Pattern of Protein Synthesis in Monkey Cells Infected by Simian Virus 40

After infection of several permanent monkey cell lines by simian virus 40 (SV40), four additional protein bands can be detected by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell extracts. These bands appear only after the onset of viral deoxyribonucleic acid (DNA) synthesis, and inhibitors of DNA synthesis prevent their appearance. Three of them correspond to three previously identified capsid components, VP1, VP2, and VP3. The fourth protein band, which does not correspond to a previously identified virion component, is induced by SV40 infection of CV-1 and BSC-1 cultures but not by infection of MA-134 cultures.     

Cleavage of Poliovirus-Specific Polypeptide Aggregates

Zonal electrophoresis resolves two aggregates of poliovirus type 2 cytoplasmic polypeptides. The more negatively charged aggregate contains mainly noncapsid viral-specific polypeptides (NCVP) 2 and x, whereas the other consists of the capsid polypeptides (VP) 0, 1, 2, and 3 (VP0, VP1, VP2, VP3). After treatment with sodium deoxycholate (DOC), the aggregates sediment at 5 to 6S. Their electrophoretic mobilities are unaffected by DOC or RNase. The capsid polypeptide aggregate is similar in mobility to virions but can be converted to a faster electrophoretic form, resembling empty capsids, by heating. If infected HeLa cells are allowed to synthesize poliovirus polypeptides in the presence of iodoacetamide, no capsid polypeptides are produced, but rather NCVP1a (the precursor to capsid polypeptides) is accumulated, along with NCVP2 and NCVPx. When analyzed by electrophoresis and centrifugation, uncleaved NCVP1a migrates with the NCVP2-x aggregate. NCVP1a can be cleaved to capsid-like polypeptides in vitro by using extracts of infected cells, but not uninfected cells, indicating either a virus-specified protease or a cellular enzyme activated during infection. After cleavage of NCVP1a by infected cell extracts, the capsid polypeptides which are produced dissociate from the NCVP2-x complex.     

Amino acid compositions of simian virus 40 structural proteins

The structural proteins of purified SV40 particles were isolated by preparative polyacrylamide gel electrophoresis and the amino acid composition of each protein was obtained. The amino acid composition of VP1 (the major coat protein) was significantly different to that of VP3 (the capsid protein most closely associated with SV40 DNA). The amino acid compositions of VP4, VP5 and VP6 indicated that these proteins were not exclusively histones.     

Proteins in intracellular simian virus 40 nucleoportein complexes: comparison with simian virus 40 core proteins.

Intracellular nucleoprotein complexes containing SV40 supercoiled DNA were purified from cell lysates by chromatography on hydroxyapatite columns followed by velocity sedimentation through sucrose gradients. The major protein components from purified complexes were identified as histone-like proteins. When analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, complex proteins comigrated with viral core polypeptides VP4, VP5, VP6, and VP7. (3H) tryptophan was not detected in polypeptides from intracellular complexes or in the histone components from purified SV40 virus. However, a large amount of (3H) tryptophan was found in the viral polypeptide VP3 relative to that incorporated into the capsid polypeptides VP1 and VP2. Intracellular complexes contain 30 to 40% more protein than viral cores prepared by alkali dissociation of intact virus, but when complexes were exposed to the same alkaline conditions, protein also was removed from complexes and they subsequently co-sedimented with and had the same buoyant density as viral cores. The composition and physical similarities of nucleoprotein complex and viral cores indicate that complexes may have a role in the assembly of virions.     

The abundant nuclear enzyme PARP participates in the life cycle of simian virus 40 and is stimulated by minor capsid protein VP3

