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1.
Database of mutations that alter the large tumor antigen in simian virus 40. 总被引:2,自引:0,他引:2
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The SV40 T antigen database is a listing of plasmids and/or viruses that express mutant forms of the virus-encoded large T antigen protein. The parental virus strain, nucleic acid sequence of the mutations, the effect of the mutation on the T antigen amino acid sequence, and key references are included in the listing. The database is available from the authors as a Macintosh FileMaker Pro file, and as a hard copy printout. 相似文献
2.
Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation 总被引:34,自引:16,他引:18
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We used site-specific mutagenesis to introduce a termination codon, TGA, into the reading frame for the polyoma virus medium T antigen. We induced this mutation in a region of the polyoma genome in which the overlapping coding regions for the large and medium TE antigens are translated in different reading frames. Therefore, the mutation terminated translation of the medium T antigen, but it caused only a single amino acid substitution in the large T antigen and did not affect the small T antigen. Cells infected by the mutant virus produced normal-size small and large T antigens. The infected cells produced a 28,000-dalton fragment of the 48,000-dalton medium T antigen, whose size and tryptic peptide map were consistent with its being a truncated N-terminal fragment terminating at the new termination codon of the mutant. Immunoprecipitates of mutant-infected cell extracts did not show medium-T-antigen-associated protein kinase activity. The mutant virus replicated normally in mouse 3T6 cells and induced cellular DNA synthesis in resting mouse 3T3 cells, but it failed to transform rat or hamster cells, as judged by focus formation and growth in agar. The mutant complemented a tsA mutant which affects the large T antigen for transformation, implying that the mutant defect for transformation was in the medium T antigen. These results imply that the small T antigen and the large T antigen together are insufficient to cause transformation and support the conclusion that the medium T antigen is essential for cell transformation by polyoma virus. 相似文献
3.
Thermally inactivated simian virus 40 tsA58 mutant T antigen cannot initiate viral DNA replication in vitro. 总被引:5,自引:4,他引:1
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The mutation in the temperature-sensitive tsA58 mutant T antigen (Ala-438----Val) lies within the presumptive ATP-binding fold. We have constructed a recombinant baculovirus that expresses large quantities of the tsA58 T antigen in infected insect cells. The mutant T antigen mediated simian virus 40 origin-containing DNA (ori-DNA) synthesis in vitro to nearly the same extent as similar quantities of wild-type T antigen at 33 degrees C. However, if wild-type and tsA58 T antigens were heated at 41 degrees C in replication extracts prior to addition of template DNA, the tsA58 T antigen but not the wild type was completely inactivated. The mutant protein displayed greater thermosensitivity for many of the DNA replication activities of T antigen than did the wild-type protein. Some of the replication functions of tsA58 T antigen were differentially affected depending on the presence or absence of ATP during the preheating period. When tsA58 T antigen was preheated in the presence of ATP at 41 degrees C for a time sufficient to completely inactivate its ability to replicate ori-DNA in vitro, it displayed substantial ATPase and normal DNA helicase activities. Conversely, when preheated in the absence of nucleotide, it completely lost both ATPase and helicase activities. Preheating tsA58 T antigen, even in the presence of ATP, led to drastic reductions in its ability to bind to and unwind DNA containing the replication origin. The mutant T antigen also displayed thermosensitivity for binding to and unwinding nonspecific double-stranded DNA in the presence of ATP. Our results suggest that the interactions of T antigen with ATP that are involved in T-antigen DNA binding and DNA helicase activities are different. Moreover, we conclude, consistent with its phenotype in vivo, that the tsA58 T antigen is defective in the initiation but not in the putative elongation functions of T antigen in vitro. 相似文献
4.
