Inhibition of vimentin synthesis and disruption of intermediate filaments in simian virus 40-infected monkey kidney cells.

The organization, synthesis, and phosphorylation of vimentin were studied at various times after infection of monkey kidney cells with simian virus 40. Late after infection (between 36 and 48 h postinfection) there is a dramatic reduction in vimentin synthesis that is paralleled by a specific disruption of the intermediate filament network. At the same time there is no apparent alteration of the organization or the synthesis of the actin-containing filaments and of the microtubules. The inhibition of vimentin synthesis is also reflected by the level of vimentin mRNA activity in the infected cells, as assayed in a cell-free in vitro translation system, and vimentin mRNA concentration as revealed by RNA blot hybridization to cloned vimentin cDNA. The level of vimentin phosphorylation also decreases dramatically but at a much earlier time after infection (between 14 and 24 h postinfection), when mitosis in the infected cells is blocked. Although the decrease in vimentin synthesis in simian virus 40-infected cells is paralleled by the alterations in the organization of the intermediate filament network, the phosphorylation of vimentin correlates with the cell cycle, as it does in other systems. A possible feedback control mechanism of vimentin synthesis by alterations in the organization of the intermediate filament network is discussed.     

Inhibition of simian virus 40 DNA synthesis in Yaba virus-preinfected cells.

The effect of Yaba virus preinfection on DNA synthesis in SV40-infected Jinet cells was studied. Time-course synthesis studies were conducted using the incorporation of labeled thymidine. Yaba virus preinfection resulted in the inhibition of SV40 DNA synthesis when the elapsed time between Yaba virus and SV40 infections was three days. This inhibition was demonstrated by hybridization studies and sedimentation analysis. In addition, the usual stimulation of cellular DNA synthesis induced by SV40 infection was inhibited. This inhibition occurred at a time in Yaba virus infection when no cytoplasmic Yaba virus-specific DNA synthesis occurred.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.     

Modification of simian virus 40 protein A.

The A protein of simian virus 40 is phosphorylated in both productive and transforming infection. The phosphorylated amino acid has been identified as serine and has been localized in a single tryptic peptide of the protein. Because the A protein synthesized in infection by A mutants is phosphorylated to the same extent and in the same peptide as in infection by wild-type virus, the functional defect of the A mutants is apparently unrelated to phosphorylation. At least three distinct forms of the A protein with apparent molecular weights of 85,000, 88,000, and 100,000 can be identified in extracts of cells infected by wild-type virus. After exposure of cells to Nonidet P-40, the 85,000- and 88,000-dalton proteins were found in varying amounts in extracts of permissive cells but not in extracts of transformed cells. This finding raised the question of the possible functional importance of the smaller proteins in productive infection. However, the virtual absence of the 85,000- and 88,000-dalton proteins in some extracts of the fully permissive CV-1 cell line indicates that a conversion of the larger to the smaller forms of the A protein is not required in significant quantity for productive infection. Furthermore, a study of extraction conditions shows that the smaller proteins are easily generated during extraction and provides an explanation for the appearance of these proteins in some cells after extraction under unfavorable conditions.     

Identification of simian virus 40 protein A.

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.     

Initiation of simian virus 40 DNA synthesis in vitro.

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of initiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin.     

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.     

Stimulation of host centriolar antigen in TC7 cells by simian virus 40: requirement for RNA and protein syntheses and an intact simian virus 40 small-t gene function

Simian virus 40 (SV 40) stimulated a host cell antigen in the centriolar region after infection of African green monkey kidney (AGMK) cells. The addition of puromycin and actinomycin D to cells infected with SV40 within 5 h after infection inhibited the stimulation of the host cell antigen, indicating that de novo protein and RNA syntheses that occurred within the first 5 h after infection were essential for the stimulation. Early viable deletion mutants of SV40 with deletions mapping between 0.54 and 0.59 map units on the SV40 genome, dl2000, dl2001, dl2003, dl2004, dl2005, dl2006, and dl2007, did not stimulate the centriolar antigen above the level of uninfected cells. This indicated that an intact, functional small-t protein was essential for the SV40-mediated stimulation of the host cell antigen. Our studies, using cells infected with nondefective adenovirus-SV40 hybrid viruses that lack the small-t gene region of SV40 (Ad2+ND1, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5), revealed that the lack of small-t gene function of SV40 could be complemented by a gene function of the adenovirus-SV40 hybrid viruses for the centriolar antigen stimulation. Thus, adenovirus 2 has a gene(s) that is analogous to the small-t gene of SV40 for the stimulation of the host cell antigen in AGMK cells.     

Stimulation of the acute-phase response in simian virus 40-hepatocyte cell lines.

Seven simian virus 40 (SV40)-hepatocyte cell lines were characterized with respect to the ability to express eight liver acute-phase genes. cDNA clones corresponding to albumin, serum amyloid A, alpha 1-acid glycoprotein, haptoglobin, alpha-, beta-, and gamma-fibrinogen, and alpha 1-major-acute-phase protein mRNAs were used in Northern (RNA) or slot blot analyses. In the noninduced state, six of the seven cell lines showed significant (i.e., liverlike) levels of constitutive expression of all genes examined except that expression of haptoglobin mRNA was considerable lower than in the normal liver. To examine whether these immortalized liver cells can respond appropriately to inflammatory mediators, cells were treated with conditioned medium from activated human monocytes or mixed lymphocyte cultures. Results showed that these SV40-hepatocyte cell lines responded to the conditioned media in culture by down-regulating albumin gene expression and up-regulating other acute-phase genes in a time- and dose-dependent manner. These results indicate that the SV40-hepatocytes retained not only the ability to express a number of acute-phase genes but also the ability to respond to external stimuli. The usefulness of these cell lines for analysis of the molecular mechanisms involved in the regulation of these acute-phase genes is discussed.     

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.     

Re-oxygenation of hypoxic simian virus 40 (SV40)-infected CV1 cells causes distinct changes of SV40 minichromosome-associated replication proteins.

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.     

Characterization of simian cells tranformed by temperature-sensitive mutants of simian virus 40.

Seven lines derived from primary African green monkey kidney cells, which had survived lytic infection by wild-type simian virus 40 (SV40) or temperature-sensitive mutants belonging to the A and B complementation groups, were established. These cultures synthesize SV40 tumor (T) antigen constitutively and have been passaged more than 60 times in vitro. The cells released small amounts of virus even at high passage levels but eventually became negative for the spontaneous release of virus. Virus rescued from such "nonproducer" cells by the transfection technique exhibited the growth properties of the original inoculum virus. Four of the cell lines were tested for the presence of altered growth patterns commonly associated with SV40-induced transformation. Although each of the cell lines was greater than 99% positive for T antigen, none of the cultures could be distinguished from primary or stable lines of normal simian cells on the basis of morphology, saturation density in high or low serum concentrations, colony formation on plastic or in soft agar, hexose transport, or concanavalin A agglutinability. However, the cells could be distinguished from the parental green monkey kidney cells by a prolonged life span, the presence of T antigen, a resistance to the replication of superinfecting SV40 virus or SV40 viral DNA, and, with three of the four lines, an ability to complement the growth of human adenovirus type 7. These properties were expressed independent of the temperature of incubation. These results indicate that the presence of an immunologically reactive SV40 T antigen is not sufficient to ensure induction of phenotypic transformation and suggest that a specific interaction between viral and cellular genes and/or gene products may be a necessary requirement.     

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.