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Activation of SV40 DNA replication in vitro by cellular protein phosphatase 2A. 总被引:30,自引:2,他引:28
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We have made use of the cell-free SV40 DNA replication system to identify and characterize cellular proteins required for efficient DNA synthesis. One such protein, replication protein C (RP-C), was shown to be involved with SV40 large T antigen in the early stages of viral DNA replication in vitro. We demonstrate here that RP-C is identical to the catalytic subunit of cellular protein phosphatase 2A (PP2Ac). The purified protein dephosphorylates specific phosphoamino acid residues in T antigen, consistent with the hypothesis that SV40 DNA replication is regulated by modulating the phosphorylation state of the viral initiator protein. We also show that purified RP-C/PP2Ac preferentially stimulates SV40 DNA replication in extracts from early G1 phase cells. This finding suggests that the activity of a cellular factor that influences the net phosphorylation state of T antigen is cell cycle dependent. 相似文献
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Polyclonal antibodies were used to assay human embryonal carcinoma (EC), differentiating EC, yolk sac carcinoma, and teratoma cells for expression of viral early antigen (T-Ag) after infection with simian virus 40 (SV40). Cells of four EC lines were induced to differentiate by cultivation at low density or by exposure to retinoic acid or dimethyl sulfoxide. After infection with SV40, T-Ag was expressed by 1%, or less, of the cells (presumed to be differentiated derivatives) in only some EC cultures whereas the antigen was synthesized by a significant percentage of the yolk sac carcinoma, teratoma, and differentiating EC cells. Also, viral late proteins were produced by EC cells infected with human adenovirus type 7 (Ad7), and SV40 T-Ag was expressed by EC cells after infection with PARA, which is an Ad7-SV40 hybrid virus containing the SV40 T-Ag sequence controlled by Ad7 late regulatory sequences. Thus, T-Ag is not synthesized by the parental EC cells infected with SV40, but it is expressed in cultures of infected differentiated derivatives. The EC cells produce T-Ag, however, when expression of the viral protein is controlled by the Ad7 regulatory sequences in PARA particles. These results demonstrate that expression of T-Ag after infection with SV40 is an indicator of EC cell differentiation and also raise the possibility that, as in mouse EC cells infected with the virus, the SV40 regulatory sequences controlling T-Ag synthesis are not active in human EC cells. 相似文献
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Hans-J?rg Riedinger Maria van Betteraey-Nikoleit Hans Probst 《European journal of biochemistry》2002,269(9):2383-2393
Hypoxia interrupts the initiation of simian virus 40 (SV40) replication in vivo at a stage situated before unwinding of the origin region. After re-oxygenation, unwinding followed by a synchronous round of viral replication takes place. To further characterize the hypoxia-induced inhibition of unwinding, we analysed the binding of several replication proteins to the viral minichromosome before and after re-oxygenation. T antigen, the 34-kDa subunit of replication protein A (RPA), topoisomerase I, the 48-kDa subunit of primase, the 125-kDa subunit of polymerase delta, and the 37-kDa subunit of replication factor C (RFC) were present at the viral chromatin already under hypoxia. The 70-kDa subunit of RPA, the 180-kDa subunit of polymerase alpha, and proliferating cell nuclear antigen (PCNA) were barely detectable at the SV40 chromatin under hypoxia and significantly increased after re-oxygenation. Immunoprecipitation of minichromosomes with T antigen-specific antibody and subsequent digestion with micrococcus nuclease revealed that most of the minichromosome-bound T antigen was associated with the viral origin in hypoxic and in re-oxygenated cells. T antigen-catalysed unwinding of the SV40 origin occurred, however, only after re-oxygenation as indicated by (a) increased sensitivity of re-oxygenated minichromosomes against digestion with single-stranded DNA-specific nuclease P1; (b) stabilization of RPA-34 binding at the SV40 minichromosome; and (c) additional phosphorylations of RPA-34 after re-oxygenation, probably catalysed by DNA-dependent protein kinase. The results presented suggest that the subunits of the proteins necessary for unwinding, primer synthesis and primer elongation first assemble at the SV40 origin in form of stable, active complexes directly before they start to work. 相似文献