Characterization of am404, an amber mutation in the simian virus 40 T antigen gene. 总被引:2,自引:2,他引:0
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We analyzed the biological activity of an amber mutation, am404, at map position 0.27 in the T antigen gene of simian virus 40. Immunoprecipitation of extracts from am404-infected cells demonstrated the presence of an amber protein fragment (am T antigen) of the expected molecular weight (67,000). Differential immunoprecipitation with monoclonal antibody demonstrated that am T antigen was missing the carboxy-terminal antigenic determinants. The amber mutant was shown to be defective for most of the functions associated with wild-type T antigen. The mutant did not replicate autonomously, but this defect could be complemented by a helper virus (D. R. Rawlins and N. Muzyczka, J. Virol. 36:611-616, 1980). The mutant failed to transform nonpermissive rodent cells and did not relieve the host range restriction of adenovirus 2 in monkey cells. However, stimulation of host cell DNA, whose functional region domain has been mapped within that portion of the protein synthesized by the mutant, could be demonstrated in am404-infected cells. A number of unexpected observations were made. First, the am T antigen was produced in unusually large amounts in a simian virus 40-transformed monkey cell line (COS-1), but overproduction was not seen in nontransformed monkey cells regardless of whether or not a helper virus was present. This feature of the mutant was presumably the result of the inability of am T antigen to autoregulate, the level of wild-type T antigen in COS-1 cells, and the unusually short half-life of am T antigen in vivo. Pulse-chase experiments indicated that am T antigen had an intracellular half-life of approximately 10 min. In addition, although the am T antigen retained the major phosphorylation site found in simian virus 40 T antigen, it was not phosphorylated. Thus, phosphorylation of simian virus 40 T antigen is not required for the stimulation of host cell DNA synthesis. Finally, fusion of am404-infected monkey cells with Escherichia coli protoplasts containing appropriate procaryotic suppressor tRNAs showed that am404 is a suppressible nonsense mutation. 相似文献
5.
Linker insertion mutants of simian virus 40 large T antigen that show trans-dominant interference with wild-type large T antigen map to multiple sites within the T-antigen gene. 总被引:12,自引:10,他引:2
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Linker insertion mutants affecting the simian virus 40 (SV40) large tumor (T) antigen were constructed by inserting a 12-base-pair oligonucleotide linker into restriction endonuclease cleavage sites located within the early region of SV40. One mutant, with the insertion at amino acid 5, was viable in CV-1p and BSC-1 cells, indicating that sequences very close to the amino terminus of large T could be altered without affecting the lytic infection cycle of SV40. All other mutants affecting large T were not viable. In complementation assays between the linker insertion mutants and either a late-gene mutant, dlBC865, or a host range/helper function (hr/hf) mutant, dlA2475, delayed complementation was seen with the 6 of the 10 nonviable mutants. Of these 10 mutants, 5 formed plaques 3 to 4 days later than in control complementations, while complementation by one of the mutants, inA2827, with an insertion at amino acid 520, was delayed more than 1 week. Most mutants which showed delayed complementation replicated less well in Cos-1 cells than did a control mutant, dlA1209, which produced no T antigen. The replication of inA2827(aa520) was reduced by more than 90%. Similar interference with viral DNA replication was seen when CV-1, HeLa, or 293 cells were cotransfected with an origin-defective plasmid encoding wild-type large T antigen and with inA2827(aa520). Only one of the mutant T antigens, inA2807(aa303), was unstable. These results indicate that some of the mutant T antigens interfered with functions of wild-type T required for viral DNA replication. However, not all of the mutants which showed delayed complementation also showed interference with viral DNA replication. This indicates that mutant large T antigens may interfere trans dominantly with multiple activities of wild-type large T antigen. 相似文献
6.
Characterization of T antigen in cells infected with a temperature-sensitive mutant of simian virus 40. 总被引:15,自引:15,他引:0
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T antigen induced in African green monkey kidney cells by a temperature-sensitive mutant of simian virus 40, defective in a function required for cell transformation, was characterized. The number of T antigen-positive cells estimated by an immunofluorescent techniques was almost equal at permissive (32.5 C) and restrictive (38.5 C) temperatures, but was slightly reduced when the infected cells were incubated at a higher temperature (40.5 C). However, a complement fixation test indicated that the amount of T antigen induced by the mutant is not significantly different from that induced by wild-type virus at 40.5 C. These results suggest that the T antigen-inducing ability of the mutant is not defective. Two distinct molecular species of T antigen were induced by the mutant at the permissive temperature, whereas only one form was observed at the restrictive temperature. The larger molecular form (14 to 15S) induced by the mutant at the permissive temperature was more heat labile than that induced by wild-type virus, suggesting that the mutated gene product is a component of the larger molecular form. 相似文献
7.
