor et al., 1942; Watson and Littlefield, 1960). In this laboratory the DNA content could not be increased by differential centrifugation to more than 8 % even after six cycles of centrifugation. However, one cycle of rate zonal density gradient sedimentation was found to yield a middle sedimenting band of material consistently containing more than 10% DNA. After the middle component was subjected to a second rate zonal centrifugation, the material was found to have about 12 % DNA (Table 1). The DNA content of the slow sedimenting component of rate zonal centrifugation was in the neighborhood of 1% or less, while the fast sedimenting component contained only slightly
OF SHOPE PAPILLOMA
VIRUS
277
TABLE 1 DNA CONTENT OF FRACTIONS OF SPV
Component Slow Middle Fast
1 cycle” 1.65% 11.2 9.1
2 cycle@ <0.5% 12.1 11 .o
0 DNA estimated from P analysis. b DNA was estimated from A260 and protein was estimated by the Method of Lowry et al. (1951), with corrections for light scattering (see Materials and Methods).
less DNA than the middle component. Slight contamination of the fast component by protein was indicated by the appearance of two small bands near a density of 1.31 in the equilibrium gradient centrifugation. Infectious DNA from SPV Preparations of DNA obtained from SPV by the phenol procedure were examined for absorbancy in the ultraviolet. The absorption curves obtained showed a maximum at 258 rnp, a minimum at 230 rnp, and ratios of maximum to minimum of 2.4 or more, making them comparable t’o the best preparations of Watson and Littlefield (1960). The infectivities of DNA preparations ranged from 0.01 to 1% that of DNA in intact SPV. The infectivity was destroyed by 30 minutes’ incubation with DNase (2 pg/ ml). After equilibrium sedimentation in CsCl, all the infectivity and the absorbancy at 260 rnp of the DNA preparation were found in a narrow band near a density of 1.7. No infectivity was found in less dense fractions where SPV or partially degraded SPV would be found. Serological Relationship of the Three Components Observed in Rate-Zonal Centrifugation In a semiquantitative precipitin test, all three density gradient components of SPV were found to have on a weight basis approximately the same precipitin end-point (about 0.2 mg/ml against undiluted immune antiSPV serum). In the comparison of antigens by diffusion in agar gel (Ouchterlony technique) the middle and fast components were serologically equivalent as shown by the
KASS AXrJn KKIGHT
Increased Digestion-
FIG. 2. Kinetics
of digestion by carboxypeptidase of detergent-treated Shope papilloma virus. Three different digestion experiments are indicated in this composite figure. Points 1 and 2 represent, results of minimal digestion using an enzyme-substrate (E-S) ratio of 1:250 for 10 and 30 minutes, respectively. Points 3 and 4 indicate results of a digestion with an E:S ratio of 1:200 for 4% hours followed by addition of the same amount of enzyme and incubation for an additional 15 hours. The maximal degree of digestion obtained is shown by points 5 and 6 which represent the results of adding enzyme at an E:S ratio of 1:200 every two hours, removing aliquots for analysis after G and 10 hours (points 5 and 6, respect)ively). joining of the arcs of precipitate with no spurs. In a comparison of the slow and middle components, arcs of precipitate formed at approximately the same distance from the antigen wells, but t’he arcs did not join even after 24 days. This failure to merge may be
attributable to a spacing of the antigen wells at too great a distance apart for the low concentrations of material employed. Terminal Amino Acids of SPV-Protein Amino-terminal analysis with fluorodinitrobenzene revealed no amino acids reactive or accessible to this reagent. The reaction was attempted with the following substrates: (a) 6 mg of SPV purified by 4 cycles of differential centrifugation; (b) 12 mg of the middle sedimenting component from ratewnal sedimentation; (c) 3 mg of protein stripped from the virus with phenol and solubilized at pH 12; (d) samples of alkali degraded SPV as large as 22 mg. NO DNP-amino acids were seen on the
paper chromatograms except in the largest sample of (d), where a number of faint spots were seen. Since t’he strongest’ of these spots, at, the locations of DSP-alanine and diDNP-lyeine, indicat,ed peptide chains of improbably large size (mol. wt., 5 X 106), it was concluded that the DNP-amino acids represent either small amounts of impurities or products resulting from slight degradation of the SPV-protein in the alkali. Carboxyl-terminal analysis by hydrazinolysis of protein st’ripped by phenol from SPV (purified by diff erent!ial centrifugation) showed only t’hreonine when analyzed on the amino acid analyzer. In analysis of two samples (6 mg and 20 mg) from different batches of virus, threonine was recovered in amounts corresponding to a peptide chain of about 230,000 in molecular weight. However, owing to dest’ruction of amino acids in the hydrazinolysis reaction (Akabori et aZ., 1956), this value is cert’ainly high. Better recovery of the C-terminal residue was obtained by treatment of the protein wit.h carboxypeptidase. From results of the sort shown in Fig. 2, a C-terminal sequence for SPV-protein of (Leu,Tyr)-Ala-Thr was deduced. However, release of C-terminal threonine by carboxypeptidase may not have been complete even under maximal conditions since the penultimate residue, alanine, was never obtained in equivalent amount. Hence, the molecular weight of 160,000 for the SPV-protein subunit, calculated from the threonine released by carboxypeptidase, may not be the true value although it does set an upper limit. It appears that the susceptibility of whole SPV to carboxypept’idase is strongly dependent on steric factors since it was essential to denature the virus in detergent before amino acids were released in significant quantities and in a sequential order. Amino Acid Analysis The results of amino acid analyses are summarized in Table 2. In general the results agree fairly well with those obtained much earlier by microbiological assay of hydrolyzates of whole virus (Knight, 1950). Differences greater than about 10 % were observed between results of the earlier assays
PURIFICATION
AND CHEMICAL
TABLE
ANALYSIS
2
presenting minimum per protein subunit,.
