Inhibition of SV40 replication by 5-azacytidine: effect on DNA synthesis and conformation.

SV40 DNA synthesis was inhibited by approximately 50% following a 3-hr treatment with 100 μg/ml 5-azacytidine (5-AzaCR). Cesium chloride-ethidium bromide analysis of SV40 DNA from drug-treated infected cultures demonstrated that SV40 DNA I banded at a higher buoyant density and therefore had a decreased ability to bind ethidium bromide. Sedimentation analysis in sucrose gradients containing various concentrations of ethidium bromide indicated that these molecules were deficient in superhelical terms. Treatment of infected cultures with 5-AzaCR was shown to inhibit protein synthesis which preceeded the inhibition of DNA synthesis. It is concluded that inhibition of SV40 DNA synthesis and the conformational alterations in DNA I result from the inhibition of protein synthesis.     

DNA and histone synthesis in butyrate-inhibited BSC-1 cells infected with SV40

The effect of butyrate on DNA synthesis, histone synthesis, and virus production in SV40-infected monkey cells has been studied. When cells in which DNA and histone synthesis have been blocked by treatment with butyrate are infected with SV40, DNA synthesis is induced to a level at least equal to that in untreated infected cells. Both viral and cellular DNA sequences are replicated. Viral DNA synthesized is predominantly SV40 Form I. The amount of viral DNA which accumulates is reduced by 10-fold in the presence of butyrate, and the yield of infectious virus is lowered 100-fold. Histone synthesis in these cells is stimulated concomitantly with DNA synthesis. The hyperacetylation of histones characteristic of butyrate-treated cells is unchanged by SV40 infection; the newly synthesized histones are hyperacetylated. Thus the mechanism by which butyrate blocks DNA and histone synthesis is not directly related to the hyperacetylation of histones.     

Specific interaction of the SV40 T antigen-cellular p53 protein complex with SV40 DNA

Three forms of the simian virus 40 (SV40) large tumor antigen (T antigen), free T antigen from infected monkey cells, T antigen-p53 complex from virus-infected mouse cells, and T antigen-p53 complex from SV40-transformed mouse cells, were examined for their abilities to bind specifically to SV40 DNA. Employing an immunoassay that detected T antigen and/or p53 protein bound to restriction fragments of viral DNA, it was shown that all three of these forms of SV40 T antigen bind preferentially to the restriction fragment of SV40 DNA containing the nucleotide sequence at the origin of viral DNA replication. Free p53 protein alone failed to bind to SV40 DNA in this assay. These results indicate that the SV40 T antigen-p53 protein complex displays the same SV40 DNA binding specificity as free T antigen obtained from a productive infection.     

Inhibition of nuclear migration of wild-type SV40 tumor antigen by a transport-defective mutant of SV40-adenovirus 7 hybrid virus

A mutant of the defective SV40-adenovirus 7 hybrid, PARA, induces the synthesis of SV40 large T-antigen (T-ag) that is not transported to the nucleus and accumulates in the cytoplasm of infected or transformed cells. The effect of this cT mutation on the transport of wild-type (WT) T-ag was examined by coinfection experiments. Immunofluorescence staining for T-ag revealed that when SV40-transformed green monkey kidney cells were infected with the cT mutant of PARA [PARA(2cT)] there was a total loss of nuclear T-ag (nT-ag) reactivity concomitant with the appearance of cytoplasmic T-ag (CT-ag) reactivity. The dominance of the cT-ag phenotype was also manifest in cells coinfected with PARA(2cT) and either SV40 or WT PARA, both of which induce nT-ag during single infection. Two lines of evidence indicate that the absence of nuclear T-ag reactivity in coinfected cells is due to a failure of migration of WT T-ag into the nucleus, rather than an inhibition of its synthesis. First, coinfection of cells with SV40 and either helper adenovirus or PARA-adenovirus populations, at the multiplicities of infection employed in these studies, did not reduce the yields of infectious SV40. Second, cells coinfected with PARA(2cT) and deletion mutants of SV40 which encode T-ag polypeptides of reduced molecular weight expressed the cT-ag phenotype, while the presence of the deleted forms of T-ag was confirmed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. The observation that the replication of infectious SV40 is not inhibited in cells coinfected with PARA(2cT) and expressing the cT phenotype indicates that levels of nuclear T-ag below the limits of detection by immunofluorescence are sufficient to promote SV40 DNA synthesis. The dominant effect of the cT mutation was specific for transport of SV40 T-ag, since normal migration of adenovirus tumor and virion antigens as well as SV40 virion antigen occurred. Several possible mechanisms for the dominance of the CT-ag phenotype are presented.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IV. Inhibition by human interferon of expression of SV40 TrAg in SV40-infected monkey cells.

Human interferon inhibits the synthesis of SV40 transplantation rejection antigen (TrAg) in SV40-infected but not in SV40-transformed monkey cells. The synthesis of SV40 tumor antigen as detected by the indirect immunofluorescence test and the large and small T antigens as detected by the immunoprecipitation with sera from tumor-bearing hamsters and electrophoresis in SDS-gels was similarly affected in SV40-infected monkey cells. These results suggest that the induction of SV40-specific TrAg in the cytolytic cycle depends upon a viral, rather than a host, message.     

