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Journal of Virology Oct 1978Polyacrylamide gel electrophoresis and tryptic peptide fingerprint analysis of the proteins made in a cell-free system derived from L-cells and immunoprecipitated with...
Polyacrylamide gel electrophoresis and tryptic peptide fingerprint analysis of the proteins made in a cell-free system derived from L-cells and immunoprecipitated with simian virus 40 (SV40) anti-T serum demonstrated that both SV40 large-T and small-T antigens are synthesized in vitro in response to mRNA isolated from productively infected CV1 CELLS. Sucrose density centrifugation in gradients containing 85% formamide showed that the mRNA's for both forms of T-antigen sediment at about 17.5S, with the mRNA for small-t sedimenting marginally, but reproducibly, ahead of the mRNA for large-T. Hybridization experiments using restriction endonuclease fragments Hae III-E and Hind II/III-B showed that all fractions active in the cell-free synthesis of both forms of T-antigen hybridized equally to both fragments. This suggests that the mRNA's for SV40 T-antigens are at least partly virus coded and that the bulk of the early SV40 mRNA contains sequence information from both ends of the early region. The data are consistent with the suggestion that the large-T mRNA is spliced. SV40 complementary RNA (the product of transcription of SV40 DNA using Escherichia coli RNA polymerase) was also translated in the L-cell system and gave two families of polypeptides which specifically immunoprecipitate with anti-T serum. One family (the small-t family) includes a polypeptide indistinguishable by gel electrophoresis and tryptic peptide fingerprinting from small-t isolated from cells. The other family (the 60K family) has a major component with molecular weight approximately 60,000 and includes other polypeptides with molecular weights ranging from approximately 14,000 to about 70,000. The 60K family has petides in common with large-T but not with small-T. Together, the peptides of the small-t and 60K families account for virtually all of the methionine peptides of SV40 large-T. We conclude from these results (i) that small-t is probably entirely, and large-T at least predominantly, virus coded; (ii) that the small-t and 60K families represent the translation products of two different portions of the early region of SV40 DNA (approximately 0.65 to 0.55 map units and 0.54 to 0.17 map units); and (iii) that although most, if not all, of the large-T and small-t peptides are present in the cell-free product, some feature of sequence arrangement of SV40 complementary RNA prevents the translation of full-length large-T and results instead in the synthesis of fragments. We suggest that the absence of a splice in the complementary RNA is responsible for this result.
Topics: Antigens, Viral; Base Sequence; Cell-Free System; L Cells; Molecular Weight; Peptide Biosynthesis; Protein Biosynthesis; RNA, Messenger; RNA, Viral; Simian virus 40; Viral Proteins
PubMed: 212600
DOI: 10.1128/JVI.28.1.154-170.1978 -
Molecular and Cellular Biology Jan 1983During the lytic infection of monkey and mouse cells with simian virus 40 and polyoma virus, respectively, the preferentially increased synthesis of two host proteins of... (Comparative Study)
Comparative Study
During the lytic infection of monkey and mouse cells with simian virus 40 and polyoma virus, respectively, the preferentially increased synthesis of two host proteins of 92,000 and 72,000 Mr was observed by 15 to 20 h after infection besides the general stimulation of most cellular proteins. The incubation of uninfected monkey and mouse cell cultures for 30 to 60 min at 43.5 degrees C induced the enhanced synthesis of at least three proteins of 92,000, 72,000 and 70,000 Mr, the last one being the major heat shock protein of mammalian cells. Two-dimensional gel electrophoresis and partial proteolytic digestion confirmed that the same 92,000- and 72,000-Mr proteins are stimulated by virus infection and thermal treatment. In simian virus 40-infected CV-1 cells, we also observed the weak stimulation of a 70,000-Mr protein comigrating in gel electrophoresis with the major heat shock protein. The 92,000-, 72,000- and 70,000-Mr proteins of monkey cells are structurally very similar to the corresponding proteins of mouse cells. In immunoprecipitations, no specific association of these proteins to simian virus 40 T antigens was noticed.