AMINO
ACID CONTENT OF RPV PROTEIN PRECIPITaTED FROM PHENOL
-
Moles
relative to glycine
I on exchange after hydrolysis
-I 24 hr
Alanine Arginine Aspartic acid Cysteine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Trypt,ophan
Other nethodi
I ;&I
VIRUS
numbers
279
of residues
Search for Lipids and Polyamines in SPV
100 moles
I Amino acid
OF SHOPE PAPILLOMA
“,fF:? sidues
Per
histidine
?I I
72 hr
3 102 102 88 88 3 182 6 182 2 56b 56 G 202 202 3 100 100 1 32 32 2 65 65 4 133 133 4 119 119 1 10 33b 33b 2 64 64 3 108 108 3 104 107 3 91 95” 21CJ 1 18” 22c 224 2 71 71 Tyrosine 61c 60c 107 3 102 107 Valine QThe HCl was removed by a slow procedure in the case of the 24-hour sample, which probably caused a greater than normal loss. b Value calculated from microbiological assay results (Knight., 1950). c Determined by a spectrophotometric method. d Determined by a calorimetric method. e Value extrapolated to zero time. Value may be low for reason given in a. 101 88 182 193 100 29 58 130 119 18 59 107 108’ 94 70
-
and those of the present analyses in the cases of alanine, methionine, serine, and tryptophan. In all cases except methionine the more recent values were higher. This result reflects improvements in hydrolytic procedures to some extent and the superior accuracy of the current assay method. An exception is in the methionine analysis which came out low owing to partial conversion of methionine to oxidation products. Therefore, the microbiological assay value for methionine is used in Table 2 in the column
Neither essential lipid nor polyamines were found in SPV that had been purified by differential centrifugation. Insensitivity of SPV to ether was reported by Andrewes and Horstmann (1949) using 12-year-old papillomas that contained minimal amounts of infectious virus. Two of our preparations, which were 500 times as infectious as those of Andrewes and Horstmann, showed no decrease in infectivity after ether treatment. Polyamines could not be detected by the paper chromatographic analysis that was sufficiently sensitive to detect a polyamine comprising 0.07 % of the virus by weight. DISCUSSION
The most highly purified preparations of SPV reported here contained aout 12 % DNA. This DNA has been recently shown to have a cyclic form with a contour length of 2.3-2.8 I-(, depending upon conditions of measurement (Kleinschmidt et al., 1965). Such a DNA particle can be estimated to have a molecular weight of about 5 X 106 (Langridge et al., 1960). The molecular weight of SPV is not yet known with certainty, but the limits of various estimations made from hydrodynamic data are from about 35 X lo6 to 47 X lo6 (Kass, 1964; Neurat’h et al., 1941). From these values and the DNA content of 12% found in the present investigation, it can be calculated that there are 4.2 to 5.6 avograms of DNA per particle of SPV. Watson and Littlefield (1960) obtained values of 4.9 and 5.3 X 106 for the molecular weight of DNA isolated from SPV and studied by the CsCl density gradient equilibrium method of Meselson. Thus it seems probable that the tota DNA of each particle of SPV is contained in a single cyclic duplex. In view of the low efficiency of infection of the phenol extracts of viruses, it has often been proposed that the infectivity is due to residual virus or partially uncoated virus particles (for discussion see Knight, 1963). In confirmation of the findings of Ito (1960), the infectivities of our phenol extracts were resistant
to heating
in neutral
saline
at 98”
KASS
280
AND
for 30 minutes, and were entirely abolished by DNase under conditions in which SW was unaffected. Furt’hermore, the infectivity of such extrack was found at the same buoyant density in CsCl (1.7) as the DNA. If residual virus were present, it would have been found in the lightest fraction of the CsCl gradient. Since no infectivit’y was found either in the lightest fraction or at intermediate levels, it may be concluded that the infectivity of the phenol ext’racts is due to DNA which is not associated with any measurable amount of protein. Although the size of the protein subunit of SPV could not be established with certainty here, the results of studies on t’he carboxyl-terminal group indicated that it could not exceed a molecular weight of about 160,000. The minimum molecular weight based on one histidine residue per subunit is about 6000. The results of the C-terminal analyses also indicate that there is only one type of protein subunit present, namely one beginning with an N-terminal group not reacting with fluorodinitrobenzene (and hence probably acetylated) and ending with C-terminal threonine. The amino acid content of t’he subunit indicates that it contains the usual amino acids found in proteins and has no unusual compositional features. REFERENCES AKABORI, S., OHNO, K., IKENAK.~, T., OIUDA, Y., HaNa~us.4, H.,HARuN~, I., TSUGITA, A., SUGAE, K., and MATSUSHIMA, T. (1956). Hydrazinolysis
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PURIFICATION
AND
CHEMICAL
ANALYSIS
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OF SHOPE
PAPILLOMA
VIRUS
281
H. K. (1951). Physical chemical studies on rabbit papilloma virus. J. Am. Chem. sot. 73, 44534455. SPIES, J. It., and CHAMBERS, D. C. (1949). Chemical determination of tryptophan in proteins. Anal. Chem. 21, 1249-1266. TAYLOR, A. R., BEARD, D., SHARP, D. G., and BEARD, J. W. (1942). Nucleic acid of the rabbit papilloma virus protein. J. Infectious Diseases SCHACHMAN,
71, 11s114. TSUGITA, A., GISH, D. T., YOUNG, J., FRAENKELCONRAT, H., KNIGHT, C. A., and STANLEY, W.
