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E Winocour

Publications and source records attributed to E Winocour.

At least 37 records · Page 2Linked to original sources
PubMed1980

Structure of a newly isolated variant of Simian virus 40 DNA containing monkey DNA and its similarity to previously isolated variants.

The structure of a newly and independently isolated defective variant of simian virus 40 that contains covalently linked monkey and SV40 DNA sequences is described. This variant, termed 290, has a structure essentially identical with a previously isolated and characterized variant named CVP8/1/P2 (Eco RI res). The structural similarities include the monkey (host) DNA segment that is combined with viral DNA sequences, the particular viral DNA segment that is present, and the arrangement of these within the defective genome. The monkey DNA segment contains sequences derived from both low and high reiteration frequency monkey DNA. The viral sequences include the origin of replication. The separate isolation of essentially identical variants suggests a high level of specificity in the events leading to the formation and amplification of this type of defective genome.

Animals↗
PubMed1980

Differential affinities of simian virus 40 large tumor antigen for DNA.

The binding of simian virus 40 (SV40) large tumor antigen (T antigen) to DNA was analyzed by using the salt-sensitive affinities of the protein for various DNAs immobilized on cellulose. At least two types of interactions could be distinguished that differed in their stability. Higher salt concentrations were required to elute T antigen from SV40 DNA than from calf thymus DNA; and even greater salt concentrations were required for the lution of T antigen from multiorigin SV 40 DNA compared to wild-type SV40 DNA. This would indicate that T antigen can bind weakly or strongly to DNA, depending on the DNA sequence. It was also found that a greater proportion of rapidly labeled or newly synthesized T antigen binds more efficiently and tightly to multiorigin SV40 DNA than to long-labeled or older forms of T antigen. This approach can be utilized not only to distinguish between different forms of T antigens which vary in their affinities for DNA but also for rapidly obtaining highly enriched T antigen preparations.

Animals↗
PubMed1980

Indiscriminate recombination in simian virus 40-infected monkey cells.

DNA transfection of African green monkey BSC-1 cells with simian virus 40 (SV40) DNA and bacterial virus phi X174 replicative form DNA ("cotransfection") yielded stocks containing SV40/phi X174 recombinant virus, which was detected by an infectious-center in situ plaque hybridization procedure and which was sensitive to anti-SV40 antiserum. The recombinant virus replicated during serial passage. Restriction endonuclease cleavage of the SV40/phi X174 DNA indicated that several different types of recombinant DNA structures had arisen. Similar SV40 DNA cotransfection experiments with polyoma virus DNA, bacterial plasmid (pBR322) DNA, and a plasmid-cloned segment of the mouse genome (coding for intracisternal type A particles) yielded stocks that generated recombinant plaques as judged by in situ plaque hybridization with the appropriate labeled probe. It appears, therefore, that an active indiscriminate recombination process, incapable of distinguishing between diverse DNAs of prokaryotic and eukaryotic origin, occurs in SV40-infected monkey cells.

Animals↗
PubMed1978

Cellular and cell-free synthesis of simian virus 40 T-antigens in permissive and transformed cells.

mRNA extracted from a variety of simian virus 40 (SV40)-infected monkey cell lines directs the cell-free synthesis of viral T-antigen polypeptides with molecular weights estimated as 90,000 and 17,000. However, the size, abundance, and distribution of these T-antigens synthesized in vivo vary greatly over a range of permissive and transformed cell lines. To establish whether differences in the size of T-antigen polypeptides can be correlated with the transformed or lytic state, recently developed lines of SV40-transformed monkey cells that are permissive to lytic superinfection were analyzed for T-antigen. In these cells, regardless of the state of viral infection, the size and pattern of T-antigen are the same. However, species differences in the largest size of T-antigen are the same. However, species differences in the largest size of T-antigen do exist. In addition to the 90,000 T-antigen, mouse SV3T3 cells contain a 94,000 T-antigen polypeptide as well. Unlike the size variations in monkey cells, which are due to modification of T-antigen polypeptides, the 94,000 SV3T3 T-antigen results from an altered mRNA, since the cell-free products of SV3T3 mRNA also contains the 94,000 T-antigen polypeptide.

Animals↗
PubMed1977

Inhibition of viral protein synthesis in monkey cells treated with interferon late in simian virus 40 lytic cycle.

We have investigated the effect of interferon on SV40 gene expression late in the lytic cycle, after early functions have been expressed and viral DNA replication has been initiated. Whereas pretreatment with interferon prior to infection reduces the amount of early SV40 RNA, post-infection treatment does not inhibit viral RNA synthesis. Viral 19S and 16S RNA species are found undiminished in quantity and poly(A) content. Despite the apparent normalcy of viral RNA classes, however, there is a marked reduction in the synthesis of their protein products, both T antigen and capsid polypeptides. The association of viral RNA with heavy polyribosomes is strongly reduced. On the other hand, there is no degradation of nonviral polyribosomes and the synthesis of most cellular proteins continues. These experiments demonstrate that late in infection, interferon treatment results in an inhibition of viral mRNA translation.

Antigens, Viral↗
PubMed1977

Cell-free translation of simian virus 40 early messenger RNA coding for viral T-antigen.

