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.     

Polyoma virus complementary RNA directs the in vitro synthesis of capsid proteins VP1 and VP2.

Polyoma virus complementary RNA, synthesized in vitro by using highly purified Escherichia coli RNA polymerase and nondefective form I polyoma DNA, was translated in a wheat germ cell-free system. Polypeptides were synthesized that comigrated on sodium dodecyl sulfate-polyacrylamide gels with the polyoma capsid proteins VP1 and VP2, although most of the cell-free products were of smaller molecular weights. The VP1-size protein specifically immunoprecipitated with anti-polyoma virus serum, and upon digestion by trypsin yielded [35S]methionine-labeled tryptic peptides that co-chromatographed with the [3H]methionine-labeled tryptic peptides of virion-derived VP1 on both cation-exchange and anion-exchange resins. The VP2-size in vitro product contained all the virion VP2 methionine-labeled tryptic peptides, as shown by cation- and anion-exchange chromatography and two-dimensional fingerprinting on cellulose. We conclude that full-length polyoma VP1 and VP2 are synthesized in response to complementary RNA and consequently that the viral capsid proteins VP1, VP2, and VP3 are entirely virus coded.     

Relationship between the methionine tryptic peptides of simian virus 40 and BK virus tumor antigens.

The monomer form of BK virus (BKV) tumor antigen (T Ag) was immunoprecipitated from extracts of BKV-transformed cells and had a molecular weight of approximately 113,000. This compared with 97,000 for the molecular weight of either BKV or simian virus 40 (SV40) T Ag from lytically infected cells. The SV40 and BKV T Ag's from productively infected cells were compared by examining their methionine-labeled tryptic peptides. Out of a total of 20 SV40-and 21 BKV-specific peptides, there were seven pairs of similar peptides on the basis of ion-exchange chromatography, These coeluting peptides contained approximately 25 to 30% of the total methionine radioactivity. Similar results were obtained when the tryptic peptides of SV40 T Ag from lytically infected cells were compared with those of BKV T Ag from virally transformed cells.     

Identification of simian virus 40 protein A.

A large simian virus 40 (SV40)-specific protein can be efficiently immunoprecipitated from infected cell extracts with antisera obtained from hamsters bearing SV40-induced tumors. The protein has an apparent molecular weight of 88,000 to 100,000 with respect to markers with known molecular weights, but behaves anomalously on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Cell lines infected by two different strains of SV40 synthesize immunoreactive proteins that differ slightly in mobility during SDS-polyacrylamide gel electrophoresis, evidence that the protein is coded for by the virus. These differences in protein size correlate with differences in the electrophoretic mobility of viral DNA fragments obtained by digestion with HindII and III restriction enzymes. The size of the viral capsid proteins VP2 and VP3 also varies with the strain of virus. dl-1001, a constructed deletion mutant that lacks part of the SV40A gene, directs the synthesis of a 33,000-dalton polypeptide that is not detected in cells infected with wild-type virus. The deletion fragment, like the larger protein, is phosphorylated. Maps of tryptic peptides from the 88,000- to 100,000-dalton protein and the 33,000-dalton fragment show common peptides and provide strong direct evidence that the proteins are products of the SV40 A gene. The deletion fragment reacts with antitumor sera and binds to double-stranded DNA in the presence of the complete A protein.     

Extraction and fingerprint analysis of simian virus 40 large and small T-antigens.

A study of simian virus 40 (SV40) T-antigens isolated from productively infected CV1 cells using a variety of different extraction procedures showed that under some conditions the highest molecular weight form of T-Ag (large-T) isolated comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with large-T from SV40-transformed H65-90B cells. Other faster-migrating forms of large-T are probably generated during the extraction procedure by a protease which is active at low pH, and such forms are probably experimental artifacts. After extraction under conditions which minimize proteolytic degradation of large-T, a further form of T-antigen was isolated; this has an apparent molecular weight in the range 15,000 to 20,000 and is referred to as small-t. Fingerprint analysis of [35S]methionine-labeled SV40 proteins showed that small-t has 10 to 12 methionine peptides whereas large-T has 15 to 18 methionine peptides. All but two of the methionine tryptic peptides present in small-t are also present in large-T. The fingerprint data also showed that T-antigens have no peptides in common with SV40 VP1. Experiments using reagents which inhibit posttranslational cleavage of encephalomyocarditis virus polyproteins showed that these reagents do not affect the synthesis of small-t and suggest that it is not made by proteolytic cleavage of large-T in vivo. An alternative model, which proposes that large-T and small-t are synthesized independently, is discussed in terms of the fingerprint data and the number of methionine tryptic peptides predicted from the primary sequence of SV40 DNA.     