The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) functions in DNA damage surveillance and repair and at the decision between apoptosis and necrosis. Here we show that PARP binds to simian virus 40 (SV40) capsid proteins VP1 and VP3. Furthermore, its enzymatic activity is stimulated by VP3 but not by VP1. Experiments with purified mutant proteins demonstrated that the PARP binding domain in VP3 is localized to the 35 carboxy-terminal amino acids, while a larger peptide of 49 amino acids was required for full stimulation of its activity. The addition of 3-aminobenzamide (3-AB), a known competitive inhibitor of PARP, demonstrated that PARP participates in the SV40 life cycle. The titer of SV40 propagated on CV-1 cells was reduced by 3-AB in a dose-dependent manner. Additional experiments showed that 3-AB did not affect viral DNA replication or capsid protein production. PARP did not modify the viral capsid proteins in in vitro poly(ADP-ribosylation) assays, implying that it does not affect SV40 infectivity. On the other hand, it greatly reduced the magnitude of the host cytopathic effects, a hallmark of SV40 infection. Additional experiments suggested that the stimulation of PARP activity by VP3 leads the infected cell to a necrotic pathway, characterized by the loss of membrane integrity, thus facilitating the release of mature SV40 virions from the cells. Our studies identified a novel function of the minor capsid protein VP3 in the recruitment of PARP for the SV40 lytic process.     

Common structural antigen of papovaviruses of the simian virus 40-polyoma subgroup.

An antigenic determinant common to the major capsid polypeptide (VP1) of simian virus 40 (SV40) and polyoma virus is described. Antisera prepared against intact viral particles reacted only with cells infected with the homologous virus by immunofluorescence tests (IF). However, antisera prepared against disrupted SV40 particles reacted in IF with both polyoma- and SV40-infected permissive cells. The cross-reaction with polyoma was localized to VP1 by the following evidence. (i) The IF cross-reaction was inhibited by preincubation of the antiserum with purified SV40 VP1; (ii) purified radiolabeled polyoma VP1 was precipitated by the cross-reactive serum, and this reaction was inhibited by unlabeled SV40 VP1; (iii) other antisera prepared against purified SV40 VP1 or polyoma VP1 reacted in IF with both SV40- and polyma-infected permissive cells. These cross-reacting antisera also reacted in IF with permissive cells infected with BK virus, rabbit kidney vacuolating virus, and the stumptailed macaque virus, suggesting that all members of the polyoma-SV40 subgroup share a common antigenic determinant located in their major capsid polypeptides.     

Immunological detection of specific proteins in total cell extracts by fractionation in gels and transfer to diazophenylthioether paper

We describe a sensitive immunological procedure for the detection of specific proteins in total cell extracts and for the comparison of antigenically related polypeptides. Proteins are fractionated in polyacrylamide gels and transferred electrophoretically to diazophenylthioether paper, to which they bind covalently. Specific proteins are identified by incubation with specific antibody and 125 I-labeled protein A from Staphylococcus aureus, followed by autoradiography. High-resolution separation of proteins prior to transfer is achieved by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate or by nonequilibrium pH gradient electrophoresis, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Further information can be obtained by limited enzymatic proteolysis of the proteins in the gel following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by gel electrophoresis at right angles to the first gel. We show the application of this technique to the detection and comparison in extracts from infected cells of proteins related immunologically to the simian virus 40 capsid proteins VP1 and VP3.     

Engineering of SV40-based nano-capsules for delivery of heterologous proteins as fusions with the minor capsid proteins VP2/3

The capsid of SV40 is regarded as a potential nano-capsule for delivery of biologically active materials. The SV40 capsid is composed of 72 pentamers of the VP1 major capsid protein and 72 copies of the minor coat proteins VP2/3. We have previously demonstrated that, when expressed in insect Sf9 cells by the baculovirus system, VP1 self-assembles into virus-like particles (VP1-VLPs), which are morphologically indistinguishable from the SV40 virion and can be easily purified. Here, we show that heterologous proteins fused to VP2/3 can be efficiently incorporated into the VP1-VLPs. Using EGFP as a model protein, we have optimized this encapsulation system and found that fusion to the C-terminus of VP2/3 is preferable and that the C-terminal VP1-interaction domain of VP2/3 is sufficient for incorporation into VLPs. The VLPs encapsulating EGFP retain the ability to attach to the cell surface and enter the cells. Using this system, we have encapsulated yeast cytosine deaminase (yCD), a prodrug-modifying enzyme that converts 5-fluorocytosine to 5-fluorouracil, into VLPs. When CV-1 cells are challenged by the yCD-encapsulating VLPs, they become sensitive to 5-fluorocytosine-induced cell death. Therefore, proteins of interest can be encapsulated in VP1-VLPs by fusion to VP2/3 and successfully delivered to cells.     