Asp-286----Asn-286 in polyomavirus large T antigen relaxes the specificity of binding to the polyomavirus origin. 总被引:9,自引:8,他引:1
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We isolated revertants of a polyomavirus whose origin of DNA replication contains a point mutation in the palindrome to which large T antigen binds. Four independent second-site revertants contain an Asp-286----Asn-286 substitution in large T antigen. This mutant large T antigen activates replication of DNAs containing the mutant polyomavirus origin as well as replication of DNAs containing the wild-type origin; however, replication of DNAs with enhancer mutations is not activated by this large T antigen. The Asn-286 mutation occurs in a positively charge region of large T antigen near the location of several mutations which inactivate DNA replication. We suggest that this region of large T antigen is responsible for recognition of specific DNA sequences at the origin and that ionic forces are important for this interaction. 相似文献
8.
A mutant SV40 large T antigen interferes with nuclear localization of a heterologous protein 总被引:7,自引:0,他引:7
A mutant SV40 genome carrying a frameshift at the carboxyl terminus of the large T antigen failed to replicate SV40 DNA and to transform rat2 cells, although the altered region is known to be dispensable for these functions. The mutant T antigen also failed to localize normally in the nucleus and interfered with nuclear localization of at least one other nuclear protein, adenovirus fiber. A double mutant carrying an additional lesion in the nuclear localization signal was also localized in the cytoplasm, but regained the ability to transform rat2 cells and no longer affected the nuclear localization of fiber protein. We suggest that the frameshift T antigen may disrupt a mechanism required for nuclear localization of proteins. 相似文献
9.
Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants. 总被引:7,自引:27,他引:7
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To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants. 相似文献
10.
Large tumor antigen (T antigen) was extracted from SV40-infected African Green Monkey cells and purified to homogeneity by immunoaffinity chromatography. The purified T antigen preparations unwind DNA duplices of greater than 120 bp in a reaction which is dependent on magnesium ions and ATP hydrolysis. Based on these and other properties of the reaction we classify this newly discovered enzymatic activity as a eukaryotic DNA helicase. The helicase and the known ATPase function of T antigen cosediment with the mono- or dimeric 4-6 S form of T antigen, but not with higher T antigen aggregates. The helicase activity seems to be an intrinsic function of SV40 T antigen. First, several different T antigen-specific monoclonal antibodies interfere with the DNA unwinding activity; monoclonals which are known to reduce the T antigen-specific ATPase most strongly inhibited the helicase reaction. Second, mutant T antigens with impaired ATPase function also showed a reduced DNA unwinding activity. 相似文献
11.
Wild-type and J domain mutant simian virus 40 large T antigens alter the cell cycle and bud morphology of Saccharomyces cerevisiae. In contrast, yeast cells expressing mutant T antigen lacking the carboxy-terminal 150 aa exhibit normal morphology, indicating that this region of T antigen is required for cell cycle disruption. 相似文献
12.
Transformation of precrisis human cells by the simian virus 40 cytoplasmic-localization mutant pSVCT3 is accompanied by nuclear T antigen. 总被引:2,自引:0,他引:2
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pSVCT3 is a cytoplasmic-localization mutant of simian virus 40 (SV40) isolated from the SV40 adenovirus 7 hybrid virus (PARA) and cloned into plasmid PBR. The large T antigen of pSVCT3 accumulates in the cytoplasm of infected monkey cells instead of being transported to the nucleus. The sole change in CT3 large T antigen is amino acid residue 128 (Lys----Asn). Transformation of precrisis rodent cells by pSVCT3 is negligible, whereas the frequency of transformation of established rodent cell lines by pSVCT3 is comparable to that of wild-type SV40. According to the model, in which transformation of precrisis cells involves the combined oncogenic action of both nuclear and cytoplasmic gene products, we predicted that pSVCT3 would localize in the cytoplasm of human cells and would therefore at most only partially and rarely transform precrisis human cells. We have found that pSVCT3 is able to transform precrisis human cells at high frequency. Furthermore, pSVCT3-transformed human precrisis cells relocalized T antigen to their nuclei. The relocalization of large T antigen was not dependent on cell growth. Wild-type and pSVCT3-transformed human cell lines both have about five copies of integrated SV40 DNA. SV40 virus-specific proteins, including the 100,000-molecular-weight super large T antigen, were expressed in pSVCT3-transformed human cells. Our results suggest that molecules in precrisis human cells, but not cells of other species, are able to complement the cytoplasmic-localization defect of the CT3 mutant large T antigen. 相似文献
13.