Comparison of Two Human Papovaviruses with Simian Virus 40 by Structural Protein and Antigenic Analysis

The proteins of simian virus 40 (SV40) and two human papovaviruses, the hemagglutinating BK virus and the non-hemagglutinating DAR virus, were analyzed and compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The virions of SV40 and DAR contain seven proteins. By molecular weight analysis the constituent proteins of SV40 and DAR are identical. Approximately 84% of the viral protein has a molceular weight of 45,000. The major protein of BK virus is 3,000 to 5,000 daltons lighter than the major proteins of SV40 and DAR viruses. The five most rapidly migrating proteins of BK virus are indistinguishable by molecular weight analysis from the corresponding proteins of SV40 and DAR viruses. Radial immunodiffusion and immunoelectrophoresis of whole virus gave lines of identity between SV40 and DAR when reacted with SV40 antibody. SV40 antiserum tested against BK virus and BK antiserum tested against SV40 virus showed no reactivity by complement fixation, immunodiffusion, or immunoelectrophoresis.     

The primary sequence of the late region of polyoma virus DNA II. The expression of the late genes and comparison with DNA sequences of SV40 and BKV.

The nucleotide sequence of the late region of the polyoma virus genome has been deduced, which codes for the major capsid protein VP1 and the C-terminal region of the minor proteins VP2 and VP3. The amino acid sequence of VP1 predicted from the nucleotide sequence is in good agreement with the partial N-terminal sequence 1 and amino acid composition of VP1 2,3. When both nucleotide and amono acid sequences are compared with their counterparts in the related viruses, SV40 4,5 and BKV (R. Young, personal communication), extensive homologies are found along the entire regions of the viral genes. Maximum homologies appear to occur in the regions which code for the C-terminal of VP1, on the contrary of the result of heteroduplex analysis 6 with 6 with SV40 and polyoma virus DNAs.     

Simian virus 40-specific polypeptides in AD2+ ND1- and Ad2+ ND4-infected cells.

A comparison of the proteins synthesized in human cells at late times after infection with adenovirus (Ad2) and with the adeno-simian virus 40 (SV40) hybrid viruses revealed polypeptides of 30,000 and 92,000 molecular weight specific for the hybrid viruses Ad2+ND1 and Ad2+ND4, respectively. Cell-free translation of SV40-specific mRNA, prepared from these cells by hybridization of total cytoplasmic RNA to SV40 DNA, showed that the mRNA's specifying these two polypeptides were at least partially encoded by the SV40 portion of the hybrid viruses. Cell-free translation of SV40-specific mRNA prepared from monkey cells infected with SV40 produced polypeptides of 40,000, 43,000, 48,500, and 92,000 molecular weight. The SV40 and Ad2+ND4 92,000-molecular-weight polypeptides made in vitro were very similar in electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels to the polypeptide precipitated by Tegtmeyer (1974) with SV40 anti-T serum.     

Correlation between genetic loci and structural differences in the capsid proteins of polyoma virus plaque morphology mutants.

Two plaque morphology variants of polyoma virus (A-2 and 208) showed marked differences in agarose gel electrophoresis of the whole particles, isoelectric focusing of the major capsid protein VP1 (45,000 daltons) and three tryptic peptides (A, B and C) of VP1. No major difference in apparent molecular weight on NaDodSO₄ gels, amino acid composition or carbohydrate detectable by Schiff staining was revealed between the capsid proteins of the two viruses.Correlations have been made between phenotype, portions of the primary amino acid sequence of VP1 and the physical map of polyoma virus DNA by analysis of this protein from large plaque A-2 virus, minute plaque 208 virus and large plaque 208 virus selected after marker rescue with a fragment of polyoma virus DNA generated by the Hpa II restriction enzyme. The interrelationship of these properties was established by taking advantage of the observations of Miller, Cooke and Fried (1976)that heterozygous markers present on heteroduplex DNA are found in 100% of selected progeny and in only 50% of unselected progeny.All five marker rescued isolates selected for large plaque morphology showed only two A-2-specific characters, the absence of peptide C in tryptic maps of VP1 and the aggregation of VP1 on isoelectric focusing. The other four characters which distinguish A-2 and 208 were present or absent in 40–60% of the five isolates, which is close to the expected 50% for unselected markers. Three of the four A-2-specific characters (the presence of peptide A, absence of peptide B and isoelectric point of VP1) have been found to occur coordinately in the marker rescued isolates. The fourth character (electrophoretic mobility of virus particles in agarose gels) segregated independently.The techniques used in this study should find wide application in correlating primary amino acid sequence, nucleotide sequence and phenotype in other systems.     