M. (1960). The complete amino acid sequence of the protein of tobacco mosaic virus. Proc. Natl. Acad. Sci. U.S. 46, 1463-1469. WATSON, J. D., and LITTLEFIELD, J. W. (1960). Some properties of DNA from Shope papilloma virus. J. Mol. Biol. 2, 161-165. WEIL, R. (1961). A quantitative assay for a subviral infective agent related to polyoma virus. ViroZogy 14, 46-53. WILLIAMS, R. C., KASS, S. J., and KNIGHT, C. A. (1960). Structure of Shope papilloma virus particles. Virology 12, 48-58.
2’7, 273-281 (1965)
Purification
and Chemical S. J. KASS’
Analysis AND
of Shope
Papilloma
Virus
C. A. KNIGHT
Department of Biochemistry and Virus Laboratory, University of California,
Berkeley, California
Accepted July 19, 1965 Rate eonal density gradient centrifugation of preparations of Shope papilloma virus revealed three components (slow, middle, and fast) which were morphologically and serologically related. The slow sedimenting component was noninfectious, contained little or no DNA, and had a buoyant density of 1.29 in CsCI. The middle and fast sedimenting components were infectious and had the same buoyant density of 1.34 in CsCl. The middle sedimenting component, yielding a single band upon equilibrium sedimentation in CsCl, was found upon analysis to contain protein and 12% DNA, but no essential lipid and no detectable polyamines. Protein end-group analyses by treatment with hydrazine or with carboxypeptidase A showed the carboxyl terminal amino acid residue to be threonine. The amino-terminal group was unreactive t,oward fluorodinitrobenzene. The viral protein was analyzed for total amino acid content. Infectious DNA was extracted from the virus by treatment with 80% phenol, and shown to be free of contaminating virus by equilibrium density gradient centrifugation in CsCl. INTRODUCTION
The chemical composition of preparations of Shope papilloma virus (SPV), obtained by differential centrifugation of wart extracts was investigated some years ago by Taylor et al. (1942) and Knight (1950). Since such preparations have subsequently been shown to contain three or four components (Schachman, 1951; Williams et al., 1960; Breedis et al., 1962) it seemed desirable to reinvestigate the chemical and physical properties of SPV. Several groups of investigators have succeeded in fractionating SPV by density gradient centrifugation and have determined something of the morphology of the particles in various fractions by electron micros1 Taken in part from a thesis submitt,ed by S. J. Kass in partial satisfaction of the requirements for the Doctor of Philosophy degree, Department of Biochemistry, University of California, Berkeley, 1964. Present address: Section of Genetics, Weizmann Institute of Science, Rehovoth, Israel. This investigation was supported in part by Public Health Service Research Grant AI 00634, from the National Institute of Allergy and Infectious Diseases.