Interactions between polyomavirus medium T antigen and three cellular proteins of 88, 61, and 37 kilodaltons. 总被引:16,自引:6,他引:10
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Affinity-purified medium T antigen of wild-type polyomavirus and dl8, a transforming mutant with a deletion in the medium T gene, is associated with three cellular proteins with apparent molecular weights of 88,000 (88K protein), 61,000 (61K protein), and 37,000 (37K protein). Medium T antigen encoded by the nontransforming hrt mutants fails to associate with these proteins, whereas medium T antigen of the nontransforming mutant dl1015 is able to do so. Medium T antigen of the nontransforming mutant dl23 binds to the 61K and 37K proteins; however, binding to the 88K protein is uncertain. The pattern of complex formation between these proteins and medium T antigen resembles that of pp60c-src and medium T antigen. The binding of medium T antigen to the 88K, 61K, and 37K proteins, as well as to pp60c-src, might represent a necessary but insufficient step in transformation. By mixing extracts from infected and uninfected cells, complex formation between medium T antigen and the 88K, 61K, and 37K proteins can be demonstrated in vitro. Pulse-chase experiments indicated that in vivo the association between medium T antigen and the 61K and 37K proteins is a slow process. The latter two proteins are probably bound to each other in uninfected cells. On two-dimensional gels of whole-cell extract, the 61K protein comigrated with a minor protein with an isoelectric point of 5.2. The 61K protein was neither phosphorylated nor glycosylated. Polyomavirus tumor serum precipitated the 61K and 37K proteins independently of medium T antigen. Therefore, the 61K protein or the 37K protein or both have the properties of a cellular tumor antigen. 相似文献
14.
Mechanisms of interference with simian virus 40 (SV40) DNA replication by trans-dominant mutants of SV40 large T antigen.
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Mutations at multiple sites within the simian virus 40 (SV40) early region yield large T antigens which interfere trans dominantly with the replicative activities of wild-type T antigen. A series of experiments were conducted to study possible mechanisms of interference with SV40 DNA replication caused by these mutant T antigens. First, the levels of wild-type T antigen expression in cells cotransfected with wild-type and mutant SV40 DNAs were examined; approximately equal levels of wild-type T antigen were seen, regardless of whether the cotransfected mutant was trans dominant or not. Second, double mutants that contained the mutation of inA2827, a strong trans-dominant mutation with a 12-bp linker inserted at the position encoding amino acid 520, and various mutations in other parts of the large-T-antigen coding region were constructed. The trans-dominant interference of inA2827 was not affected by second mutations within the p105Rb binding site or the amino or carboxy terminus of large T antigen. Mutation of the nuclear localization signal partially reduced the trans dominance of inA2827. The large T antigen of mutant inA2815 contains an insertion of 4 amino acids at position 168 of large T; this T antigen fails to bind SV40 DNA but is not trans dominant for DNA replication. The double mutant containing the mutations of both inA2815 and in A2827 was not trans dominant. The large T antigen of dlA2433 lacks amino acids 587 to 589, was unstable, and failed to bind p53. Combining the dlA2433 mutation with the inA2827 mutation also reversed the trans dominance completely, but the effect of the dlA2433 mutation on trans dominance can be explained by the instability of this double mutant protein. In addition, we examined several mutants with conservative point mutations in the DNA binding domain and found that most of them were not trans dominant. The implications of the results of these experiments on possible mechanisms of trans dominance are discussed. 相似文献
15.
Cell transformation in vivo seems to be a multistep process. In in vitro studies certain combinations of two oncogenes, a cytoplasmic gene product together with a nuclear gene product, are sufficient to transform primary rodent cells. Polyoma virus large T antigen can immortalize and, in cooperation with polyoma virus middle T antigen, transform primary cells. On the other hand mutant mouse p53 can also immortalize and, in cooperation with an activated Ha-ras oncogene, transform primary cells. In the present study we analyzed whether mutant p53 can replace polyoma virus large T antigen in a cell transformation assay with polyoma virus middle T antigen. Transfection of mutant p53 alone resulted in a cell line which had retained the actin cable network, grew poorly in medium with low concentration of serum, and failed to grow in semisolid agar. Cotransfection of mutant p53 together with polyoma virus middle T led to cells which grew in medium containing low serum concentration, grew well in semisolid agar, and displayed an altered morphology with the tendency to overgrow the normal monolayer. By these criteria these cells were considered fully transformed. The rate of p53 synthesis was similar in both cell lines. However, only p53 from the transformed cell line turned out to be stable. Cells transformed by mutant p53 and polyoma virus middle T expressed nearly the same amount of the c-src-encoded pp60c-src protein as cells transformed by the same p53 and cotransfected activated Ha-ras oncogene. However, only the polyoma virus middle T/p53-transformed cells exhibited an elevated level of pp60c-src-specific tyrosine kinase activity. Thus, despite different mechanisms leading to cell transformation, mutant p53 can replace polyoma virus large T antigen and polyoma virus middle T can replace the activated Ha-ras oncogene in cell transformation. 相似文献
16.