Gangliosides are receptors for murine polyoma virus and SV40

Polyoma virus (Py) and simian virus 40 (SV40) travel from the plasma membrane to the endoplasmic reticulum (ER) from where they enter the cytosol and then the nucleus to initiate infection. Here we demonstrate that specific gangliosides can serve as plasma membrane receptors for these viruses, GD1a and GT1b for Py and GM1 for SV40. Binding and flotation assays were used to show that addition of these gangliosides to phospholipid vesicles allowed specific binding of the respective viruses. The crystal structure of polyoma VP1 with a sialic acid-containing oligosaccharide was used to derive a model of how the two terminal sugars (sialic acid-alpha2,3-galactose) in one branch of GD1a and GT1b are recognized by the virus. A rat cell line deficient in ganglioside synthesis is poorly infectible by polyoma and SV40, but addition of the appropriate gangliosides greatly facilitates virus uptake, transport to the ER and infection. Lipid binding sites for polyoma are shown to be present in rough ER membranes, suggesting that the virus travel with the ganglioside(s) from the plasma membranes to the ER.     

Comparison of the serology, transforming ability, and polypeptide composition of human papovaviruses isolated from urine.

Four isolates of human papovaviruses (RF, GS, DW, and MG viruses) obtained from the urine specimens of renal allograft recipients in widely separated locations were compared with BK virus. Hemagglutination inhibition tests and plaque neutralization assays showed that all were antigenically related to BK virus. All isolates transformed baby hamster kidney cells, transformation being determined by the ability of the cells to plate in soft agar. Purified preparations of each isolate were iodinated with chloramine T and the polypeptide compositions were compared by electrophoresis of disrupted viruses in polyacrylamide gels containing sodium dodecyl sulfate. The gel patterns of all isolates were similar to that of BK virus. The tryptic digests of two major iodinated virion proteins, VP1 and VP3, were analyzed on an ion exchange column. The peptide patterns of GS, DW, and BK virus were identical; those of RF and MG virus closely resembled the patterns of the above three with only minor differences in some peptides. The results show that the four isolates and BK virus are antigenically closely related, have similar onocogenic potential, and are distinguishable from simian virus 40 and JC virus.     

Structural polypeptides of rabbit, bovine, and human papillomaviruses.

The number and apparent molecular weight of the structural polypeptides of Shope rabbit papilloma virus (RPV), bovine papilloma virus (BPV), and human papilloma virus (HPV) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Up to 10 polypeptides were detected in highly purified BPV and HPV full particles; a close homology was found between the polypeptide composition of both viruses. Purified RPV virions gave a similar polypeptide pattern. The main components of the three papillomaviruses are the major polypeptide (VP1) with a mol wt of approximately 54,000 and the three smaller polypeptides (VP8, 9, 10) with mol wt of about 16,500, 15,500 and 12,500, respectively. VP8, VP9, and VP10 are never detected in empty capsids. When BPV virions were disrupted with alkaline buffer, the six lower-molecular-weight polypeptides (VP5 to 10) remained associated with viral DNA. This suggests that they are internal components of the virions and that the four higher-molecular-weight polypeptides (VP1 to 4) may represent external components. The polypeptide compositions of BPV and polyoma virus, another papovavirus, have been compared. The number of BPV and polyoma virus components (10 and 6, respectively) and the molecular weight of their major polypeptide (54,000 and 44,500, respectively) are different; however, the three main DNA-associated polypeptides of BPV (VP8, 9, 10) and the three histone-like components of polyoma virus (VP4, 5, 6) were shown to have identical apparent molecular weights. The possibility that some of the minor components of papillomaviruses may be proteolytic degradation products or cell protein contaiminants is discussed.     

Evidence for Simian Virus 40 (SV40) Coding of SV40 T-Antigen and the SV40-Specific Proteins in HeLa Cells Infected with Nondefective Adenovirus Type 2-SV40 Hybrid Viruses