copy (Williams et al., 1960; Breedis et uZ., 1962; Crawford and Crawford, 1963). Some other properties of these fractions have been determined as well. From these observations, it appears that most of the material in a preparation of SPV purified by differential centrifugation is virus-related in the sense that it consists mainly of a mixture of whole virus particles, virus particles lacking DNA, of the and aggregates or mixed aggregates first two species. In the present study, some of the earlier investigations on purification of SPV have been reexamined and expanded; the results obtained, together with new findings about the physical and chemical properties of SPV preparations, are presented here. MATERIALS
AND
METHODS
Isolation and puri$cation of virus. Isolation of SPV from naturally occurring papillomas -was described earlier (Williams et al., 1960). Modification of the preparation by homogenizing the papillomas with the following buffers at pH 7.1-7.3 produced virus in essentially the same yield and with approximately the same content of aggregated 273
274
HAS
AND KNIGHT
material and empty (nucleic acid-free) capsids: 0.05 dP phosphate, Veronal-NaCl (0.02 A&O.2 M), and 0.1 dl et,hylenedinmine tetraacetate (EDTA). Other buffer mixtures were t,ested and found inferior t’o t’hose just listed, either because they were less efficient in the extraction of virus or because Obey extracted excessive amounts of nonviral materials along with the virus (Kass, 1964). Likewise, heating homogenates at 50”60” for 30 minutes was tried as a purification step, but was abandoned when it showed no advantage. Therefore, virus was routfinely isolated and purified by differential centrifugation in the presence of one of t’he three buffers listed above. Density gradient centrifugation of purified SPV. In rate zonal centrifugation, purified virus obtained by differential centrifugation was layered on a preformed gradient of glycerol or sucrose of density 1.17-1.05 and centrifuged in a swinging-bucket rotor as described earlier (Williams et al., 1960). In order to achieve greater efficiency of separation, materials from the visible bands of the first density gradient centrifugation were diluted with an equal volume of water and sedimented at about 62,000 g for 2 hours; the resulting pellet was resuspended and subjected to another cycle of rate zonal sedimentation. Since mechanical losses were rather great in this process, further cycles of rate zonal sedimentation were not attempted. For equilibrium density gradient centrifugation, virus preparations were mixed with CsCl (Maywood Co., optical grade) at a final density of 1.30 g/ml and centrifuged in the SW-39 Spinco swinging-bucket rot,or at 34,000 rpm for 18 hours (Meselson et al., 1957). The components appearing as a result of this procedure were located and collected as in the rate zonal method. Density of the fractions was measured by weighing aliquots in a 50-~1 pipette (H. Pederson, Carlsberg Laboratories, Copenhagen). Infectivity titrations. Infectivity measurements were made by placing test solutions on l-inch-square areas of depilated skin of domestic rabbits and spreading and injecting the material in a regular pattern with an electric tattoo needle. Owing to large varia-
t,ions in susceptibility among diff crent animals (Beard, 1956), comparisons of infectivit#y of virus fractions were performed by inoculating a series of dilutions on different areas of the same animal, and noting the number and size of the papillomas produced in a given time (usually 35-40 days). Estimation of protein. Routine protein estimation was made by the calorimetric procedure of Lowry et al. (1951), using crystalline bovine serum albumin as a standard. These result,s in turn were standardized in terms of virus by analyzing t’he latter for nitrogen and use of the relationship : virus = Nessler nitrogen X 6.1. The nitrogen content of SPV was calculated to be 16.4%, based on a composition of 12 % DNA with an N content of 15.2 % (sodium salt: Chargaff, 1955) and 88 % protein of t’he amino acid composition reported here. Extraction of DNA. SPV purified by repeated cycles of differential centrifugation was shaken for 5-10 minutes at room temperature with an equal volume of redistilled 80% phenol in the presence of 0.1 M NaCI, 0.15 M neutralized trichloroacetat,e, and 1O-2 &r EDTA (see Weil, 1961). Phenol was removed from the aqueous phase with ether, and et’her was removed with a st,ream of nitrogen gas. In some experiments, separation of DNA from other materials in the aqueous phase of the extraction mixture was accomplished by equilibrium centrifugation in CsCl of density 1.70 at 34,000 rpm in the SW-39 rotor for 40 hours at 11”. Fractions were removed by puncturing the bottom of the tube and were assayed for infectivity both undiluted and diluted 1: 10 in buffered saline. CsCl appeared to interfere with the infectivity assay, since the diluted material produced the same number as or more papillomas than t’he undiluted material. The density of the fractions was measured by weighing, as described above, and the DNA was located by the absorbancy at 260 rnp. Estimation of DNA. The DNA content of SPV fractions was estimated as phosphorus X 10.9 (Chargaff, 1955). However, when the quantities of material available were insufficient for P analysis, DNA was estimated from the ultraviolet absorption, using cor-
PURIFICATION
AND
CHEMICAL
ANALYSIS
rections obtained with intact virus for light scattering (Bonhoeffer and Schachman, 1960) and for ultraviolet absorption by protein. Phosphorus was estimated by a micromodification of the method of Nakamura (1952) in which digestion, color development, and dilution were done in a 1.0 ml volumetric test tube. Serological tests. Semiquantitative precipitin tests were made on serial twofold dilutions of virus materials incubated in 1 ml test tubes with a constant amount of precipitating antiserum (Bryan and Beard, 1941; Knight, 1946). Incubation was for 2 hours at room temperature, and overnight in the refrigerator, after which the sizes of precipitates were noted. The qualitative relationship of antigens was tested by the Ouchterlony technique of diffusion in agar gel (Le Bouvier, 1957). Terminal amino acids of SPV protein. Attempts were made to determine the amino terminal amino acid of SPV-protein by applying the fluorodinitrobenzene method (Fraenkel-Conrat et al., 1955) to the virus using either 1% sodium bicarbonate at neutral pH or sodium carbonate-NaOH at pH 12. The latter buffer disrupts the virus particles, but does not interfere with the coupling reaction. The carboxyl terminal amino acid was investigated by hydrazinolysis and by digestion with carboxypeptidase. Hydrazinolysis was by a modification (Tsugita et al., 1960) of the method of Niu and Fraenkel-Conrat (1955). Protein which had been stripped from purified SPV with phenol and washed with methanol (Anderer, 1959) was heated in vacua with 200-500 times its weight of hydrazine (Eastman, 95+ %). After removal of the unreacted hydrazine in vacua over H2S04, the residue was analyzed without further treatment for neutral and acidic amino acids in the Spinco amino acid analyzer. Digestion of whole virus with carboxypeptidase (Worthington 2 X crystallized carboxypeptidase A-DFP, solubilized with alkali) was performed by the method of Guidotti using 1% sodium dodecyl sulfate (Duponol C recrystallized from ethanol) to disrupt the virus particles (Guidotti, 1960;