Topics: Animals; Antigens, Viral; Cells, Cultured; Chlorocebus aethiops; Heat-Shock Proteins; Hot Temperature; Mice; Molecular Weight; Polyomavirus; Protein Biosynthesis; Proteins; Simian virus 40; Tumor Virus Infections
PubMed: 6298601
DOI: 10.1128/mcb.3.1.1-8.1983 -
Proceedings of the National Academy of... May 1978Large and small tumor (T)antigens of simian virus 40 were synthesized in vitro with L-cell extracts that had been treated by the method of Palmiter to prevent...
Large and small tumor (T)antigens of simian virus 40 were synthesized in vitro with L-cell extracts that had been treated by the method of Palmiter to prevent amino-terminal acetylation of nascent proteins. Partial amino-terminal amino acid sequences of both forms of T-antigen were determined and found to be identical. Methionine residues were located at positions 1 and 14, a lysine residue at position 3, and leucine residues at positions 5, 11, 13,16, 17, and 19. These amino acid sequence data match perfectly the amino acid sequence predicted from a sequence of nucleotides in the E strand of simian virus 40 DNA which begins near the junction between HindII/III fragments A and C at about 0.65 map units. This strongly suggests that the sequence coding for the amino terminus of both proteins is located at this position. Furthermore, the data are consistent with a model for the synthesis of both forms of T-antigen that predicts that (i) small T-antigen is coded for by a sequence of nucleotides from the 5' end of the early region and (ii) large T-antigen is coded for by nucleotide sequences from two noncontiguous regions of simian virus 40 DNA.
Topics: Amino Acid Sequence; Antigens, Neoplasm; Antigens, Viral; Chromosome Mapping; Molecular Weight; Simian virus 40; Viral Proteins
PubMed: 209456
DOI: 10.1073/pnas.75.5.2165 -
Journal of Bacteriology Oct 1966Takemoto, K. K. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), R. L. Kirschstein, and K. Habel. Mutants of simian virus 40 differing in plaque...
Takemoto, K. K. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), R. L. Kirschstein, and K. Habel. Mutants of simian virus 40 differing in plaque size, oncogenicity, and heat sensitivity. J. Bacteriol. 92:990-994. 1966.-Three mutants of simian virus 40 were isolated on the basis of the type of plaques produced in primary cultures of African green monkey kidney cells and designated as L (large), S (small), and M (minute) strains. Significant differences in oncogenicity for hamsters were observed, with the 50% oncogenic dose being 10(4.5) for the L, 10(5.2) for the S, and 10(5.8) for the M strains. All three strains were capable of transforming human diploid cells (W138 strain). At temperatures up to 41 C, the S and M mutants were capable of multiplying to titers almost equivalent to those obtained at 37 C. In contrast, infectious virus was not produced when cells were infected with the L mutant and were incubated at temperatures above 39 C, although complement-fixing viral and tumor antigens were formed. The temperature-sensitive phase of replication of the L strain was shown to be a late stage in viral maturation or assembly.
Topics: Antigens; Complement Fixation Tests; Culture Techniques; Hot Temperature; Mutation; Oncogenic Viruses; Simian virus 40; Virus Cultivation
PubMed: 4288804
DOI: 10.1128/jb.92.4.990-994.1966 -
Virology Feb 2009Duplication of the simian virus 40 (SV40) genome is the best understood eukaryotic DNA replication process to date. Like most prokaryotic genomes, the SV40 genome is a... (Review)
Review
Duplication of the simian virus 40 (SV40) genome is the best understood eukaryotic DNA replication process to date. Like most prokaryotic genomes, the SV40 genome is a circular duplex DNA organized in a single replicon. This small viral genome, its association with host histones in nucleosomes, and its dependence on the host cell milieu for replication factors and precursors led to its adoption as a simple and powerful model. The steps in replication, the viral initiator, the host proteins, and their mechanisms of action were initially defined using a cell-free SV40 replication reaction. Although our understanding of the vastly more complex host replication fork is advancing, no eukaryotic replisome has yet been reconstituted and the SV40 paradigm remains a point of reference. This article reviews some of the milestones in the development of this paradigm and speculates on its potential utility to address unsolved questions in eukaryotic genome maintenance.