HeLa cells infected with the nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2(+)ND1, Ad2(+)ND2, Ad2(+)ND4, and Ad2(+)ND5) synthesize SV40-specific proteins ranging in size from 28,000 to 100,000 daltons. By analysis of their methionine-containing tryptic peptides, we demonstrated that all these proteins shared common amino acid sequences. Most methionine-containing tryptic peptides derived from proteins of smaller size were contained within the proteins of larger size. Seventeen of the 21 methionine-containing tryptic peptides of the largest SV40-specific protein (100,000 daltons) from Ad2(+)ND4-infected cells were identical to methionine-containing peptides of SV40 T-antigen immunoprecipitated from extracts of SV40-infected cells. All of the methionine-containing tryptic peptides of the Ad2(+)ND4 100,000-dalton protein were found in SV40 T-antigen immunoprecipitated from SV40-transformed cells. All SV40-specific proteins observed in vivo could be synthesized in vitro using the wheat germ cell-free system and SV40-specific RNA from hybrid virus-infected cells that was purified by hybridization to SV40 DNA. As proof of identity, the in vitro products were shown to have methionine-containing tryptic peptides identical to those of their in vivo counterparts. Based on the extensive overlap in amino acid sequence between the SV40-specific proteins from hybrid virus-infected cells and SV40 T-antigen from SV40-infected and -transformed cells, we conclude that at least the major portion of the SV40-specific proteins cannot be Ad2 coded. From the in vitro synthesis experiments with SV40-selected RNA, we further conclude that the SV40-specific proteins must be SV40 coded and not host coded. Since SV40 T-antigen is related to the SV40-specific proteins, it must also be SV40 coded.     

Multiple forms of polyoma virus tumor antigens from infected and transformed cells.

At least three distinct forms of polyoma virus tumor antigens were isolated from productively infected and transformed hamster cells by immunoprecipitation with anti-T serum. These proteins had approximate molecular weights of 105,000 (large T antigen), 63,000 (middle T antigen), and 20,000 (small T antigen) as estimated by acrylamide gel electrophoresis. An examination of the appearance of these antigens in polyoma-infected mouse cells showed that all three polypeptides were synthesized maximally at approximately the same time after infection. Analysis of the methionine-containing tryptic peptides of these proteins indicated that the large, middle, and small forms of polyoma T antigens contained five similar or identical peptides. In addition, the 63,000- and 20,000-dalton antigens contained two other methionine peptides absent from the large T-antigen species. Other methionine peptides were found only in the large or middle T-antigen forms. These results and results obtained previously suggested that the three T-antigen species have the same NH2-terminal end regions but different COOH termini. A model is presented describing the synthesis of these polypeptides from different regions of the polyoma virus genome.     

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.     

Characterization of different tumor antigens present in cells transformed by simian virus 40.

In addition to large T and small t antigens, cells transformed by simian virus 40 (SV40) commonly contain other proteins which specifically immunoprecipitate with SV40 anti-T serum and which are not detected in untransformed cells. The additional tumor antigens (T-Ags) fall into two groups: those having a close structural relationship with normal SV40 T-Ags, and those unrelated to large T and small t. The latter are probably nonviral T-Ags (NVT-Ags). The NVT-Ags comprise a family of proteins of molecular weight 50,000-55,000. Fingerprint analysis shows that NVT-Ags have few if any peptides in common with large T or small t, and that they lack the amino terminal tryptic peptide and the peptides unique to small t. NVT-Ags from different species have different fingerprints, but those isolated from different transformants of the same cell line are identical. The size of NVT is unaltered in cells transformed by mutants of SV40 with deletions in the region 0.60-0.55 map units. The mRNA for NVT does not hybridize to SV40 DNA. The other forms of T-Ag isolated from transformed cells fall into three classes: shortened forms of large T (truncated large T); multiple species of T-Ag with molecular weights very similar to, but distinct from, those of normal large T (large T doublets and triplets); and elongated forms of large T (super T). These proteins all contain the normal amino terminus of SV40 T-Ags, and the truncated forms of large T lack peptides from the carboxy terminal half of large T. One species of super T (molecular weight 130,000) contains only those methionine tryptic peptides present in normal large T, although it may contain some peptides in more than one copy.     

Identification of Virus-Induced Proteins in Cells Productively Infected with Simian Virus 40

The number and molecular weight of the structural polypeptides of highly purified simian virus 40 (SV40) were determined by polyacrylamide gel electrophoresis. Six different polypeptides were found, two of which (VP1 and VP2) comprise the bulk of the viral capsid proteins. The pattern of protein synthesis in productively infected CV-1 cells was studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Identification of virus-induced proteins in the infected CV-1 cells was achieved in double-labeling experiments by electrophoresis with purified labeled SV40 capsid proteins. Four of these proteins (VP1 and VP4) could be classified as components of the virion because their synthesis occurred after the onset of viral deoxyribonucleic acid (DNA) replication and because they were inhibited by arabinofuranosylcytosine (ara-C). Appearance of two other virus-induced proteins was not prevented by ara-C; one of them did not comigrate in the electrophoresis with purified virion polypeptides, and both could be detected before the onset of viral DNA synthesis. These latter two proteins were classified on the basis of these criteria as nonvirion capsid proteins (NCVP1 and NCVP2).     

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.