scholarly journals Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

1985 ◽  
Vol 5 (8) ◽  
pp. 2019-2028 ◽  
Author(s):  
T Michaeli ◽  
C Prives
Keyword(s):  
Xenopus Laevis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Oocyte Nucleus ◽  
Xenopus Laevis Oocytes ◽  
Large T Antigen ◽  
Viral Dna ◽  
Infected Monkey ◽  
Sv40 Dna

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.

Regulation of simian virus 40 gene expression in Xenopus laevis oocytes

1985 ◽  
Vol 5 (8) ◽  
pp. 2019-2028
Author(s):  
T Michaeli ◽  
C Prives
Keyword(s):  
Xenopus Laevis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Oocyte Nucleus ◽  
Xenopus Laevis Oocytes ◽  
Large T Antigen ◽  
Viral Dna ◽  
Infected Monkey ◽  
Sv40 Dna

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.

Bidirectional promoter elements of simian virus 40 are required for efficient replication of the viral DNA

1986 ◽  
Vol 6 (10) ◽  
pp. 3513-3522
Author(s):  
G Z Hertz ◽  
J E Mertz
Keyword(s):  
Base Pair ◽  
Simian Virus 40 ◽  
Bidirectional Promoter ◽  
Simian Virus ◽  
T Antigen ◽  
Viral Dna ◽  
Promoter Elements ◽  
Frameshift Deletion ◽  
Efficient Replication ◽  
Sv40 Dna

Mutants of simian virus 40 (SV40) lacking parts of the 72- and 21-base-pair repeat regions were made deficient in large T antigen by recombination with dlA 4000, a mutant containing a frameshift deletion near the amino terminus of the T antigen genes. These double mutants were transfected into COS cells, and the amounts of replicated viral DNA were measured at various times thereafter. It was found that deletion of either the 72- or 21-base-pair repeat region did not significantly reduce the accumulation of viral DNA. However, cells transfected with mutants lacking both of these promoter elements accumulated 100-fold less viral DNA than cells transfected with wild-type SV40. This indicates that the 72- and 21-base-pair repeat regions are each sufficient for supplying a function required for efficient replication of SV40 DNA. In addition, the ability of either of these regions to support efficient replication was gradually reduced as the number of promoter elements within each was decreased. Since the 72- and 21-base-pair repeat regions bidirectionally induce transcription, our results indicate that bidirectional promoter elements play a role in the replication of viral DNA. However, fewer of these elements are required for efficient replication than for efficient transcription.

Kinetics of Accumulation and Processing of Simian Virus 40 RNA in Xenopus laevis Oocytes Injected with Simian Virus 40 DNA

1982 ◽  
Vol 2 (12) ◽  
pp. 1581-1594
Author(s):  
Timothy J. Miller ◽  
Donald L. Stephens ◽  
Janet E. Mertz
Keyword(s):  
Xenopus Laevis ◽  
Transcription Termination ◽  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
Xenopus Laevis Oocytes ◽  
Sequence Specificity ◽  
The Stability ◽  
Sv40 Dna ◽  
Kinetics Of

We examined the kinetics of accumulation and processing of simian virus 40 (SV40) RNA in stage 6 oocytes of Xenopus laevis microinjected intranuclearly with SV40 DNA. The rates of synthesis and degradation, cellular distribution, size, and sequence specificity of radiolabeled SV40-specific and endogenous oocyte RNA were determined. The kinetics of accumulation of SV40 RNA were biphasic, with greater than 90% of the viral RNA turning over in the nucleus with a half-life of 20 to 40 min. Although most of the primary transcription products were multigenomic in length, some stable polyadenylated SV40-specific RNA similar in size and sequence to late 19S mRNA accumulated in the cytoplasm with time. Differences in strand preference, efficiencies of transcription termination and polyadenylation, and the splice sites used in the synthesis and processing of SV40 RNA in Xenopus oocytes and monkey cells were noted. However, these differences were quantitative, rather than qualitative, in nature. Consequently, they are probably due to regulatory rather than mechanistic differences between the two cell types. We therefore conclude that Xenopus oocytes may be a useful system for studying both mechanistic and cell type-specific regulatory aspects of mRNA biogenesis from cloned DNAs. However, since only a small percentage of the initially synthesized RNA ends up in stable mRNA, it will be important to determine whether mutants of cloned DNAs that produce abnormal amounts of stable mRNAs are altered in promotion and initiation of RNA synthesis, transcription termination, RNA processing, or the stability of the resultant mRNAs.

scholarly journals Simian Virus 40 Large T Antigen Can Specifically Unwind the Central Palindrome at the Origin of DNA Replication

2009 ◽  
Vol 83 (7) ◽  
pp. 3312-3322 ◽  
Author(s):  
Weiping Wang ◽  
Daniel T. Simmons
Keyword(s):  
Dna Replication ◽  
Critical Role ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Central Origin ◽  
Sv40 Dna