OF SHOPE
PAPILLOMA
VIRUS
275
Light, 1962). To compensate for the partial inactivation of the protease in detergent (Light, 1962), the enzyme was added in portions of ?&,o the weight of the substrate every 30 minutes for 4% hours. The inactivation of carboxypeptidase was somewhat variable, and the conditions not’ed above did not result in t’he release of as much total amino acids as did another digestion by addition of f$cc enzyme every 2 hours for 10 hours. Under all conditions t’ested, the C-terminal amino acid was released in greater quantity t’han the penultimate residue. To stop the digestion and to precipitate protein the pH was lowered to 2.0 and the amino acids in the supernatant, fluid were analyzed in the amino acid analyzer. Since basic amino acids were not detected in qualitative tests (paper chromatography) after treatment of SPV-protein with carboxypeptidase B (Worthington) alone or together with carboxypeptidase A, quantitative assays for basic amino acids were not made in subsequent carboxyl-terminal st’udies. Amino acid composition of SPV protein. Protein stripped from SPV with phenol was hydrolyzed in vacua with constant boiling HCl (5.7 N) at 108” for 24 and 72 hours. The HCI was rapidly removed on a rotary evaporator (Ikawa and Snell, 1961) and the residue, taken up in the appropriate buffer, was analyzed on the long and short columns of the Spinco amino acid analyzer. Tyrosine and tryptophan were estimated directly in a sample of protein by a spectrophotometric method (Goodwin and Morton, 1946). Tryptophan was also determined in the same sample by a calorimetric method (Spies and Chambers, 1949). Tests for polyamines in SPV. Two microliters of SPV at a concentration of 20 mg/ml were applied to the origin of Whatman No. 1 paper and chromatographed along with reference polyamines in the solvent isopropanolHCl-HZ0 (170:44:36) (Ames et al., 1958). After drying for 24 hours the paper was sprayed with 0.1% ninhydrin in 95 % ethanol-acetic acid-collidine (160:75: 10). The limit of detection of spermidine (a typical polyamine) by t’his procedure was observed to be somewhat less than 0.03 pg.
KASS AND KNIGHT
2iG
mmI
h’ther sensitivity of SPI’. Ether sensitivity of SPV was t’ested by the mebhod of Andrewes and Horstmann (1949). After incuhation with >< volume of et’hyl ether for 20 hours at 4” t,he ether was permitted to evaporate in an open dish at room temperature and dilutions of treated and control virus were tested for infectivit’y on the same rabbits.
T
t--l
I SUPERNATANT
RESULTS
Rate Zonal Centrifugation When an SPV preparation which had been purified by several cycles of differential centrifugation was sedimented for 2 hours
on a glycerol or sucrose density gradient, the material was resolved into three visible bands (Fig. 1) and a small pellet. Since the pellet material separated into the characteristic three bands when resuspended and subjected to another gradient sedimentation, it was assumed to be a mixed aggregate or an artifact of sedimentation in cylindrical tubes. The three banding components, called slow, middle, and fast with reference to their
FIG. 1. A sketch of the appearance of a centrifuge tube after rate zonal density gradient sedimentation of SPV. A 0.8 cm (3.5 ml) layer of SPV purified by two cycles of differential centrifugation was placed carefully on the top of the gradient, and centrifuged for 2.5 hours at 24,000 rpm in the SW-25 swinging-bucket rotor. The density range of the glycerol gradient is indicated at the left, along with a distance scale.
sedimentation
tivity present in the preparation. The fast sedimenting component of the rate zonal centrifugation banded in CsCl chiefly at a density of 1.34 although two small bands were observed near density 1.31. The buoyant densities of the three components are consistent with the chemical observat’ions that the slow sedimenting component is essentially protein, the middle sedimenting component contains the purest SPV (highest content of DNA), while the fast sedimenting component consists largely of SPV with a minor protein contaminant.
rates on the density
gradient,
were found to be stable to a second cycle of zonal sedimentation thus providing highly purified discrete fractions for further characterization. The absorbancies in the ultraviolet of the rate zonal fractions and the appearance of the particles of these fractions in the electron microscope were about the same as reported earlier (Williams et al., 1960). Equilibrium
Density Gradient Centrifugation
The material from each of the three zones of the rate zonal centrifugation was centrifuged to equilibrium in CsCl. The middle sedimenting, highly infectious component of the rate zonal separation gave rise in CsCl almost exclusively to a narrow, intense band at density 1.34. Traces of material in the middle component were seen at densities of 1.29 and 1.31. The material comprising the slowest sedimenting zone was nearly all found in a broad, diffuse band in a region of density centering around I.29 g/ml. A trace of material was seen at density 1.34, and t,his nrobablv ” accounts for the slight, infec-_
Analytical
Ultracentrifugation
ultracentrifuge Analytical runs with schlieren optics were made on the slow and middle sedimenting components. The middle component purified by one cycle of rate zonal centrifugation was markedly enriched in the 280 S component relative to the 180 S and 390 S components (H. K. Schachman, personal communication). The slow sedimenting component purified by 2 cycles of equilibrium sedimentation contained a sharp peak
with
a sedimentation
rate
of 192 S.
