VIROLOGY 68, 245-255

(1975)

Decapsidation

of Polyoma Subviral

ERIC FROST’ Dhpartement

de Microbiologic.

AND

Virus:

Identification

of

Species1 PIERRE

BOURGAUX3

Centre Hospitalier Uniuersitaire, Uniuersit6 Qutibec, Canada Jl H 5N4

de Sherbrooke,

Sherbrooke,

Accepted June 20, 1975 Mouse embryo cells that had been exposed to polyoma virus (Py) labeled in its protein and/or DNA moiety were fractionated at various times after infection. Sedimentation analysis of the cytoplasm in 1 M NaCl solution revealed the presence of a labeled viral DNA-protein complex, termed NP, which is absent from virus preparations used for infection. This complex has a sedimentation coefficient of about 135 S and a DNA/protein ratio higher than that of virus, reflecting primarily loss of histone-like polypeptides. Another newly recognized species, a DNA-free complex (DFC) sedimenting at about 185 S, was identified in the cytoplasm. It contains reduced amounts of histone-like polypeptides and little or no DNA, as compared with virus. Unlike the material sedimenting like “full” virus particles which was also found in the cytoplasm, neither NP or DFC was stable in 2.7 M CsCl.Histone-likepolypeptides lost from the above-mentioned species were detected in the cytoplasm as slowly sedimenting material. Radioactive material sedimenting as free DNA in 1 M NaCl solution was recovered from the nucleus and, occasionally, from the cytoplasm. It never represented more than 1.5% of the labeled DNA molecules present in virus at the time of infection. We propose a scheme for the decapsidation of Py in the cell. As the viron meanders in the cytoplasm, histone-like polypeptides are lost and NP is generated. The ensuing step is transfer of DNA to the nucleus. with a simultaneous reorganization of the capsid proteins into DFC. The viral DNA in the nucleus presumably is instrumental in directing the infectious process.

(Fraser and Crawford 1965; Mattern et al., 1966), as with most other viruses (Dales, 1973), is viropexis. Engulfed singly or in groups in cytoplasmic vacuoles, Py meanders in the cytoplasm, while infecting particles are never seen in the nucleus (Fraser and Crawford, 1965; Mattern et al., 1966). Autoradiographic studies have also shown that the DNA of Py, but very little or none of its protein, entered the nucleus (Khare and Consigli, 1965). It thus appeared likely that uncoating of Py takes place in the cytoplasm. Rather surprisingly, the very similar papovavirus simian virus 40 (SV40), has been reported to have quite a different fate (Barbanti-Brodano et al., 1970; Hummeler et al., 1970). It was found that most of the inoculum does not enter the cell but re-

INTRODUCTION

Much attention has been devoted to the replication of polyoma virus (Py) and to the role of the viral genes in transformation (Benjamin, 1972; Eckhart, 1972; Sambrook, 1972). In contrast, little is known about how the virus invades the cell, and particularly the nucleus, where its DNA replicates (Frost and Bourgaux, 1973). The principal means of entry of Py into the cell ‘This work was supported by the Medical Research Council of Canada. It is part of a thesis submitted by E. F. in partial fulfillment of the requirements for a Ph.D. degree. *Research Fellow of the National Cancer Institute of Canada. Present address: MRC Experimental Virus Research Unit, Institute of Virology, University of Glasgow, Glasgow, Scotland GII 5JR. 3 To whom all correspondence should be addressed. 245 CoppriEhtO 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