Topics: Antigens, Polyomavirus Transforming; DNA Replication; DNA, Viral; Genome, Viral; Simian virus 40; Virus Replication
PubMed: 19101707
DOI: 10.1016/j.virol.2008.11.038 -
Journal of Virology May 1993Simian virus 40 (SV40) can be disassembled under mild conditions by reducing disulfide bonds in the capsid and removing calcium ions. The nucleoprotein complexes formed,...
Simian virus 40 (SV40) can be disassembled under mild conditions by reducing disulfide bonds in the capsid and removing calcium ions. The nucleoprotein complexes formed, analyzed by electron microscopy, were circular and made up of 59 +/- 4 subunits, each with a diameter of about 10 nm. The complexes contained the viral DNA, histones, and the viral capsid proteins. The complexes had much-reduced infectivities compared with intact SV40. Addition of calcium ions to the disrupted virus caused the nucleoprotein complexes to refold into virus-like structures which sedimented at the same rate as intact SV40 and regained infectivity. Treatment of the disrupted SV40 with a high concentration of salt dissociated the viral proteins from the DNA. Lowering stepwise the salt concentration, removing the reducing agent, and adding calcium ions allowed structures to be reformed, and these structures sedimented, like SV40, at 240S and were infectious. The plaque-forming ability of the reconstituted particles was between that of the dissociated components and that of intact SV40. The addition of purified DNA of polyomavirus to the dissociated SV40 before the lowering of the salt concentration showed that virus-like structures could be formed from SV40 proteins and a foreign DNA.
Topics: Capsid; DNA Replication; DNA, Viral; Genetic Vectors; Macromolecular Substances; Nucleic Acid Conformation; Polyomavirus; Protein Conformation; Simian virus 40; Viral Plaque Assay; Virion; Virulence; Virus Replication
PubMed: 8386277
DOI: 10.1128/JVI.67.5.2779-2786.1993 -
Journal of Virology Jan 2019Host range (HR) mutants of simian virus 40 (SV40) containing mutations in the C terminus of large T antigen fail to replicate efficiently or form plaques in restrictive...
Host range (HR) mutants of simian virus 40 (SV40) containing mutations in the C terminus of large T antigen fail to replicate efficiently or form plaques in restrictive cell types. HR mutant viruses exhibit impairments at several stages of the viral life cycle, including early and late gene and protein expression, DNA replication, and virion assembly, although the underlying mechanism for these defects is unknown. Host protein FAM111A, whose depletion rescues early and late gene expression and plaque formation for SV40 HR viruses, has been shown to play a role in cellular DNA replication. SV40 viral DNA replication occurs in the nucleus of infected cells in viral replication centers where viral proteins and cellular replication factors localize. Here, we examined the role of viral replication center formation and DNA replication in the FAM111A-mediated HR phenotype. We found that SV40 HR virus rarely formed viral replication centers in restrictive cells, a phenotype that could be rescued by FAM111A depletion. Furthermore, while FAM111A localized to nucleoli in uninfected cells in a cell cycle-dependent manner, FAM111A relocalized to viral replication centers after infection with SV40 wild-type or HR viruses. We also found that inhibition of viral DNA replication through aphidicolin treatment or through the use of replication-defective SV40 mutants diminished the effects of FAM111A depletion on viral gene expression. These results indicate that FAM111A restricts SV40 HR viral replication center formation and that viral DNA replication contributes to the FAM111A-mediated effect on early gene expression. SV40 has served as a powerful tool for understanding fundamental viral and cellular processes; however, despite extensive study, the SV40 HR mutant phenotype remains poorly understood. Mutations in the C terminus of large T antigen that disrupt binding to the host protein FAM111A render SV40 HR viruses unable to replicate in restrictive cell types. Our work reveals a defect of HR mutant viruses in the formation of viral replication centers that can be rescued by depletion of FAM111A. Furthermore, inhibition of viral DNA replication reduces the effects of FAM111A restriction on viral gene expression. Additionally, FAM111A is a poorly characterized cellular protein whose mutation leads to two severe human syndromes, Kenny-Caffey syndrome and osteocraniostenosis. Our findings regarding the role of FAM111A in restricting viral replication and its localization to nucleoli and viral replication centers provide further insight into FAM111A function that could help reveal the underlying disease-associated mechanisms.