Structural Proteins of Simian Virus 40

Sodium dodecyl sulfate acrylamide gel electrophoresis of the solubilized proteins from purified simian virus 40 (SV40) virions revealed two major and two minor structural polypeptide components. The major components which comprise over 75% of the total virion were shown to be the capsid proteins by immunological and isoelectric focusing fractionation analysis. These two polypeptides have estimated molecular weights of 45,000 daltons as determined by gel electrophoresis. One of the two minor components was identified as the nucleocapsid protein and has an approximate molecular weight of 16,000. The other unidentified minor component has an average molecular weight of 29,000.     

Structural proteins of simian virus 40. I. Histone characteristics of low-molecular-weight polypeptides.

The DNA-associated polypeptides of simian virus 40 (SV40), VP4 (mol wt 14,000), VP5 (mol wt 12,000), and VP6 (mol wt 11,000), have several properties characteristic of cell histones. After extraction from purified SV40 with dilute acids, these three polypeptides co-electrophoresed on low pH polyacrylamide gels with monkey-kidney cell histones F3, F2b, and F2a1. No virus polypeptide co-electrophoresed with histone F1. Polypeptides VP4, 5, and 6 lacked tryptophan, and only VP4 contained cysteine, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis of virus labeled in vivo with (3H)lysine and either (14C)tryptophan or (35S)cystine. All of the capsid polypeptides VP1, 2, and 3 contained tryptophan whereas only VP1 and 2 contained cysteine. In addition, VP4, 5, and 6 are rich in arginine and lysine when compared with virus labeled with a mixture of amino acids. Analysis of virus grown in cells labeled prior to infection showed that VP4, 5 and 6 were labeled fivefold greater than the major capsid polypeptide, VP1, which indicates that they were partially derived from preexisting cell histones. Based on these data and on previously determined molecular weight estimates, we conclude that VP4, 5, and 6 are histones F3, F2b, and F2a1, respectively, although the possibility that SV40 contains a small amount of F2a2 could not be excluded.     

Resolution of simian virus 40 proteins in whole cell extracts by two-dimensional electrophoresis: heterogeneity of the major capsid protein.

The major capsid protein (VP1) of simian virus 40 (SV40) has been analyzed by two-dimensional electrophoresis. This system separates protein according to isoelectric point by isoelectric-focusing, and according to molecular weight by sodium dodecylsulphate electrophoresis (O'Farrell, 1975). VP1 synthesis in infected CV-1 cells can be monitored directly by analysis of unfractionated whole cell extracts; the resolution of VP1 from cellular proteins allows its detection as early as 13 hr after infection. The two-dimensional separation of VP1 reveals that it is heterogeneous, consisting of one major protein (molecular weight 47,000 daltons and isoelectric point of approximately pH 6.8) and five minor protein components. The minor forms of VP1 are 10% of the total VP1 and differ from the major form of VP1 both in molecular weight (by approximately 500 daltons) and isoelectric point (ranging from approximately pH 6.7 to pH 6.9). Evidence is presented to show that two of the minor forms are phosphorylated derivatives of VP1, and it is further suggested that all the different forms of VP1 are the result of modifications of the primary product of translation. A temperature-sensitive mutant of the BC complementation group (BC11) of SV40 results in the synthesis of VP1 with an altered electrophoretic mobility; both the major form of VP1 and the minor forms are shifted in their isoelectric points. In addition to the specific case of SV40, two aspects of these studies should be generally significant to investigators studying eucaryotic gene expression by two-dimensional gel electrophoresis: first, the genetic origin of a protein can be determined by a temperature-sensitive mutation which causes a charge change in the resultant protein; and second, two or more protein spots on a two-dimensional separation may be the products of a single gene.     

Cell-free synthesis of polyoma virus capsid proteins VP1 and VP2.

Polyadenylated RNA isolated from the cytoplasm of mouse 3T6 cells 28 h after infection with polyoma virus has been isolated and translated in vitro. Polyoma capsid proteins VP1 and VP2 have been identified in the cell-free product by polyacrylamide gel electrophoresis, specific immunoprecipitation, and tryptic peptide fingerprinting. Polyoma mRNA species have been isolated by preparative hybridization to purified viral DNA immobilized on cellulose nitrate filters and shown to code for both VP1 and VP2. These experiments establish conditions for the isolation of late polyoma mRNA and the cell-free synthesis of polyoma capsid proteins and indicate that the active mRNA species are at least partially virus coded.     