PURIFICATION
AND CHEMICAL
ANALYSIS
No material could be detected sedimenting faster than the peak material, but there was some inhomogeneous slower sedimenting material with a broad peak near 115 S. The isolated fast sedimenting zone was not examined in the analytical ultracentrifuge. From its behavior in gradient sedimentation and its chemical and biological properties it is quite likely that it is a dimer of the virus and corresponds to the minor fast component (390 S) observed in the analytical ultracentrifugation (Schachman, 1951). Markham (1962) has found that dimers of spherical viruses have a sedimentation rate 1.4 times that of the monomer, which is the relationship between 280 S and 390 S components. Infectivity
of the Density Gradient Fractions
End-point dilution tests for infectivity showed that about 5 X 1O-g g of the two faster sedimenting components (rate zonal centrifugation) produced papillomas, while more than 250 times this amount of the slow sedimenting component was required to produce the same response. The specific infectivities of the two faster sedimenting components appeared to be identical, but the infectivity tests are not accurate enough to reflect the small differences in purity which were detected in buoyant density and in DNA content. DNA Content of SPV The reported values for the DNA content of SPV range from 6.6 to 8.7% (Taylor et al., 1942; Watson and Littlefield, 1960). In this laboratory the DNA content could not be increased by differential centrifugation to more than 8 % even after six cycles of centrifugation. However, one cycle of rate zonal density gradient sedimentation was found to yield a middle sedimenting band of material consistently containing more than 10% DNA. After the middle component was subjected to a second rate zonal centrifugation, the material was found to have about 12 % DNA (Table 1). The DNA content of the slow sedimenting component of rate zonal centrifugation was in the neighborhood of 1% or less, while the fast sedimenting component contained only slightly
OF SHOPE PAPILLOMA
VIRUS
277
TABLE 1 DNA CONTENT OF FRACTIONS OF SPV
Component Slow Middle Fast
1 cycle” 1.65% 11.2 9.1
2 cycle@ <0.5% 12.1 11 .o
0 DNA estimated from P analysis. b DNA was estimated from A260 and protein was estimated by the Method of Lowry et al. (1951), with corrections for light scattering (see Materials and Methods).
less DNA than the middle component. Slight contamination of the fast component by protein was indicated by the appearance of two small bands near a density of 1.31 in the equilibrium gradient centrifugation. Infectious DNA from SPV Preparations of DNA obtained from SPV by the phenol procedure were examined for absorbancy in the ultraviolet. The absorption curves obtained showed a maximum at 258 rnp, a minimum at 230 rnp, and ratios of maximum to minimum of 2.4 or more, making them comparable t’o the best preparations of Watson and Littlefield (1960). The infectivities of DNA preparations ranged from 0.01 to 1% that of DNA in intact SPV. The infectivity was destroyed by 30 minutes’ incubation with DNase (2 pg/ ml). After equilibrium sedimentation in CsCl, all the infectivity and the absorbancy at 260 rnp of the DNA preparation were found in a narrow band near a density of 1.7. No infectivity was found in less dense fractions where SPV or partially degraded SPV would be found. Serological Relationship of the Three Components Observed in Rate-Zonal Centrifugation In a semiquantitative precipitin test, all three density gradient components of SPV were found to have on a weight basis approximately the same precipitin end-point (about 0.2 mg/ml against undiluted immune antiSPV serum). In the comparison of antigens by diffusion in agar gel (Ouchterlony technique) the middle and fast components were serologically equivalent as shown by the
KASS AXrJn KKIGHT
Increased Digestion-
FIG. 2. Kinetics
of digestion by carboxypeptidase of detergent-treated Shope papilloma virus. Three different digestion experiments are indicated in this composite figure. Points 1 and 2 represent, results of minimal digestion using an enzyme-substrate (E-S) ratio of 1:250 for 10 and 30 minutes, respectively. Points 3 and 4 indicate results of a digestion with an E:S ratio of 1:200 for 4% hours followed by addition of the same amount of enzyme and incubation for an additional 15 hours. The maximal degree of digestion obtained is shown by points 5 and 6 which represent the results of adding enzyme at an E:S ratio of 1:200 every two hours, removing aliquots for analysis after G and 10 hours (points 5 and 6, respect)ively). joining of the arcs of precipitate with no spurs. In a comparison of the slow and middle components, arcs of precipitate formed at approximately the same distance from the antigen wells, but t’he arcs did not join even after 24 days. This failure to merge may be
attributable to a spacing of the antigen wells at too great a distance apart for the low concentrations of material employed. Terminal Amino Acids of SPV-Protein Amino-terminal analysis with fluorodinitrobenzene revealed no amino acids reactive or accessible to this reagent. The reaction was attempted with the following substrates: (a) 6 mg of SPV purified by 4 cycles of differential centrifugation; (b) 12 mg of the middle sedimenting component from ratewnal sedimentation; (c) 3 mg of protein stripped from the virus with phenol and solubilized at pH 12; (d) samples of alkali degraded SPV as large as 22 mg. NO DNP-amino acids were seen on the