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Debrah A. Fine Orit Rozenblatt-Rosen Megha Padi Anna Korkhin Robert L. James Guillaume Adelmant Rosa Yoon Luxuan Guo Christian Berrios Ying Zhang Michael A. Calderwood Soundarapandian Velmurgan Jingwei Cheng Jarrod A. Marto David E. Hill Michael E. Cusick Marc Vidal Laurence Florens Michael P. Washburn Larisa Litovchick James A. DeCaprio 《PLoS pathogens》2012,8(10)
The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT. 相似文献
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Angelo Taronna Elisa Mazzoni Alfredo Corallini Ilaria Bononi Silvia Pietrobon Giovanni Guerra Caterina Palmonari Caterina Borgna-Pignatti Manola Comar Massimo Bovenzi Ferruccio Casali Roberto Marci Giovanni Rezza Giuseppe Barbanti-Brodano Mauro Tognon Fernanda Martini 《PloS one》2013,8(4)
At present Simian virus 40 (SV40) infection in humans appears to be transmitted independently from early contaminated vaccines. In order to test the spread of SV40 infection in children, an immunologic assay employing specific SV40 synthetic peptides corresponding to its viral protein (VP) antigens was employed to estimate the seroprevalence of this polyomavirus in Italian infants and adolescents. Serum samples from 328 children and adolescents, up to 17 years, were investigated. Serum antibodies against SV40 VPs were detected by indirect enzyme-linked immunosorbent assays. The seroprevalence of this polyomavirus was calculated after stratifying the subjects by age. Anti-viral capsid protein 1-2-3 SV40 IgG antibodies were detected in 16% of the study participants. The prevalence of antibodies against SV40 VPs tended to increase with age in children, up to 10 year old (21%). Then, in the cohort of individuals aged 11–17 years, the prevalence decreased (16%). A higher prevalence rate (23%) of SV40 VP antibodies was detected in the cohorts of 1–3 year and 7–10 year old children, than in children and adolescents of the other age groups. This age corresponds to children starting nursery and primary school, respectively, in Italy. IgM antibodies against SV40 VP mimotopes were detected in 6–8 month old children suggesting that SV40 seroconversion can occur early in life. SV40 VP antibodies are present at low prevalence in Italian children (16%), suggesting that SV40 infection, although acquired early in life, probably through different routes, is not widespread. The low SV40 seroprevalence suggests that SV40 is less transmissible than other common polyomaviruses, such as BKV and JCV. Alternatively, our immunologic data could be due to another, as yet undiscovered, human polyomavirus closely related to SV40. 相似文献
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A DNA replication-positive mutant of simian virus 40 that is defective for transformation and the production of infectious virions. 总被引:8,自引:7,他引:1
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K W Peden S L Spence L C Tack C A Cartwright A Srinivasan J M Pipas 《Journal of virology》1990,64(6):2912-2921
Simian virus 40 (SV40) mutant 5002 carries base pair substitutions of C-5109----T and C-5082----T. These mutations lie in a region of the genome that encodes amino acids common to the large and small viral tumor antigens (T and t antigens, respectively) and result in amino acid substitutions of Leu-19----Phe and Pro-28----Ser. In contrast to wild-type SV40, which produces large plaques that are clearly visible 8 days postinfection, mutant 5002 is defective for productive infection, producing tiny plaques that arise at around 21 days postinfection. However, 5002 is capable of replicating viral DNA and producing normal amounts of capsid proteins, indicating that the mutations alter an activity of T antigen that is required subsequent to DNA synthesis, such as maturation, viral assembly, or release of virions. The mutant T antigen has normal ATPase activity, is phosphorylated in a manner that is indistinguishable from that of the wild-type T antigen, and retains the ability to oligomerize. 5002 complements mutants defective in T antigen host range-adenovirus helper function for productive infection. Thus, T antigen encodes two activities that affect at least two different steps in viral infection other than DNA replication, one inactivated by mutations in the host range-adenovirus helper domain and one inactivated by the mutations present in 5002. The 5002-encoded T antigen is also defective for transformation of REF52 cells when expressed from the normal SV40 early promoter, although this defect can be partially overcome by expressing the protein from stronger promoters. 相似文献
