Simian Virus 40-infected Monkey Cells Research Articles

Efficient expression of small RNA polymerase III genes from a novel Simian virus 40 vector and their effect on viral gene expression

In the past, simian virus 40 (SV40) has been used as a cloning vehicle to clone foreign genes by substituting portions of the viral genome vital for viral replication. Propagation of these defective viruses required a helper virus and the recombinant viruses obtained could be grown only as a mixture. In this study, we describe a novel nondefective SV40 vector to clone small RNA polymerase III genes. Two small RNA polymerase III genes, an amber suppressor human serine tRNA gene and the adenovirus (Ad) VAI RNA gene, were cloned in the intron region of the large-T antigen gene of SV40 after deleting DNA sequences coding for the small-t polypeptide. The recombinant viruses grew to wild type levels and showed no growth defects. When CV-1p cells were infected with these viruses, the cloned RNA polymerase III genes were expressed at high levels at late times. Interestingly, large amounts VAI RNA in CV-1p cells infected with SV40-VA recombinant virus, did not enhance translation of viral mRNAs significantly but did lead to a 3 to 4 fold increase in the steady state levels of large-T mRNA suggesting a novel function for VAI RNA in SV40 infected monkey cells. Furthermore, VAI mutants which fail to function in Ad infected human cells also failed to enhance the levels of large-T mRNAs in monkey cells infected with SV40. The simple SV40 vector described here may be useful to study the structure and function of small RNA polymerase III genes in the context of a eucaryotic chromosome. In addition, the nondefective recombinant SV40 which expresses the suppressor tRNA gene at high levels may provide a useful helper system to propagate animal viruses with amber mutations in essential genes.

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.

Subcellular Localization of Simian Virus 40 Large Tumor Antigen

The distribution of simian virus 40 large tumor antigen in subcellular fractions from simian virus 40-transformed hamster (H-50) and mouse (VLM) cells and from simian virus 40-infected monkey cells was determined. Solubilized [(35)S]-methionine- or (32)P(i)-labeled surface membrane and nuclear fractions were prepared, immunoprecipitated with hamster anti-T serum, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor antigen with an apparent molecular weight of approximately 96,000 was detected in both subcellular fractions. Minor components of approximately 68,000 and approximately 56,000 with anti-T reactivity which labeled with [(35)S]methionine were also detected in both fractions from H-50 cells, as were components of approximately 140,000 and approximately 56,000 from VLM cells. The 56,000 component appeared to be greatly reduced in (32)P(i)-labeled surface membrane fractions. Normal cells or cells transformed with a heterologous agent, such as polyoma virus or a chemical carcinogen, lacked immunoprecipitable tumor antigen. Cell fractionation was monitored by [(3)H]thymidine labeling, NADH-diaphorase activity, and Na(+)-K(+)-dependent ATPase activity. These analyses revealed only trace contamination of surface membranes by nuclei, extremely low levels of nuclear rupture during homogenization, and an approximate 10-fold enrichment of surface membrane. Reconstruction experiments demonstrated that soluble tumor antigen failed to associate or copurify with surface membranes during fractionation procedures. These results indicate the presence of a protein in the plasma membrane of cells transformed or infected by simian virus 40 that is immunologically indistinguishable from nuclear tumor antigen.

A procedure for the quantitation of relaxed closed circular DNA in the presence of superhelical DNA: An improved fluorometric assay for nicking-closing enzyme

The procedure described here takes advantage of the recently discovered single-strand-specific endonuclease activity of snake-venom phosphodiesterase to convert supercoiled PM2 DNA (DNA I′), but not relaxed DNA (DNA I′), to open forms of DNA. The DNA I' was quantitated using a fiuorometric method for covalently closed circular DNA ( A. R. Morgan and D. E. Pulleyblank, 1974, Biochem. Biophys. Res. Commun. 61, 396–403). The percentage of DNA I′ in mixtures of DNAs I and I′ can be determined to ±l%. The procedure was used as an assay for a nicking-closing enzyme activity partially purified from simian virus 40-infected monkey cells. The assay is linear from 0 to 0.4 μg DNA I′ produced and reproducible to ±0.01 μg DNA I′.