Simian virus 40 host range/helper function mutations cause multiple defects in viral late gene expression.

Simian virus 40 (SV40) deletion mutants dlA2459 and dlA2475 express T antigens that lack the normal carboxy terminus. These mutants are called host range/helper function (hr/hf) mutants because they form plaques at 37 degrees C on BSC-1 and Vero monkey kidney cell lines but not on CV-1p monkey kidney cells. Wild-type SV40 can provide a helper function to permit growth of human adenoviruses in monkey kidney cells; the hr/hf mutants cannot. Progeny yields of hr/hf mutants are also cold sensitive in all cell lines tested. Patterns of viral macromolecular synthesis in three cell lines (Vero, BSC-1, and CV-1) at three temperatures (40, 37, and 32 degrees C) were examined to determine the nature of the growth defect of hr/hf mutants. Mutant viral DNA replication was similar to that of the wild type in all three cell lines, indicating that the mutations affect late events in the viral lytic cycle. In mutant-infected Vero cells, in which viral yields were highest, late mRNA levels were similar to those observed during wild-type infection. Levels of viral late mRNA from mutant-infected CV-1 and BSC-1 cells at 32 and 37 degrees C were reduced relative to those of wild-type-infected cells. The steady-state level of the major viral capsid protein, VP1, in mutant-infected CV-1 cells was reduced to the same extent as was late mRNA. The synthesis of agnoprotein could not be detected in mutant-infected CV-1 cells but was readily detected in CV-1 cells infected by wild-type SV40. Primer extension analyses indicated that most late mRNAs from mutant-infected CV-1 cells utilize start sites downstream from the major wild-type cap site (nucleotide 325) and the agnoprotein initiation codon (nucleotide 335). These results indicate that deletion of the carboxyl-terminal domain of T antigen affects viral late mRNA production, both quantitatively and qualitatively. The agnoprotein is detected late in the wild-type SV40 lytic cycle and is thought to play a role in the assembly or maturation of virions. Reduced hr/hf progeny yields could result from decreased capsid protein synthesis and, in the absence of detectable levels of agnoprotein, from inefficient use of available capsid proteins.     

Respiratory syncytial virus proteins: identification by immunoprecipitation.

The proteins of respiratory syncytial virus have not been clearly identified due to the lability of the virus and difficulties in its purification. We have pulse-labeled respiratory syncytial virus with [35S]methionine and [35S]cysteine and analyzed cell lysates by polyacrylamide gel electrophoresis. Five 35S-labeled viral proteins ranging in molecular weight from 21,000 to 73,000 (VP73, VP44, VP35, VP28, and VP21) were easily discernable above background cellular proteins. Treatment of the infected cells with 0.15 M NaCl before labeling suppressed host cell protein synthesis and allowed clearer visualization of the five viral proteins by polyacrylamide gel electrophoresis. Three glycoproteins (VGP 92, VGP 50, and VGP 17) were also identified after labeling with [3H]glucosamine. Five of these polypeptides (VP51, VP44, VP35, VP28, and VGP92) were shown to be antigenically active because they could be immunoprecipitated with anti-respiratory syncytial virus antibody produced in New Zealand white rabbits, cotton rats, and humans before analysis by polyacrylamide gel electrophoresis.     

Characterization of cricket paralysis virus-induced polypeptides in Drosophila cells

Cricket paralysis virus purified from Galleria mellonella larvae was shown to be similar to virus purified from Drosophila melanogaster cells. Cricket paralysis virus contained three major structural polypeptides of similar molecular weight (around 30,000), had a buoyant density of 1.344 g/ml, and had a capsid diameter of 27 nm. Twenty virus-induced polypeptides could be detected in CrPV-infected Drosophila cells. Two major polypeptides found in the infected cells corresponded to two structural viral polypeptides (VP1 and VP3), whereas the third major intracellular polypeptide was the apparent precursor of the third viral structural polypeptide (VP2). Three of the primary virus-induced polypeptides had molecular weights of 144,000, 124,000, and 115,000. These and other polypeptides were chased into lower-molecular-weight proteins when excess cold methionine was added after a short [³⁵S]methionine pulse. Although cricket paralysis virus has a number of characteristics in common with the mammalian enteroviruses, the extremely fast processing of high-molecular-weight polypeptides into viral proteins seems atypical. Also, no VP4 (8,000 to 10,000 molecular weight) has been found in the virus particles.     