246

FROST AND ROUHGAIX

mains attached to the cytoplasmic membrane. Subsequently, transfer to the nucleus is very rapid, intact virions being present in the nucleus 1 hr after infection. Finally, uncoating proceeds in the nucleus until complete dissociation of the viral genome from the protein coat is accomplished. Viral material, which could represent uncoating intermediates of SV40. is found with a buoyant density intermediate between that of virions and of free DNA. No similar subviral species have been reported for Py. In order to further our understanding of the early events in the Py-cell interaction, we addressed ourselves to the problem of uncoating and transfer to the nucleus of Py labeled both in its protein and nucleic acid. Studies on the fate of doubly labeled preparations of vaccinia virus (Sarov and Joklik, 1972) and reovirus (Chang and Zweerink, 1971; Silverstein et al., 1972) have revealed several uncoating intermediates for which the identification of viral polypeptides was possible. The principal hindrance to such studies has been the lack of radioactive virus of sufficiently high specific activity. One of us (Frost, 1975; Frost, manuscript in preparation) has recently developed an iodination procedure whereby Py can be labeled in its protein moiety to high specific activity, with no changes in its biological or physical properties. Using virus labeled by this procedure, we were able to monitor the intracellular fate of virus after infection of mouse cells at a relatively low input multiplicity, similar or even inferior to those generally required for the study of virus replication. In conclusion to our experiments, we present a model for Py decapsidation in mouse embryo cells. The important feature of this model is the transfer of free DNA to the nucleus via a newly recognized subviral particle. MATERIALS

AND METHODS

Virus. Py was grown in secondary cultures of whole mouse embryos as already described (Frost and Bourgaux, 1975), except that infected cultures were incubated at 37” for 1 week before virus harvest. It was purified from receptor-destroying enzyme extracts (Crawford, 1962) by Freon

extraction (Girardi. 1959), treatment with ribonuclease, deoxyribonuclease and trypsin, followed by banding in buoyant CsCl solution (Thorne and Wardle, 1973). The purified virus was pelleted and finally suspended in phosphate-buffered saline (PBS) containing 0.1%’ bovine serum albumin (BSA). Labeling with 32P. After infection, cell monolayers in rolling bottles were first washed in phosphate-free medium, and then incubated twice for at least 1 hr in the same medium to remove residual phosphate. Next, each bottle received 2-3 mCi of carrier-free 32P (ICN) in 50-75 ml of fresh phosphate-free medium supplemented with antibiotics and 2.5% serum. After 1 week, labeled virus was harvested and purified as described above. Iodination. The purified virus (0.05 ml) was added to a mixture containing 10 ~1 each of: 1 M Na phosphate buffer, pH 6.9; 0.1 N HCl; carrier-free ‘Y (200-500 PCi, ICN) in 0.1 N NaOH. Iodination was initiated by addition of 4 pg of chloramine T. After 30-45 set at room temperature, the reaction was stopped with 12.5 pg of K,S,O,. Labeled virus was separated from unreacted iodine and iodine-labeled BSA by velocity centrifugation in a sucrose solution (see below) containing 0.1 M rather than 1 M NaCl. The peak of labeled material sedimenting as expected for “full” virus particles was collected and diluted in Dulbecco’s modified Eagle’s medium, buffered with HEPES N-2-hydroxyethylpiperazine-l\r’-2-ethanesulfonate, GIBCO. In the latter medium, the concentration of Ca2+ and Mg2+ was doubled to account for the chelating effect of the EDTA from the sucrose solution. The preparation was used for cell infection within the first 24 hr after labeling. Infection with labeled virus. Confluent monolayers of secondary mouse embryo cells in petri dishes were infected at a multiplicity of l-10 PFU of virus/cell. After incubation for 90 min at 37”, the inoculum was removed. The cells were then washed and reincubated with the preinfection medium or with fresh medium containing 1% serum. Cell fractionation. Cytoplasmic extract: The cells were collected and disrupted