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Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells IV. Two Different Requirements for Protein Synthesis During Simian Virus 40 Deoxyribonucleic Acid Replication 总被引:4,自引:4,他引:0
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The replication of simian virus 40 (SV40) deoxyribonucleic acid (DNA) was inhibited by 99% 2 hr after the addition of cycloheximide to SV40-infected primary African green monkey kidney cells. The levels of 25S (replicating) and 21S (mature) SV40 DNA synthesized after cycloheximide treatment were always lower than those observed in an infected untreated control culture. This is consistent with a requirement for a protein(s) or for protein synthesis at the initiation step in SV40 DNA replication. The relative proportion of 25S DNA as compared with 21S viral DNA increased with increasing time after cycloheximide treatment. Removal of cycloheximide from inhibited cultures allowed the recovery of viral DNA synthesis to normal levels within 3 hr. During the recovery period, the ratio of 25S DNA to 21S DNA was 10 times higher than that observed after a 30-min pulse with (3)H-thymidine with an infected untreated control culture. The accumulation of 25S replicating SV40 DNA during cycloheximide inhibition or shortly after its removal is interpreted to mean that a protein(s) or protein synthesis is required to convert the 25S replicating DNA to 21S mature viral DNA. Further evidence of a requirement for protein synthesis in the 25S to 21S conversion was obtained by comparing the rate of this conversion in growing and resting cells. The conversion of 25S DNA to 21S DNA took place at a faster rate in infected growing cells than in infected confluent monolayer cultures. A temperature-sensitive SV40 coat protein mutation (large-plaque SV40) had no effect on the replication of SV40 DNA at the nonpermissive temperature. 相似文献
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Essential role of the Vp2 and Vp3 DNA-binding domain in simian virus 40 morphogenesis. 总被引:4,自引:3,他引:1
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Both a DNA-binding domain and a Vp1 interactive determinant have been mapped to the carboxy-terminal 40 residues of the simian virus 40 (SV40) minor capsid proteins, Vp2 and Vp3 (Vp2/3), with the last 13 residues being necessary for these activities. The role of this DNA-binding domain in SV40 morphogenesis and the ability to separate these two signals were investigated by mutagenesis and assessment of the activity and viability of the mutants. The carboxy-terminal 40 residues of Vp2/3 were expressed as a polyhistidine fusion protein, and five basic residues at the extreme carboxy terminus (Vp3 residues K226, R227, R228, R230, and R233) were mutagenized. The wild-type fusion protein bound DNA with a Kd of 3 x 10(-8) identical to that of the full-length Vp3. Mutant proteins containing either one to three or four amino acid substitutions bound DNA 4- to 7-fold or 20- to 30-fold less well, respectively, than the wild-type protein did. The most severe point mutants showed residual DNA binding similar to that of a truncated protein which lacks the entire 13 carboxy-terminal residues. All of the point mutants were able to interact with Vp1, indicating that the two signals within this region are mediated by different residues. When the mutations were placed into the context of the viral DNA and introduced into cells, all the structural proteins were expressed and localized correctly. Not all, however, were viable: mutant genomes whose Vp2/3 bound DNA with intermediate affinities formed plaques just as well as wild-type SV40 DNA did, but three mutants showing greatly reduced DNA binding failed to form plaques at all. These results are consistent with the hypothesis that Vp2/3 plays an essential role in SV40 virion assembly in the nucleus. 相似文献
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Immunoprecipitation of SV40 replicating minichromosomes complexed with bacteriophage T4 gene 32 protein.