ABSTRACT The hydrophilic channels between helicase domains of simian virus 40 (SV40) large T antigen play a critical role in DNA replication. Previous mutagenesis of residues in the channels identified one class of mutants (class A: D429A, N449S, and N515S) with normal DNA binding and ATPase and helicase activities but with a severely reduced ability to unwind origin DNA and to support SV40 DNA replication in vitro. Here, we further studied these mutants to gain insights into how T antigen unwinds the origin. We found that the mutants were compromised in melting the imperfect palindrome (EP) but normal in untwisting the AT-rich track. However, the mutants' defect in EP melting was not the major reason they failed to unwind the origin because supplying an EP region as a mismatched bubble, or deleting the EP region altogether, did not rescue their unwinding deficiency. These results suggested that specific separation of the central palindrome of the origin (site II) is an essential step in unwinding origin DNA by T antigen. In support of this, wild-type T antigen was able to specifically unwind a 31-bp DNA containing only site II in an ATPase-dependent reaction, whereas D429A and N515S failed to do so. By performing a systematic mutagenesis of 31-bp site II DNA, we identified discrete regions in each pentanucleotide necessary for normal origin unwinding. These data indicate that T antigen has a mechanism to specifically unwind the central palindrome. Various models are proposed to illustrate how T antigen could separate the central origin.

scholarly journals Interaction of the Transcription Factor TFIID with Simian Virus 40 (SV40) Large T Antigen Interferes with Replication of SV40 DNA In Vitro

1999 ◽  
Vol 73 (2) ◽  
pp. 1099-1107 ◽  
Author(s):  
Utz Herbig ◽  
Klaus Weisshart ◽  
Poonam Taneja ◽  
Ellen Fanning
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Cell Extracts ◽  
Sv40 Dna ◽  
Replication Activity

ABSTRACT Simian virus 40 (SV40) large tumor (T) antigen is the major regulatory protein that directs the course of viral infection, primarily by interacting with host cell proteins and modulating their functions. Initiation of viral DNA replication requires specific interactions of T antigen bound to the viral origin of DNA replication with cellular replication proteins. Transcription factors are thought to stimulate initiation of viral DNA replication, but the mechanism of stimulation is poorly understood. Since the transcription factor TATA-binding protein (TBP) binds to sequences within the origin of replication and interacts specifically with T antigen, we examined whether TBP complexes stimulate SV40 DNA replication in vitro. On the contrary, we found that depletion of TBP complexes from human cell extracts increased their ability to support viral DNA replication, and readdition of TBP complexes to the depleted extracts diminished their activity. We have mapped the sites of interaction between the proteins to residues 181 to 205 of T antigen and 184 to 220 of TBP. Titration of fusion proteins containing either of these peptides into undepleted cell extracts stimulated their replication activity, suggesting that they prevented the T antigen-TBP interaction that interfered with replication activity. TBP complexes also interfered with origin DNA unwinding by purified T antigen, and addition of either the T antigen or the TBP fusion peptide relieved the inhibition. These results suggest that TBP complexes associate with a T-antigen surface that is also required for origin DNA unwinding and viral DNA replication. We speculate that competition among cellular proteins for T antigen may play a role in regulating the course of viral infection.

Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation

1984 ◽  
Vol 4 (6) ◽  
pp. 1125-1133
Author(s):  
M M Manos ◽  
Y Gluzman
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Atpase Activity ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Oncogenic Transformation ◽  
Viral Dna ◽  
Viral Dna Replication

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.

Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants

1984 ◽  
Vol 4 (8) ◽  
pp. 1653-1656
Author(s):  
K Van Doren ◽  
Y Gluzman
Keyword(s):  
Cell Lines ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Origin Of Replication ◽  
Viral Dna ◽  
Early Region ◽  
Transformed Cell Lines ◽  
Sv40 Dna

The origin-defective simian virus 40 (SV40) mutant 6-1 has been useful in transforming human cells (Small et al., Nature [London] 296:671-672, 1982; Nagata et al., Nature [London] 306:597-599, 1983). However, the low efficiency of transformation achieved by DNA transfection is a major drawback of the system. To increase the efficiency of SV40-induced transformation of human fibroblasts, we used recombinant adenovirus-SV40 virions which contain a complete SV40 early region including either a wild-type or defective (6-1) origin of replication. The SV40 DNA was cloned into the adenovirus vector in place of early region 1. Cell lines transformed by viruses containing a functional origin of replication produced free SV40 DNA. These cell lines were subcloned, and some of the subclones lost the ability to produce free viral DNA. Subclones that failed to produce free viral DNA were found to possess a mutated T antigen. Cell lines transformed by viruses containing origin-defective SV40 mutants did not produce any free DNA. Because of the high efficiency of transformation, we suggest that the origin-defective chimeric virus is a convenient system for establishing SV40-transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5.

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