DECAPSIDATION

using Tween 80 as already described (Frost and Bourgaux, 1973). This detergent was employed, because even at a concentration tenfold higher than that used for cell fractionation it had been found to exert little or no effect on the physical properties of purified virus (Frost, 1975). Briefly. the cell monolayer, representing 3-5 x lo6 cells, was washed with distilled water, scraped in 1 ml of TET (0.1% Tween 80. 0.01 M EDTA, 0.01 M Tris-Hcl, pH 7.4) and transferred to a centrifuge tube. After shaking for 1 min on a Vortex Junior mixer, the nuclei were sedimented at 2400 rpm for 4 min in a refrigerated Sorvall HG-4 centrifuge. The supernatant fluid, termed cytoplasmic extract, contained less than 5% of the newly synthesized viral DNA (Frost and Bourgaux, 1973). Distribution of pulse-labeled protein and RNA indicated recovery of 90-95% of the cytoplasmic material in this fraction (Frost, 1973). Nuclear fractions: The pelleted nuclei, having a normal morphology and being surrounded by both nuclear membranes (Frost, 1973), were washed with either TET or a Triton X-100 solution (1% Triton X-100, 0.1 M KCl, 0.01 M Mg acetate, 0.01 M Tris-HCl, pH 7.0) yielding a nuclear wash. These washed nuclei were broken with Carborundum (Frost and Bourgaux, 1973) and their debris washed in succession with TET and with a solution containing 0.25% Na deoxycholate (DOC), 0.02 M EDTA, 0.02 M Tris-HCl, pH 8.6. The washes, separated from the nuclear debris by clarification for 15 min at 4500 rpm, were termed nuclear debris wash 1 and nuclear debris wash 2, respectively. The pellet obtained after the second wash was treated with Na sarcosinate and Pronase (Frost and Bourgaux, 1973). Centrifugations. Sucrose gradient velocity sedimentation was performed as follows. The sample, representing one-fortieth of the liquid column, was layered over a 5-20% linear sucrose gradient (1 M NaCl, 0.001 M EDTA, 0.01 M Tris-HCl, pH 7.4), formed on top of a O.l-ml cushion (56% CsCl, 25% sucrose, 0.02 M EDTA, 0.02 M Tris-HCl, pH 7.4). Centrifugation was performed in an International B60 ultracentrifuge for either 48 min at 35,000 rpm (SB405 rotor) or 72 min at 35,000 rpm(SB283 rotor).

247

OF POLYOMA

Density gradient centrifugation was performed as follows. The sample was layered over a CsCl solution (0.02 M EDTA, 0.02 M Tris-HCl, pH 7.4) of density 1.35 g/ml containing 0.03% BSA. Centrifugation was for 16 hr at 30,000 rpm in an SB405 rotor. For all sedimentations, fractions were collected dropwise from the bottom of the tube.

Polyacrylamide-gel electrophoresis (PAGE). Samples were dialyzed against 0.001 M Na Phosphate buffer, pH 6.9, and concentrated by blowing air past the dialysis tubing. They were dissociated by boiling for at least 2 min in 2% sodium dodecyl sulfate (SDS), 2% mercaptoethanol. Phenol red, used as a tracking dye, and 57~ glycerol were added after cooling. Samples were then analyzed on &cm-long 10% polyacrylamide gels, as described for the SDSphosphate system (Maizel, 1971). After electrophoresis, the gels were frozen at -90” and cut with a Hoefer gel slicer. The dried slices were counted in a Nuclear Chicago gamma counter. RESULTS

Properties

of

Iodinated Virus

Although the characteristics of iodinated virus are described in greater detail elsewhere (Frost, 1975; Frost, manuscript in we summarize here those preparation), that are considered essential. The virus used in this work sedimented as one radioactive peak, coinciding exactly with marker virus, in sucrose and CsCl gradients. As a result of iodination, its hemagglutinating activity was unchanged, while over 70% of its infectivity was retained. More important, comparing the intracellular fate of 32P-labeled virus to that of the same virus after iodination suggested that iodination does not affect uncoating. The histone-like polypeptides (VP4-6) were proportionally more effectively labeled (see, for instance, Fig. 4, left) by our iodination techniques than by the usual technique of in vivo incorporation of radioactive amino acids (Roblin et al., 1971). This peculiarity of iodinated virus turned out to be advantageous, as it facilitated quantitation of some of the minor virus

248

FROST

AND BOURGAUX

constituents. The ratio of the radioactivity in VP4-6 to that in the major capsid polypeptide (VPl), however, varied somewhat from one iodinated preparation to the other, while it was found to be relativel!, constant in otherwise-labeled Py (Roblin et al., 1971; Gibson, 1974). This variation may reflect the relatively inefficient iodination of VP1 in intact virus already reported for both SV40 (Huang et al., 1972a) and Py (Gibson, 1974). Adsorption In preliminary experiments, the early virus-cell interaction was investigated by comparing the content of the virus preparations used for infection with that of the fluid recovered from the monolayers at the end of the adsorption period. When doubly labeled virus was used, a change in the ratio of 32P to lz51 was observed, indicating in the latter fluid a 20% relative enrichment in lz51-labeled material. PAGE analysis demonstrated that lz51 in VP4-6, when related to lz51 in VPl, was also increased by 20% in the same fluid. Analysis of the virus remaining associated with the cells indicated a corresponding decrease in VP4-6. Although other explanations are possible, these changes in polypeptide composition probably result from the loss of histone-like polypeptides from virus particles entering the cells (see below). Distribution Cells