Structural Polypeptides of Simian Virus 40

To determine the number and molecular weights of the structural polypeptides of simian virus 40, we have analyzed purified virus by electrophoresis on 14% polyacrylamide gels containing sodium dodecyl sulfate. Full virus purified by several different methods showed six distinct bands with molecular weights of approximately 43,000 (VP1, containing 70% of virion protein), 32,000 (VP2, 9%), 23,000 (VP3, 10%), 14,000 (VP4, 6%), 12,500 (VP5, 4%), and 11,000 (VP6, 3%) both by analysis of radioactively labeled virions and by visualization of the polypeptide bands after staining. “Empty” virions contain decreased amounts of VP4, 5, and 6. The approximate molecular ratios of the polypeptides were 6.0, 1.0, 1.5, 1.5, 1.1, and 1.0. When virus degraded in an alkaline buffer was analyzed by velocity centrifugation in sucrose gradients, the two larger polypeptides (VP1 and VP2) remained at the top of the gradient, whereas the three smallest polypeptides (VP4, 5, and 6) sedimented as a complex with the viral deoxyribonucleic acid. VP3 was found in association with either VP1 and 2 or VP4, 5, and 6, depending on the conditions of degradation. Presumably, VP1 and VP2, comprising about 80% of the protein, form the capsid of the virus. VP4, 5, and 6 may form a nucleoprotein in the virion, and VP3 may serve as an intermediate structural component.     

Intracellular synthesis of measles virus-specified polypeptides.

The intracellular synthesis of measles-specified polypeptides was examined by means of polyacrylamide gel electrophoresis of cell extracts. Since measles virus does not efficiently shut off host-cell protein synthesis, high multiplicities of infection were used to enable viral polypeptides to be detected against the high background of cellular protein synthesis. The cytoplasm of infected cells contained viral structural polypeptides with estimated molecular weights of 200,000, 80,000, 70,000, 60,000, 41,000, and 37,000. All of these structural polypeptides, with the exception of P1, the only virion glycoprotein (molecular weight congruent to 80,000), were also found in the nuclei. In addition, two nonstructural polypeptides with estimated molecular weights of 74,000 and 72,000 were also present in the cytoplasm of infected cells. The initial synthesis of the smaller, nonstructural polypeptide began later in infection than the structural polypeptides. Pulse-chase experiments failed to detect any precursor-product relationships. The intracellular glycosylation and phosphorylation of the viral polypeptides were found to be similar to those found in purified virions.     

Simian virus 40 chromatin interaction with the capsid proteins

It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin. We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin. To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB. (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1). We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40 degrees C) and thus, share characteristics in common. For example, the tsC mutants accumulate only the 75 S chromatin. Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100-160 S and contain all the three capsid proteins VP1, VP2, and VP3. These nucleoproteins can be distinguished morphologically, however. Under the electron microscope, the tsBC 100-160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions. In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40 degrees C, in agreement with genetic analysis. Our observations favor the hypothesis that the VP1 protein contains three discrete domains. We speculate that each domain may play a specific function in SV40 assembly. To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT. In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA. This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.     

Structural organization of human simian virus 40 chromosomes. I. Heterogeneity and composition of nuclear DNA-protein complexes at different stages of lytic infection

Extraction of the purified nuclei of SV40 infected cells reveals a heterogeneous set of viral DNA-protein complexes. Earlier, the authors have shown the possibility of nuclear particles extraction being indistinguishable from mature SV40 virions. In the present work, structural intermediates of virus maturation from free minichromosomes through replicative complexes to immature virion particles have been analyzed. The fractionation of viral complexes by non-denaturing agarose gel electrophoresis has been employed. The protein composition of the complexes as determined by two-dimensional gel electrophoresis indicates that five histone fractions including H1 are present during minichromosome maturation to the chromosome of the mature virion.