Simian virus 40 (SV40) mRNA was isolated by hybridization of cytoplasmic RNA, from SV40-infected BS-C-1 monkey cells early in lytic infection, to SV40 DNA immobilized on Sepharose. The early viral mRNA, when added to a wheat-germ translation system, directed the synthesis of a unique class of products including a 90,000 molecular weight (Mr) polypeptide. It was found that this 90,000 Mr product as well as a prominent 17,000 Mr polypeptide could be specifically immunoprecipitated with hamster antiserum to SV40 T-antigen, but not with hamster control serum. Similar immunoprecipitation of extracts of SV40-infected cells with hamster anti-T serum yielded 90,000 Mr and 17,000 Mr polypeptides; these polypeptides were not found in immunoprecipitates of uninfected cell extracts. SV40 cRNA, prepared by asymmetric transcription of plaque-purified SV40 DNA, directed the cell-free synthesis of several products, including a 70,000 Mr polypeptide that could be specifically immunoprecipitated with anti-T serum. However, no T-antigen-related polypeptide was found in infected cells that corresponded in size to the major immunoprecipitated cRNA product.

Animals↗
PubMed1977

Properties of permissive monkey cells transformed by UV-irradiated simian virus 40.

African green monkey cells (CV1 line) were infected with UV-irradiated simian virus 40 (SV40), and permissive lines of stably transformed cells were established. These cell lines display the SV40 T-antigen and the growth characteristics typical of nonpermissive transformed cells (e.g., reduced cell density inhibition, reduced serum dependence, ability to overgrow normal cells, and colony formation in soft agar). The level of permissiveness to superinfecting SV40 is fully comparable with that of nontransformed CV1 and BSC-1 lines. The transformed monkey lines also support SV40 plaque production under agar. By Cot analysis, the transformed permissive cells contain, on an average, 1 to 2 SV40 genome equivalents, and the majority of the viral sequences are associated with the high-molecular-weight cellular DNA. No spontaneous production of infectious SV40 has been observed. The transformed permissive monkey cells failed to support the replication of SV40 tsA mutants at the restrictive temperature. To account for this, it is suggested that the gene A product has separate functions for transformation and initiation of viral DNA synthesis, and only the former function is expressed in the transformed permissive monkey cells.

Animals↗
PubMed1977

Recombination between endogenous and exogenous simian virus 40 genes. I. Rescue of a simian virus 40 temperature-sensitive mutant by passage in permissive transformed monkey lines.

Passage of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 at the permissive temperature in each of three permissive lines of SV40-transformed monkey CV1 cells resulted in the emergence of temperature-insensitive virus, which plated like wild-type SV40 at the restrictive temperature on normal CV1 cells. In independent experiments, the amount of temperature-insensitive virus that appeared after passage on transformed cells was from 10(3)- to 10(6)-fold greater than the amount of ts-revertant virus that appeared after an equal number of passages in nontransformed CV1 cells. The virus rescued by passage on transformed cells bred true upon sequential plaque purification, plated on normal CV1 cells with single-hit kinetics at the restrictive temperature, and displayed no selective growth advantage on transformed cells compared to non-transformed cells. Hence, the reversion of the ts phenotype is neither due to complementation effects nor to the selection of preexisting revertants, which grow better on transformed cells. In the accompanying article (T. Vogel et al., J. Virol. 24:541-550, 1977), we present biochemical evidence that the rescue of tsD202 mediated by passage on transformed cells is due to recombination with the resident SV40 genome. Parallel experiments in which tsA, tsB, and tsC SV40 mutants were passaged in each of the three permissive lines of SV40-transformed monkey cells resulted in either only borderline levels of rescue (tsA mutants) or no detectable rescue (tsB and tsC mutants). Evidence is presented that the resident SV40 genome of the transformed monkey lines is itself a late ts mutant, and we suggest that this accounts for the lack of detectable rescue of the tsB and tsC mutants. We furthermore suggest that the borderline level of rescue observed with two tsA mutants is related to a previous finding (Y. Gluzman et al., J. Virol. 22:256-266, 1977) which indicated that the resident SV40 genome of the permissive transformed monkey cells is defective in the function required for initiation of viral DNA synthesis.

Cell Line↗
PubMed1977

Recombination between endogenous and exogenous simian virus 40 genes. II. Biochemical evidence for genetic exchange.

The genome of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 rescued by passage on transformed permissive monkey lines (see accompanying paper [Y. Gluzman et al., J. Virol. 24:534-540, 1977]) was analyzed by restriction endonuclease cleavage mapping to obtain biochemical evidence that the rescue of the ts phenotype results from recombination with the resident SV40 genome of the transformed cell. It was demonstrated that the endonuclease R. HaeIII cleavage site, which is located at 0.9 map unit in the standard viral genome (and which is in the proximity of the known map position of the tsD lesion), is missing in the DNAs of the parental tsD202 virus and of three independent revertants of tsD202. In contrast, this cleavage site was shown to be present in the DNAs of four out of five independently derived rescued D202 populations and in the DNA of the SV40 strain, 777, used to transform the monkey cells. Comparison of the endonuclease R. Hin(II + III) cleavage patterns of SV40 strain 777 DNA and tsD202 DNA revealed differences in the electrophoretic mobilities of Hin fragments A, B, and F. However, the corresponding Hin fragments from all four rescued D202 genomes were identical in their mobilities to those of tsD202 DNA, indicating that these regions of the rescued D202 genome are characteristic of the tsD202 parent. We conclude, therefore, that the genome of the rescued D202 virus is a true recombinant, since it contains restriction endonuclease cleavage sites characteristic of both parents, the endogenous resident SV40 genome of the transformed monkey cells and the exogenous tsD202 mutant.

Cell Line↗