of

Viral

Label

in Infected

At different times following infection with labeled Py, cells were fractionated and aliquots from the various fractions counted to determine the distribution of the viral proteins and nucleic acid (data not shown). Throughout the course of infection, most of the viral material was found in the cytoplasmic extract. The acidinsoluble radioactivity in this fraction decreased with time, while the acid-soluble radioactivity increased slightly in the cytoplasmic extract and markedly in the medium. Radioactivity in the nuclear fractions generally increased with time until 30 hr and then decreased. In experiments where doubly labeled virus was used, we found no important difference in the distri-

bution of the 32P and lz51 labels. Total recovery of both 32P and lz51 in all cellular fractions and the medium was generally higher than 60%. These observations are consistent with those reported previously for virus labeled with 32P only (Bourgaux, 1964). Zdentification and Characterization toplasmic Components

of Cy-

Cytoplasmic extracts were fractionated by velocity sedimentation through sucrose gradients (Fig. 1). In addition to material pelleting on the cushion (CyO), four, or sometimes five, viral components were arbitrarily identified on the basis of sedimentation coefficient and 32P/1251ratio. Material cosedimenting with marker “full” virus particles was designated Cyl. A slowersedimenting material, Cy2, with a 3zP/1251 ratio smaller than that of Cyl was incompletely separated from Cyl. This component, which appeared as relatively minor in the cytoplasm, was quite prominent in nuclear fractions and sedimented at about 185 S (see below). The third component, Cy3, had a sedimentation coefficient of about 135 S and displayed a high “P/‘*‘I ratio when compared with Cyl. In addition, some iodine-labeled material was consistently found near the top of the gradient (CY4). In some experiments, a clearly distinguishable peak of 32Pactivity, with no corresponding lz51 peak, was found in the cytoplasmic extract (Fig. 1). This material, referred to as D, was always present in the nuclear fractions and occasionally in the cytoplasmic extract. Accordingly, it will be considered with the nuclear components (see below). When components Cyl, Cy2, Cy3 and Cy4 were recentrifuged under identical conditions, they displayed unchanged sedimentation properties. In contrast, CyO yielded material sedimenting as the aforementioned components after treatment with the detergent DOC. This suggests that CyO represents membrane-bound or aggregated viral material. The distribution of the lz51 radioactivity among the various cytoplasmic components was determined in several experiments. Most of the radioactivity was found