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E I Miranda E Garrido-Guerrero A Garcia-Carranca P Gariglio 《Nucleic acids research》1992,20(4):903-907
Simian Virus 40 (SV40) DNA replication is a useful model to study eukaryotic cell DNA replication because it encodes only one replication protein and its genome has a nucleoprotein structure ('minichromosome') indistinguishable from cellular chromatin. Late after infection SV40 replicating DNA molecules represent about 5% of total viral minichromosomes. Since gene 32 protein (P32) from bacteriophage T4 interacts with single-stranded DNA and SV40 replication complexes are expected to contain single-stranded regions at the replication forks, we asked whether P32 might be used to isolate replicating SV40 minichromosomes. When nuclear extracts from SV40 infected cells were treated sequentially with P32 and anti-P32 antibodies, pulse-labeled minichromosomes were selectively immunoprecipitated. Agarose gel electrophoresis analysis confirmed that immunoprecipitated material corresponded to SV40 replicative intermediates. Protein analysis of the pelleted material revealed several proteins of viral and cellular origin. Among them, T antigen and histones were found to be complexed with at least other three proteins from cellular origin, to the replicative complexes. Additionally, anti-P32 antibodies were able to detect three cellular proteins of approximately 70, 32 and 13 kDa in western blots. These proteins could correspond to those found as part of an eukaryotic multisubunit single-stranded DNA binding protein. The use of P32 and anti-P32 antibodies thus allows the separation of replicating from mature SV40 minichromosomes and can constitute a novel method to enrich and to study replicative active chromatin. 相似文献
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K Rundell J K Collins P Tegtmeyer H L Ozer C J Lai D Nathans 《Journal of virology》1977,21(2):636-646
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. 相似文献
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Hybrid genomes of the polyomaviruses JC virus, BK virus, and simian virus 40: identification of sequences important for efficient transformation. 总被引:13,自引:7,他引:6
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Hybrid viral genomes were used to investigate the influence of specific polyomavirus sequences on the transforming behavior of JC virus (JCV). One set of chimeric DNAs was made by exchanging the regulatory regions between JCV and simian virus 40 (SV40) or JCV and BK virus (BKV). A second set of constructs was produced that expressed hybrid JCV-BKV T proteins under the control of either JCV or BKV regulatory signals. Transformation of Rat 2 cells with the parental and chimeric DNAs indicated that both the JCV regulatory signals and the sequence encoding the amino terminus of T protein contributed to the restricted transforming behavior of this virus. Analysis of the viral proteins in the transformed rat cells indicated that the large T antigens of JCV and BKV were less stable than their SV40 counterpart, that small t protein was produced in JCV transformants, and that the subpopulation of T antigen that forms a stable complex with cellular p53 protein was smaller in JCV-transformed cells than in SV40- or BKV-transformed cells. 相似文献
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Replication of simian virus 40 origin-containing DNA during infection with a recombinant Autographa californica multiple nuclear polyhedrosis virus expressing large T antigen.