paper chromatograms except in the largest sample of (d), where a number of faint spots were seen. Since t’he strongest’ of these spots, at, the locations of DSP-alanine and diDNP-lyeine, indicat,ed peptide chains of improbably large size (mol. wt., 5 X 106), it was concluded that the DNP-amino acids represent either small amounts of impurities or products resulting from slight degradation of the SPV-protein in the alkali. Carboxyl-terminal analysis by hydrazinolysis of protein st’ripped by phenol from SPV (purified by diff erent!ial centrifugation) showed only t’hreonine when analyzed on the amino acid analyzer. In analysis of two samples (6 mg and 20 mg) from different batches of virus, threonine was recovered in amounts corresponding to a peptide chain of about 230,000 in molecular weight. However, owing to dest’ruction of amino acids in the hydrazinolysis reaction (Akabori et aZ., 1956), this value is cert’ainly high. Better recovery of the C-terminal residue was obtained by treatment of the protein wit.h carboxypeptidase. From results of the sort shown in Fig. 2, a C-terminal sequence for SPV-protein of (Leu,Tyr)-Ala-Thr was deduced. However, release of C-terminal threonine by carboxypeptidase may not have been complete even under maximal conditions since the penultimate residue, alanine, was never obtained in equivalent amount. Hence, the molecular weight of 160,000 for the SPV-protein subunit, calculated from the threonine released by carboxypeptidase, may not be the true value although it does set an upper limit. It appears that the susceptibility of whole SPV to carboxypept’idase is strongly dependent on steric factors since it was essential to denature the virus in detergent before amino acids were released in significant quantities and in a sequential order. Amino Acid Analysis The results of amino acid analyses are summarized in Table 2. In general the results agree fairly well with those obtained much earlier by microbiological assay of hydrolyzates of whole virus (Knight, 1950). Differences greater than about 10 % were observed between results of the earlier assays
PURIFICATION
AND CHEMICAL
TABLE
ANALYSIS
2
presenting minimum per protein subunit,.
AMINO
ACID CONTENT OF RPV PROTEIN PRECIPITaTED FROM PHENOL
-
Moles
relative to glycine
I on exchange after hydrolysis
-I 24 hr
Alanine Arginine Aspartic acid Cysteine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Trypt,ophan
Other nethodi
I ;&I
VIRUS
numbers
279
of residues
Search for Lipids and Polyamines in SPV
100 moles
I Amino acid
OF SHOPE PAPILLOMA
“,fF:? sidues
Per
histidine
?I I
72 hr
3 102 102 88 88 3 182 6 182 2 56b 56 G 202 202 3 100 100 1 32 32 2 65 65 4 133 133 4 119 119 1 10 33b 33b 2 64 64 3 108 108 3 104 107 3 91 95” 21CJ 1 18” 22c 224 2 71 71 Tyrosine 61c 60c 107 3 102 107 Valine QThe HCl was removed by a slow procedure in the case of the 24-hour sample, which probably caused a greater than normal loss. b Value calculated from microbiological assay results (Knight., 1950). c Determined by a spectrophotometric method. d Determined by a calorimetric method. e Value extrapolated to zero time. Value may be low for reason given in a. 101 88 182 193 100 29 58 130 119 18 59 107 108’ 94 70
-
and those of the present analyses in the cases of alanine, methionine, serine, and tryptophan. In all cases except methionine the more recent values were higher. This result reflects improvements in hydrolytic procedures to some extent and the superior accuracy of the current assay method. An exception is in the methionine analysis which came out low owing to partial conversion of methionine to oxidation products. Therefore, the microbiological assay value for methionine is used in Table 2 in the column
Neither essential lipid nor polyamines were found in SPV that had been purified by differential centrifugation. Insensitivity of SPV to ether was reported by Andrewes and Horstmann (1949) using 12-year-old papillomas that contained minimal amounts of infectious virus. Two of our preparations, which were 500 times as infectious as those of Andrewes and Horstmann, showed no decrease in infectivity after ether treatment. Polyamines could not be detected by the paper chromatographic analysis that was sufficiently sensitive to detect a polyamine comprising 0.07 % of the virus by weight. DISCUSSION
The most highly purified preparations of SPV reported here contained aout 12 % DNA. This DNA has been recently shown to have a cyclic form with a contour length of 2.3-2.8 I-(, depending upon conditions of measurement (Kleinschmidt et al., 1965). Such a DNA particle can be estimated to have a molecular weight of about 5 X 106 (Langridge et al., 1960). The molecular weight of SPV is not yet known with certainty, but the limits of various estimations made from hydrodynamic data are from about 35 X lo6 to 47 X lo6 (Kass, 1964; Neurat’h et al., 1941). From these values and the DNA content of 12% found in the present investigation, it can be calculated that there are 4.2 to 5.6 avograms of DNA per particle of SPV. Watson and Littlefield (1960) obtained values of 4.9 and 5.3 X 106 for the molecular weight of DNA isolated from SPV and studied by the CsCl density gradient equilibrium method of Meselson. Thus it seems probable that the tota DNA of each particle of SPV is contained in a single cyclic duplex. In view of the low efficiency of infection of the phenol extracts of viruses, it has often been proposed that the infectivity is due to residual virus or partially uncoated virus particles (for discussion see Knight, 1963). In confirmation of the findings of Ito (1960), the infectivities of our phenol extracts were resistant