DECAPSIDATION

OF POLYOMA

249

density of either full or empty virus particles; most of the label was found in the peak near the top of the gradient. From the velocity and equilibrium sedimentations, we tentatively concluded that, in addition to contaminating full and empty virus particles, Cy2 and Cy3 consisted of subviral species sedimenting at different rates in 1 M NaCl solution, but both were unstable in 2.7 A4 CsCl solution. When doubly labeled virus was used for infection, Cy2 was found to contain little 32P, possibly fraction no. present in the contaminating full virus FIG. 1. Viral components in cytoplasmic extracts particles. In contrast, the 32P/1251ratio was of mouse cells fractionated (see Materials and higher for Cy3 than for Cyl. Presumably, Methods) 24 hr after infection with virus labeled with the subviral species in Cy2 lacked DNA, “P and ‘*‘I. Radioactive profile registered after sediwhile that in Cy3 contained DNA. Hetice, mentation of the cytoplasmic extract through a suthese species were termed DFC (for DNAcrose gradient (1 M NaCl, 0.001 M EDTA, 0.01 M free complex) and NP (for nucleoprotein), Tris-HCl, pH 7.4). Centrifugation was for 72 min at respectively. 35,000 rpm in an SB2E3 rotor of an International PAGE analysis of Cy2 and Cy3 suggested ultracentrifuge. The fractions under the bars were that both contained the polypeptides presnamed as indicated. In a similar centrifugation, marker virus cosedimented with Cyl. Sedimentation ent in virus (or Cyl) but with reduced is from right to left. amounts of VP4-6 (Fig. 2). In 13 or 14 experiments, radioactivity in VP4-6, when in Cyl, while the relative amounts of the related to that in VPl, was lower in Cy3 different species varied little with time than in Cyl; the average reduction and after the first 4 hr of infection. At first, this 95% confidence interval in VP4-6 in all would seem to indicate that none of the determinations was 27.6 & 8.5%. From the components described represents the final 32P/‘251 ratios, we calculated that Cy3 product of the uncoating process. Such a missed some 20% of the 125I radioactivity conclusion might not be valid however, for present in Cyl. Since about 60% of this earlier results (Bourgaux, 1964) have difference was accounted for by the loss of shown that, under such circumstances, high specific activity VP4-6, we estimated input viral material is continuously lost the total mass of polypeptides in NP would from the cytoplasm to both the extracellustill represent 90% or more of that of full lar compartment and the nuclear fraction. virus particles. In seven of eight determiSamples from Cyl, Cy2, and Cy3, la- nations, radioactivity in VP4-6 was also beled with lz51, were mixed with 32P- less in Cy2 than in Cyl, but the average relabeled virus used as a marker and sub- ducticn was only 16.5 + 10.6%. This is not jected to density gradient centrifugation in surprising, for Cy2, unlike Cy3, was ex(see Materials and CsCl solution pected to be markedly contaminated with Methods). For all components, the lz51 Cyl. In contrast, radioactive polypeptides label was recovered as three distinct bands, migrating as VP3 and VP4-6 were found in ranging from the bottom to the top of the increased amounts in Cy4 in 14 experitube: One band with the density of “full” ments (Fig. 2d). When analyzed by PAGE, virus particles, one with the density of both the virus used for infection and CyO “empty” particles, and the third near the appeared rather similar to Cyl, with occatop of the gradient. Radioactivity was very sionally a somewhat higher amount of raprominent in the densest band in the case dioactivity in VP4-6. of Cyl. We may thus conclude that this component is comprised principally of full Analysis of Nuclear Fractions Conceivably, virus particles. In Cy2, and even more so in cytoplasmic Py species Cy3, little label was found to have the could be found in nuclear fractions as a

250

FROST I3

12

“PI

AND BOURGAUX

b

24

/i

were sedimented for times longer than usual, no lz51-labeled material was found to cosediment with D, suggesting the latter component indeed was free of capsid protein (Fig. 4). Yet, D was not extensively degraded, for it sedimented as a mixture of supercoiled and nicked circular Py-DNA. This labeled viral DNA truly represented input parent molecules rather than recycling of label in progeny DNA, as indicated by the following evidence. First, small amounts of D were already present at 4 hr after infection (Fig. 3a and Table l), prior to the onset of viral DNA synthesis. Sec-

4

FIG. 2. PAGE analysis of cytoplasmic components. Viral components, obtained after velocity sedimentation of cytoplasmic extracts (see Fig. I), were boiled for 2 min in dissociating buffer (2%’ SDS, 2% mercaptoethanol, 0.01 M phosphate buffer, pH 6.9). Samples were applied to 10% polyacrylamideeSDSphosphate gels (Maizel, 1971). After electrophoresis the frozen gels were sliced, dried and counted in a gamma counter. The radioactivity profiles are from Cyl (a), Cy2 (b), Cy3 (c) and Cy4 (d). Migration is from left to right.

result of either contamination by cytoplasmic material or in oiuo adsorption of viral material to the nuclear membrane (Fraser and Crawford, 1965; Mattern et al., 1966). Thus, it is not surprising that components with the sedimentation coefficients (Fig. 3) and PAGE profiles of those found in the cytoplasm were found in all nuclear fractions. Component Cy2 (DFC) however was very conspicuous in these fractions, whereas Cy3 (NP) was very much reduced or absent (Fig. 3). This is what would be expected if splitting of NP into DNA and DFC took place, either within the nucleus or in the vicinity of the nuclear membrane. Another conspicuous species in the nuclear fractions was D (Fig. 3), which, as already mentioned, appeared labeled with 32P exclusively and was often absent from the cytoplasm. When nuclear fractions

-i 0

Y ’

E 6 a 0”

3 I

2

4

1

0

I fraction

no.