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Autographica californica multiple nuclear polyhedrosis virus (AcMNPV) has been shown to encode many of the enzymes involved in the replication of its own DNA. Although the AcMNPV genome contains multiple sets of reiterated sequences that are thought to function as origins of DNA replication, no initiator protein has yet been identified in the set of viral replication enzymes. In this study, the ability of a heterologous origin initiator system to promote DNA replication in AcMNPV-infected cells was examined. A recombinant AcMNPV that expressed the simian virus 40 (SV40) large T antigen was surprisingly found to induce the efficient replication of a transfected plasmid containing an SV40 origin. This replication was subsequently found to involve three essential components: (i) T antigen, since replication of SV40 origin-containing plasmids was not induced by wild-type AcMNPV which did not express this protein; (ii) an intact SV40 core origin, since deletion of specific functional motifs within the origin resulted in a loss of replicative abilities; and (iii) one or more AcMNPV-encoded proteins, since viral superinfection was required for plasmid amplification. Characterization of the replicated DNA revealed that it existed as a high-molecular-weight concatemer and underwent significant levels of homologous recombination between inverted repeat sequences. These properties were consistent with an AcMNPV-directed mode of DNA synthesis rather than that of SV40 and suggested that T antigen-SV40 origin complexes may be capable of initiating DNA replication reactions that can be completed by AcMNPV-encoded enzymes. 相似文献
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Isolation and characterization of defective simian virus 40 genomes which complement for infectivity. 总被引:1,自引:1,他引:0
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A new variant of simian virus 40 (EL SV40), containing the complete viral DNA separated into two molecules, was isolated. One DNA species contains nearly all of the early (E) SV40 sequences, and the other DNA contains nearly all of the late (L) viral sequences. Each genome was encircled by reiterated viral origins and termini and migrated in agarose gels as covalently closed supercoiled circles. EL SV40 or its progenitor appears to have been generated in human A172 glioblastoma cells, as defective interfering genomes during acute lytic infections, but was selected during the establishment of persistently infected (PI) green monkey cells (TC-7). PI TC-7/SV40 cells contained EL SV40 as the predominant SV40 species. EL SV40 propagated efficiently and rapidly in BSC-1, another line of green monkey cells, where it also formed plaques. EL SV40 stocks generated in BSC-1 cells were shown to be free of wild-type SV40 by a number of criteria. E and L SV40 genomes were also cloned in the bacterial plasmid pBR322. When transfected into BSC-1 cell monolayers, only the combination of E and L genomes produced a lytic infection, followed by the synthesis of EL SV40. However, transfection with E SV40 DNA alone did produce T-antigen, although at reduced frequency. 相似文献
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JC virus-simian virus 40 genomes containing heterologous regulatory signals and chimeric early regions: identification of regions restricting transformation by JC virus. 总被引:2,自引:0,他引:2
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The papovavirus JC virus (JCV) is highly oncogenic in experimental animals but, unlike simian virus 40 (SV40), is severely restricted in its ability to transform cells in culture. We exploited the close genetic relatedness of these two viruses to delimit region(s) of the T protein which can restrict transforming activity. Novel chimeric genomes were produced by exchanging various segments of the JCV and SV40 T-protein-coding regions. These DNA constructs specified early proteins with in-frame substitutions of analogous amino acid sequences. A second set of genomes was prepared which, in addition to chimeric early proteins, contained substituted regulatory regions. The transformation efficiencies of these chimeric genomes were intermediate between those of SV40 and JCV, with the source of T protein exerting a greater effect than that of the regulatory region. The ability of certain constructs to induce efficient transformation required the presence of an SV40 regulatory region or specific sequences within the SV40 early coding region. Cloned cell lines prepared from representative transformants were characterized; the ability to form colonies in soft agarose was investigated, and the presence of viral T and cellular p53 proteins was determined. The various T proteins differed in amount, stability, and the ability to form stable complexes with p53. 相似文献
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G/U lesions are efficiently corrected to G/C in SV40 DNA 总被引:1,自引:0,他引:1
Cytosine spontaneously deaminates to form uracil, generating G/U pairs in DNA. We studied the repair of these lesions by introducing specific G/U pairs into the genome of SV40 and determining the fate of the mispaired bases in Simian cells. Analysis of 135 plaques obtained after transfection of the modified viral DNA indicates that G/U lesions were repaired to G/C in every case. This result indicates that G/U lesions are corrected with greater efficiency and specificity than any combination of DNA base/base mispairs, in transfected SV40 DNA. 相似文献