to heating
in neutral
saline
at 98”
KASS
280
AND
for 30 minutes, and were entirely abolished by DNase under conditions in which SW was unaffected. Furt’hermore, the infectivity of such extrack was found at the same buoyant density in CsCl (1.7) as the DNA. If residual virus were present, it would have been found in the lightest fraction of the CsCl gradient. Since no infectivit’y was found either in the lightest fraction or at intermediate levels, it may be concluded that the infectivity of the phenol ext’racts is due to DNA which is not associated with any measurable amount of protein. Although the size of the protein subunit of SPV could not be established with certainty here, the results of studies on t’he carboxyl-terminal group indicated that it could not exceed a molecular weight of about 160,000. The minimum molecular weight based on one histidine residue per subunit is about 6000. The results of the C-terminal analyses also indicate that there is only one type of protein subunit present, namely one beginning with an N-terminal group not reacting with fluorodinitrobenzene (and hence probably acetylated) and ending with C-terminal threonine. The amino acid content of t’he subunit indicates that it contains the usual amino acids found in proteins and has no unusual compositional features. REFERENCES AKABORI, S., OHNO, K., IKENAK.~, T., OIUDA, Y., HaNa~us.4, H.,HARuN~, I., TSUGITA, A., SUGAE, K., and MATSUSHIMA, T. (1956). Hydrazinolysis
of peptides and proteins. II. Fundamental studies on the determination of the carboxylends of proteins. Bull. Chem. Sot. Japan 29, 507618. AMES, B. N., DUBIN, D. T., and ROSENTHAL, 8. M. (1958). Presence of polyamines in certain bacterial viruses. Science 127, 814-816. ANDERER, F. A. (1959). Das Molekulargewicht der Peptideinheit im Protein des Tabakmosaikvirus. 2. Naturforsch. 14b, 24-28. ANDREWES, C. H., and HORSTMANN, D. M. (1949). The susceptibility of viruses to ethyl ether. J. Gen. Microbial. 3, 290-297. BEARD, J. W. (1956j. The fallacy of the concept of virus “masking.” Cancer Res. 16, 279-291. BONHOEFFER, F., and SCHACHMAN, H. K. (1960) Studies on the organization of nucleic acids within nucleoproteins. Biochem. Biophys. Res. Comm. 2,366-371.
KSIGHT BEEEDIS,
C., BER~I~K,
(1962). Fractionation in cesi[lm chloride 17, 81-94.
L., and ANDEIISOX, T. F. of Shope papilloma virus density gradients. T’iloloyy
W. I~., alld BEARD, J. W. (1941). Studies on the purification and properties of the rabbitpapilloma-virrls-protein. J. Ad7atl. Cancer Inst. 1, 607-673. CAARG.\FF, E. (1955). Isolation and composition of the deoxypentose nucleic acids and of t,he corresponding nucleoproteins. In “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. I, pp. 307-371. Academic Press, New York. CU~P~RD, L. V., and CIU~FORD, E. RI. (1963). B comparative study of polyoma and papilloma viruses. Virology 21, 258-263. FRAENKEL-CONRAT, H., HARRIS, J. I., and LEVY, A. I. (1955). Recent developments in techniques for terminal and sequence studies in peptides and proteins. In “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. II, pp. 359425. Wiley (Interscience), New York. GOODWIN, T. W., and MORTON, R. A. (1946). The spectrophotometric determination of tyrosine and tryptophan in proteins. Biochem. J. 40, 628632. GUIDOTTI, G. (1960). The action of carboxypeptidases A and B on the separated (Y and p chains of normal adult human hemoglobin. Biochim. Biophys. Acta 42, 177-179. IKAWA, M., and SNELL, E. E. (1961). Artifact production through esterification of glutamic acid during analytical procedures. J. Biol. Chem. 236, 1955-1959. ITO, Y. (1960). A tumor-producing factor extracted by phenol from papillomatous tissue (Shope) of cottontail rabbits. vi’irology 12, 596-601. KASS, S. J. (1964). Structure and chemical composition of Shope papilloma virus. Thesis (Ph.D.), University of California, Berkeley. KLEINSCHMIDT, A. K., KASS, S. J., WILLIAMS, R. C. A. (1965). Contour Lengths C., and KNIGHT, of cyclic DNA of Shope papilloma virus. J. Mol. BioZ. 13, in press. KNIGHT, C. A. (1946). Precipitin reactions of highly purified influenza viruses and related materials. J. &ptZ. Med. 83, 281-294. KNIGHT, C. A. (1950). Amino acids of the Shope papilloma virus. Proc. Sot. Exptl. Biol. Med. 75, 843-846. KNIGHT, C. A. (1963). Chemistry of viruses. Protoplasmat~logia IV, 2, 1-177. LANGRIDGE, R., WILSON, H. R., HOOPER, C. W., and WILKINS, M. H. F. (1960). The molecular configuration of deoxyribonucleic acid. I. X-ray diffraction study of a crystalline form of the lithium salt. J. Mel. Biol. 2, 1937. LEBOUVIER, G. L. (1957). Poliovirus precipitins; B~ty.\x,
PURIFICATION
AND
CHEMICAL
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