Fro. 3. Velocity sedimentation analysis of nuclear fractions. TET washes of isolated nuclei (see Materials and Methods), obtained either 4 (a). 28 (b) or 50 (cl hr after infection of mouse cells with doubly labeled virus, were sedimented through sucrose gradients (see Fig. 1). The arrow indicates the position of “full” virus particles (Cyl) in such gradients. Sedimentation was for 48 min at 35,006 rpm in an SB405 rotor (a, c) or for 72 min at 35,000 rpm in an SB283 rotor (b). Sedimentation is from right to left.

DECAPSIDATION

fraction

251

OF POLYOMA

no.

FIG. 4. Free DNA in cellular

extracts. Cells were fractionated (see Materials and Methods) 24 hr after infection with virus labeled with both 32P and ‘psI. “C-labeled Py-DNA (Bourgaux and Bourgaux-Ramoisy, 19’72b) was added to the cytoplasmic extract (left panel) and to the nuclear debris wash 2 (right panel) prior to centrifugation through a sucrose gradient (see Fig. 1) for 84 min at 60,000 rpm in an SB405 rotor. The arrows indicate the position of “C-labeled Py-DNA form I (1) and form II (2) in the gradients. Sedimentation is from right to left.

ond, the amount of D did not increase with time, while progeny viral DNA was synthesized between 24 and 50 hr postinfection (Table 1). Third, little or no 32P label was found in fast-sedimenting, cellular DNA when the cells were harvested 24 hr after infection (Fig. 5). The latter observation also suggested that little of the DNA from the virus used for infection was integrated into the host DNA. The percent of the total radioactivity associated with the cell which was found in the form of free DNA was always less than 6% (Table 1). Assuming adsorption of about 25% of the virus used for infection (Bourgaux, 1964), we concluded that less than 1.5% of the viral DNA molecules used to infect the cells were present in their nuclei at any given time.

notte and A. A. Qureshi, unpublished observations). Second, subviral particles like NP or DFC could not be generated from purified virus either by incubation with cytoplasmic extracts from uninfected cells (unpublished observations) or by treatment with 1 M NaCl and a variety of

DISCUSSION

We report here the detection and partial characterization of subviral species, DFC and NP, produced by Py as it enters the cell. Four lines of evidence indicate that NP and DFC are particles generated during virus uncoating rather than artifactual associations of degraded virus or viral protein with cellular constituents. First, no progeny nucleoprotein complexes could be isolated from productively infected cells which would sediment rapidly in sucrose containing high NaCl concentrations (Green et al., 1971; P. Bourgaux, J. Bur-

fraction

no

FIG. 5. Lack of recycling of 32P label from infecting virus. Cells infected with 32P-labeled virus were labeled from 18 to 24 hr after infection with [3H]thymidine (2 rCi/ml). At 24 hr postinfection, the cells were fractionated (see Materials and Methods). The nuclear debris was digested with Na sarcosinate and Pronase and mixed with ‘“C-labeled supercoiled PgDNA prior to analysis by velocity centrifugation (see Fig. 4). An arrow marks the position at which the “C-labeled DNA was recovered in the gradient. Sedimentation is from right to left.

252

FROST AND BOlJRGAUX

detergents (Frost, 1975; Frost and Bourgaux, manuscript in preparation). Third, the properties we assigned to NP and DFC TABLE

1

PARENT VIRAL DNA IN CELLULAR FRACTIONS’ Time of fractionation (hr) 4 24 50

Percent of 32P as free DNA” Expt 1

Expt 2

Expt 3

1.5 4.2 1.4

2.3 2.4 1.4

2.3 5.6 2.0

“Cells were fractionated (see Materials and Methods) at different times after infection, and both the acid-insoluble and acid-soluble 32P radioactivities were determined for all cell fractions. The latter were analyzed by velocity sedimentation (see Figs. 2 and 5) to determine the percent of radioactivity in free DNA (D). From these percentages and from the total radioactivity in every fraction, the percentage of the total radioactivity associated with the cell present in the form of free DNA was calculated. At all times, over 80% of this free DNA was found in the nuclear fractions. TABLE