17.
Dimers and complexes with p53 are the prevalent oligomeric forms of a transforming nonkaryophilic T antigen of simian virus 40. 总被引:1,自引:0,他引:1
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The oligomers formed by a mutant nonkaryophilic large T antigen of simian virus 40, which lacks residues 110 through 152 of normal large T antigen and transforms only established cells (L. Fischer-Fantuzzi and C. Vesco, Proc. Natl. Acad. Sci. USA 82:1891-1895, 1985), were found to consist predominantly of dimers. Anti-p53 antibodies precipitated 14 to 16S complexes containing the mutant nonkaryophilic large T antigen and p53 from extracts of transformed cells, and anti-p53 indirect immunofluorescence stained these cells in the cytoplasm. 相似文献
18.
Mapping the transcriptional transactivation function of simian virus 40 large T antigen 总被引:25,自引:19,他引:6
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T antigen is able to transactivate gene expression from the simian virus 40 (SV40) late promoter and from several other viral and cellular promoters. Neither the mechanisms of transactivation by T antigen nor the regions of T antigen required for this activity have been determined. To address the latter point, we have measured the ability of a set of SV40 large T antigen mutants to stimulate gene expression in CV-1 monkey kidney cells from the SV40 late promoter and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Transactivation, although reduced, was retained by an N-terminal 138-amino-acid fragment of T antigen. Mutants with alterations at various locations within the N-terminal 85 amino acids transactivated the RSV LTR promoter less well than did wild-type T antigen. Most of these were also partially defective in their ability to transactivate the SV40 late promoter. Two mutants with lesions in the DNA-binding domain that were unable to bind to SV40 DNA were completely defective for transactivation of both promoter, while a third mutant with a lesion in the DNA-binding domain which retained origin-binding activity transactivated both promoters as well as did wild-type T antigen. Only a low level of transactivation was seen with mutant T antigens which had lesions in or near the zinc finger region (amino acids 300 to 350). Mutations which caused defects in ATPase activity, host range/helper function, binding to p53, binding to the retinoblastoma susceptibility protein, or nuclear localization had little or no effect on transactivation. These results suggest that N-terminal portion of T antigen possesses an activation activity. The data are consistent with the idea that the overall conformation of T antigen is important for transactivation and that mutations in other regions that reduce or eliminate transactivation do so by altering the conformation or orientation of the N-terminal region so that its ability to interact with various targets is diminished or abolished. 相似文献
19.
20.
Functional characterization of temperature-sensitive mutants of simian virus 40 large T antigen.
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We investigated the molecular properties of eight temperature-sensitive mutants of simian virus 40 large T antigen (tsA mutants). The mutants have single amino acid substitutions that block DNA replication at 39 to 41 degrees C in vivo. In vitro, five of the mutant proteins were highly sensitive to a brief heat shock at 39 degrees C, while the three remaining proteins were only partially sensitive at 41 degrees C. We characterized the five most defective mutant proteins, using a variety of biochemical assays for replication functions of T antigen. Heat shock of purified T antigen with a mutation at amino acid 422 significantly impaired the oligomerization, origin-binding, origin-unwinding, ATPase, and helicase functions of T antigen. In contrast, substitution of amino acid 186, 357, 427, or 438 had more selective, temperature-sensitive effects on T-antigen functions. Our findings are consistent with the conclusion that T antigen functions via a hierarchy of interrelated domains. Only the ATPase activity remained intact in the absence of all other functions. Hexamer formation appears to be necessary for core origin-unwinding and helicase activities; the helicase function also requires ATPase activity. All five tsA mutants were impaired in functions important for the initiation of DNA replication, but three mutants retained significant elongation functions. 相似文献