Topics: Animals; Antigens, Viral, Tumor; Cell Cycle Proteins; Cell Line; Cell Nucleus; Chlorocebus aethiops; DNA, Viral; Gene Expression Regulation, Viral; Host Specificity; Humans; Mutation; Phenotype; Simian virus 40; Virus Replication
PubMed: 30333173
DOI: 10.1128/JVI.01330-18 -
Journal of Virology Aug 1972Deoxyribonucleic acid-ribonucleic acid (RNA) hybridization in formamide was used to isolate simian virus 40-specific RNA. Early in the lytic cycle, a 19S viral RNA...
Deoxyribonucleic acid-ribonucleic acid (RNA) hybridization in formamide was used to isolate simian virus 40-specific RNA. Early in the lytic cycle, a 19S viral RNA species was observed. Late in the lytic cycle, 16S and 19S viral species were found. The 16S and 19S species of viral RNA were localized in the cytoplasm. High-molecular-weight heterogeneous RNA, containing viral sequences, was isolated from the nuclear fraction of infected cells late in the lytic cycle. This RNA may contain non-viral sequences linked to viral sequences. The formamide hybridization technique can be used to isolate intact late lytic viral RNA which is at least 99% pure.
Topics: Animals; Cell Line; Cell Nucleus; Centrifugation, Density Gradient; Chemical Precipitation; Cytarabine; Cytoplasm; DNA, Viral; Dimethyl Sulfoxide; Electrophoresis, Polyacrylamide Gel; Formamides; Haplorhini; Kidney; Molecular Weight; Nucleic Acid Hybridization; Phosphorus Isotopes; RNA, Viral; Simian virus 40; Tritium; Uridine
PubMed: 4342237
DOI: 10.1128/JVI.10.2.193-201.1972 -
Journal of Virology Feb 2012The nonenveloped polyomavirus simian virus 40 (SV40) is taken up into cells by a caveola-mediated endocytic process that delivers the virus to the endoplasmic reticulum...
The nonenveloped polyomavirus simian virus 40 (SV40) is taken up into cells by a caveola-mediated endocytic process that delivers the virus to the endoplasmic reticulum (ER). Within the ER lumen, the capsid undergoes partial disassembly, which exposes its internal capsid proteins VP2 and VP3 to immunostaining with antibodies. We demonstrate here that the SV40 genome does not become accessible to detection while the virus is in the ER. Instead, the genome becomes accessible two distinct detection procedures, one using anti-bromodeoxyuridine antibodies and the other using a 5-ethynyl-2-deoxyuridine-based chemical reaction, only after the emergence of partially disassembled SV40 particles in the cytoplasm. These cytoplasmic particles retain some of the SV40 capsid proteins, VP1, VP2, and VP3, in addition to the viral genome. Thus, SV40 particles undergo discrete disassembly steps during entry that are separated temporally and topologically. First, a partial disassembly of the particles occurs in the ER, which exposes internal capsid proteins VP2 and VP3. Then, in the cytoplasm, disassembly progresses further to also make the genomic DNA accessible to immune detection.
Topics: Animals; Cell Line; Chlorocebus aethiops; Cytoplasm; Endoplasmic Reticulum; Genome, Viral; Microscopy, Fluorescence; Simian virus 40; Virus Assembly
PubMed: 22090139
DOI: 10.1128/JVI.05753-11 -
Journal of Virology Mar 1992
Review
Topics: Amino Acid Sequence; Antigens, Polyomavirus Transforming; DNA Replication; Molecular Sequence Data; Simian virus 40; Virus Replication
PubMed: 1310750
DOI: 10.1128/JVI.66.3.1289-1293.1992