do not make them unusual structures. A number of Py-DNA-protein complexes have been studied displaying a range of DNA to protein ratios and sedimentation coefficients (Table 2). Most, including NP and DFC, have been reported to be unstable in concentrated CsCl solution. Last, although they clearly are not virus particles, NP and DFC contain all the polypeptides present in virus. In neither NP nor DFC, were labeled polypeptides identified migrating differently from those of virus and indicating cleavage of structural polypeptides. The formation of these species therefore does not seem to involve extensive proteolytic degradation of the virus. These subviral particles present the interesting property, without precedent (see Table 2), of containing reduced amounts of histone-like polypeptides. Although the latter could clearly have been partially liberated in our extraction conditions from in uiuo subviral species from which NP and DFC would be derived, this loss of histones 2

COMPARISON OF PY-PROTEIN AND PY-DNA-PROTEIN DNA: protein ratio” 1. Virus “Full” particles “Empty” particles 2. Uncoating intermediates NP DFC 3. In vitro subviral complexes Reassembled DNA-protein complex Protein shell derived from it DNA “cores”

l l

25 1

100 50

7

25

3

90 60

>l

we consider these ratios to be 1 for “full”

COMPLEXES

viral particles.

References

Murakami Murakami (1971)

et al. (1968) et al. (1968); Roblin et al.

Friedmann (1971); Friedman11 and David (1972) Friedmann (1971); Friedmann and David (1972) Friedmann and David (1972); McMLllen and Consigli, 1974 Frost (1975) Green et al. (1971); Goldstein et al. (1973); McMillen and Consigli (1974) Bourgaux and Bourgaux-Ramoisy (1972a) Frost and Bourgaux (1973) Seebeck and Weil(l974)

DECAPSIDATION

may be of some significance. Histones are thought to repress transcription of both mammalian chromosomes (Allfrey et al., 1963; Huang and Bonner, 1962) and SV40DNA (Huang et al., 1972b). The loss of VP4-6 from NP could be a preliminary step to the transcription of its DNA. Removal of these polypeptides could also result in conformational changes that would account for the reduced sedimentation coefficient of NP and facilitate the release of its DNA into the nucleus. The labeled polypeptides with the electrophoretic mobility of histones which we found in the form of slow-sedimenting material (in Cy4) may have been liberated from decapsidation intermediates either in uiuo or after extraction. There is some suggestion that the presence of at least some of this material results from an in vivo process. Indeed, Cy4 contains a relatively high amount of labeled material with the mobility of VP3, contrasting with normal or reduced amounts of labeled VP1 and VP2 (see Fig. 2d). Since we found no evidence for the loss of VP3 from any of the rapidly sedimenting subviral components, we feel that this VP3-like material might be cleaved in vivo from VP1 or VP2 or from both (Friedmann, 1974; Gibson, 1974) during or after the release of the latter polypeptides from some of the subviral species. The free Py-DNA we found in the nucleus is likely to be instrumental in continuation of the lytic cycle. Of course, what we call free DNA may actually carry unlabeled cellular or viral protein that does not alter its sedimentation properties or alternatively carry, in uivo, protein that has been removed by the extraction procedure. This free DNA always accounted for less than 1.5% of the DNA in the virus used for infection. Additional viral sequences, although probably in lesser amounts than the free viral DNA, could also have been present in the nucleus, integrated in the cell DNA (Ralph and Colter, 1972). The small amount of DNA reaching the nucleus could have a direct bearing on the high particle to PFU ratio reported for Py (Smith and Benyesh-Melnick, 1961; Crawford, 1969; Fried, 1974). Taking into consideration our data, as