Nucleoprotein complexes from simian virus 40-infected monkey cells: Association of viral DNA with histones and the major viral structural protein

Nucleoprotein complexes containing progeny SV40 DNA were isolated at 48 hr postinfection from infected permissive cells incubated in normal growth medium ( +), in growth medium lacking arginine (Arg −), and in normal growth medium containing 5-bromodeoxyuridine (BrdU +) and characterized. Virus production in both Arg − and Brdu + cells is markedly inhibited. The complexes were purified by two successive sucrose gradient sedimentations to remove cellular proteins and then by sedimentation onto a cushion of saturated KBr solution to remove contaminating virus and empty capsids. The complexes extracted from Arg +, Arg −, and BrdU + cells were found to have the same sedimentation coefficient of 57 S and the same density of 1.43 g/cm 3 after glutaraldehyde fixation and to contain supercoiled SV40 DNA. The major protein species of the complex are histones and the major viral capsid protein (VP1). The Arg + complex contains predominately histones, whereas the BrdU + complex contains predominately the major capsid protein. The Arg − complex contains substantial amounts of both histones and the major capsid protein. Complexes were also isolated from Arg + cells at different times (24, 34, and 48 hr) after infection and characterized. More VPl, relative to histones, was found in the complex isolated at early than at late time after infection. Thus, the BrdU + and Arg − complexes may represent complexes formed at different times in a lytic infection.

Mechanism of Interferon Action: Further Evidence for Transcription as the Primary Site of Action in Simian Virus 40 Infection

Interferon inhibits the replication of simian virus 40 (SV40) in monkey cells and reduces markedly the formation of both early virus protein (i.e. SV40 T antigen) and early SV40 RNA. This suggests that in SV40 infection interferon acts primarily by inhibiting transcription. To test this conclusion further, we examined alternative mechanisms which might explain these results and made the following observations. (1) The quantity of input SV40 DNA in the nucleus 24 h post infection (p.i.) was the same in interferon-treated and control cells. Thus interferon does not appear to diminish the quantity of SV40 DNA template available for transcription. (2) Chemical inhibitors of protein synthesis did not mimic the selective inhibition of early SV40 RNA formation induced by interferon, indicating that the transcription of early SV40 RNA is not dependent upon the prior synthesis of any virus-induced protein. Thus a block in translation cannot readily explain the reduced formation of early SV40 RNA in interferon-treated cells. (3) Fractionation of SV40 infected cells after a one-hour labelling period showed that interferon produced a comparable reduction in the quantity of early SV40 RNA in the nucleus and the cytoplasm. Thus the observed inhibition of early SV40 RNA is not due solely (if at all) to enhanced cytoplasmic degradation. These results indicate that the primary effect of interferon in SV40 infected monkey cells is either to inhibit the transcription of early virus RNA or to enhance its turnover in the nucleus.

Gel electrophoresis, isoelectric focusing, and localization of thymidine kinase in normal and simian virus 40-infected monkey cells.

Following SV40 infection of CV-1 monkey cells, cytosol thymidine (dT) kinase activity increased 4- to 10-fold. This enzyme was not significantly different from the cytosol dT kinase of uninfected cells with respect to mobility, isoelectric point, sedimentation coefficient, and phosphate donor specificity. Monkey mitochondria contain a distinctive dT kinase isozyme localized in the mitochondrial matrix. The distinctive mitochondrial isozyme had a higher electrophoretic mobility, lower isoelectric point, and smaller sedimentation coefficient than the cytosol dT kinase, and could utilize either ATP or UTP as phosphate donor; it did not increase after SV40 infection. Mitochondria also contained a cytosol-like dT kinase which was enhanced in infected cells. These findings suggest that SV40 infection derepresses the normal cytosol dT kinase of host cells.

Protein synthesis in simian virus 40-infected monkey cells

Protein synthesis in simian virus 40-infected monkey cells

Correction: Protein Synthesis in Simian Virus 40-Infected Monkey Cells

Correction: Protein Synthesis in Simian Virus 40-Infected Monkey Cells

Protein synthesis in Simian virus 40-infected monkey cells.

The proteins of purified Simian virus 40 (SV40) were examined by sodium dodecyl sulfate-acrylamide gel electrophoresis and compared with the polypeptides synthesized in SV40-infected monkey cells. Purified virions contain two major components, with molecular weights of 44,000 and 31,000. Together they make up 83% of the total virion proteins. In addition, the virus contains 12 minor polypeptides, which are believed to be cellular proteins, or peptides derived from proteolytic degradation of the 44,000 molecular weight polypeptide. Pulse-label experiments show that about 90% of the polypeptides synthesized after SV40 infection are host-cell proteins; 10% represent the two major structural components of the virion. A small fraction (about 0.5%) consists of three polypeptides (molecular weights 70,000, 60,000, and 8,000) that are neither part of the virion nor detectable in uninfected cells. They are either virus-induced cellular proteins or, more likely, proteins coded for by the SV40 genome.