253

OF POLYOMA

well as those of others previously obtained using the electron microscope (Fraser and Crawford, 1965; Mattern et al., 1966), we propose the following model for the fate of Py in mouse embryo cells (Fig. 6). Virus is adsorbed and penetrates the cell membrane by viropexis, losing some VP4-6. This initial loss, which is not sufficient to affect the physical properties of the particle, may involve molecules located at the surface of the capsid. This possibility appears worthy of consideration, since we have observed that histones adsorb effectively to purified polyoma virus and, once bound, cannot be removed completely by conventional purification procedures (Frost and Bourgaux, manuscript in preparation). Further loss of VP4-6, possibly coupled with release of other polypeptides, results in the formation of NP. Electron microscopy has shown that Py particles tend to accumulate in the cytoplasm at the periphery of the nucleus. It is thus likely that some NP would be generated there, and this would facilitate DNA transfer to the cell nucleus. Free DNA would then promote virus replication or cell transformation. Simultaneously, the polypeptides of NP would reorganize into DFC, with a “,,*,one,

: NP ,n ‘C”3’ OFC ,” ‘&q

.:s$ij .‘.:. I.... .:>..y: (.. ... .y.:::. :. t&SC&,

FIG. 6. Proposed decapsidation mouse cells.

membmne

scheme for Py in

254

FROST AND BOlTRGAlTX

corresponding increase in sedimentation coefficient from 135 to 185 S. This is not too surprising, for it has already been shown that an in vitro Py-DNA-protein complex sedimenting at 100 S can be converted, by deoxyribonuclease treatment, to an empty reassembled particle sedimenting at 140-180 S (Friedmann, 1971). As it was observed for these in vitro species, the various alterations in sedimentation coefficient undergone by the viral material during uncoating are likely to be due to conformational changes. Finally, the polypeptides from DFC would be degraded and/or rejected into the medium. Obviously, the model we propose here cannot pretend to be more than a plausible interpretation of our results. Alternative pathways could be constructed between the subviral species we identified. For instance, one could conceive that instead of DFC being generated from NP, both species could derive directly from virus. That NP and DFC would be produced independently however appears somewhat unlikely, considering that a single mutation in the gene(s) coding for the capsid polypeptides appears sufficient to cause a block in the formation of both species (Frost et al., 1975; see below). It would be difficult to argue that NP and DFC are not produced in sufficient amounts in the cell to be seen under the electron microscope. The structure they assume may, however, render their detection difficult. In this respect, it should be recalled that some of the DNA-rich subviral particles of vaccinia virus could be detected only by following the fate of doubly labeled virus (Sarov and Joklik, 1972). Finally, it should be stressed that the results reported here provide suggestive evidence but do not guarantee that the various decapsidation products assume biological significance. As indicated earlier, the intact viral DNA molecules ultimately found in the nucleus represent a fairly small percentage of those initially present in the particles entering the cell. Quite clearly, this suggests that, for instance, most of the input viral material which we detected in the cytoplasm plays

no role in virus replication. However, given the lack of evidence for structural heterogeneity in the population of DNA-containing particles, we have no reason to suspect the existence of distinct pathways of decapsidation, one for the few particles that will turn out to be successful in initiating replication and one for all the other particles. Positive evidence for the biological relevance of the intermediates we described would be still more convincing. This we provide in the accompanying note (Frost et al., 1975), where we present data on the decapsidation of conditional lethal mutants of Py. In the case of one of these mutants, already suspected to be affected in uncoating (Eckhart and Dulbecco, 1974), we conclusively show that the formation of NP and DFC intermediates is impaired. Correspondingly, no viral DNA is found to reach the nucleus. ACKNOWLEDGMENTS We thank Miss Roberte Leblanc and Mr. Andre Lacroix for excellent technical assistance. Dr. R. Sheinin and Dr. E. Bradley are also thanked for helpful comments on the presentation of this work. REFERENCES ALLFREY, V. G., L~ITAL, V. C., and MIHSKY, A. E. (1963). On the role of histones in regulating ribonucleic acid synthesis in the cell nucleus. Proc. Nat. Acad. Sci. USA 49, 414-421. BARRANTI-BRODANO, G., S~ETLY, P., and KOPH~~SKI, H. (1970). Early events in the infection of permissive cells with simian virus 40: Adsorption, penetration, and uncoating. J. Viral. 6, 78-86. BENJAMIN, T. L. (1972). Physiological and genetic studies of polyoma virus. Curr. Top. Microbial. Immunol. 59, 1077133. BOUR~A~.X, P. (1964). The fate of polyoma virus in hamster, mouse, and human cells. Virology 23, 46-55.

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