Association of SV40 large tumor antigen and cellular proteins on the surface of SV40-transformed mouse cells

A cellular protein with a molecular weight of about 53,000 (53K) and histocompatibility antigens (mouse H-2 antigens) have been reported to be associated with viral-specified proteins in transformed cells. We investigated whether such associations could be detected on the surface of SV40-transformed mouse cells. A differential immunoprecipitation technique was adapted so that surface-associated antigens could be detected independently from intracellular antigens. Cells grown as monolayers were enzymatically labeled with ¹²⁵I-Na using a lactoperoxidase-catalyzed reaction, or metabolically labeled with either [³⁵S]methionine or ³²P_i, and were then incubated with antisera against mouse H-2 antigens or SV40 large T-antigen (T-ag) or with monoclonal antibodies against mouse 53K nonviral T-antigen (nvT-ag). The cells were then disrupted with an NP40 solution, the extracts were clarified by centrifugation, and the immune complexes in the supernatant fluids adsorbed with protein A-containing Staphylococcus aureus. Internal antigens, present in the cell lysates, were precipitated by a second incubation with antiserum and the antigen-antibody complexes collected again with immunoadsorbent. The precipitated proteins were eluted and analyzed by SDS-polyacrylamide gel electrophoresis. Reconstruction experiments established that T-ag released from the nucleus during the extraction procedure was not combining with free antigen-binding sites on antibodies bound to the cell surface in the external reaction, that nuclear unbound T-ag was not exchanging with bound surface antigen during extraction, and that the surface reaction was not due to nuclear T-ag released from dead cells and nonspecifically adsorbed onto the surface of living cells. Iodinated 94K T-ag was specifically immunoprecipitated by T antibody during the external reaction; an iodinated 53K polypeptide was coprecipitated. Conversely, labeled T-ag and 53K were coprecipitated from surface-iodinated transformed cells by monoclonal antibodies against mouse 53K nvT-ag. Thus, it appears that SV40 large T-ag and cellular 53K protein are associated on the surface as well as within SV40-transformed mouse cells. In contrast, no detergent-stable complex between T-ag and mouse H-2 antigens was detected on the transformed cells. The possibility that molecular interactions between viral- and cell-coded proteins could be involved in determining some of the observed transformation-related cellular phenotypic changes is discussed.     

SV40 antitumor sera that directly immunoprecipitate small-t-associated cellular proteins

Two cellular proteins, 56K and 32K, are immunoprecipitated in association with SV40 small-t antigen using antisera from animals bearing SV40-induced tumors. Antitumor sera have now been identified that immunoprecipitate these proteins directly from uninfected, as well as infected, cells.     

Alternative interactions of the SV40 A protein with DNA

We have characterized the DNA binding properties of SV40 A protein (T antigen) from permissive cells. The viral protein protects 110- to 115-, 60- to 65-, 40- to 45-, and 30- to 35-bp fragments of SV40 DNA from an excess of DNase 1. The 60- to 65-bp fragment extends some 30–35 by to either side of the BglI site at the origin of replication. DNase footprinting confirms the location of this binding region and shows that it is the most completely protected DNA binding site under our conditions. The A protein selectively recognizes the 60- to 65-bp origin fragment in the complete absence of adjacent DNA and binds to it in three alternative ways that protect 60–65, 40–45, or 30–35 by from nuclease. Thus these three interactions with isolated origins recapitulate the binding of viral protein to intact SV40 DNA. We propose that the alternative interactions of SV40 A protein with the 60- to 65-bp origin fragment may represent three different conformational alignments of protein with DNA rather than a simple repetition of a single kind of binding interaction. Viral protein also protects pBR322 and λ phage DNA from nuclease to a limited extent. Similar interactions could possibly alter the function of DNA control regions of host cells.     

Inhibition of host cellular ribonucleic acid synthesis by glycoprotein of mumps virus

After infection with mumps virus, cellular ribonucleic acid synthesis of a murine lymphoma cell line, EL4, was appreciably depressed. The inactivation of viral infectivity by ultraviolet irradiation or the treatment of cells with mouse interferon did not abolish the inhibiting effect, suggesting that virus replication is not required for the depressed RNA synthesis. Envelope glycoproteins isolated from disrupted mumps virus caused inhibition of cellular RNA synthesis. The addition of low concentrations of specific antibody enhanced the inhibitory effect, probably through the formation of aggregates of glycoproteins. On the contrary, the glycoproteins showed no effect on RNA synthesis in the presence of cytochalasin D.     

Inhibition of cellular protein synthesis by vaccinia virus surface tubules

The infection of HEp-2 cells with vaccinia virus results in a rapid and selective inhibition of cellular protein synthesis. This effect appears to be due to a structural component(s) of the infecting virion. Experiments investigating the nature of the inhibitory principle showed that a vaccinia virion component, the surface tubule (ST), inhibits protein synthesis in HEp-2 cells without affecting either RNA or DNA synthesis. Furthermore, ST added to a rabbit reticulocyte cell-free system inhibited the incorporation of radiolabeled amino acids into acid-insoluble protein. ST inhibitory activity was destroyed by heat (56° for 60 min) and by prior incubation with specific anti-ST serum. A decrease in the polyribosome complement of cells exposed to purified ST, with a concomitant increase in the free ribosome pool, suggests that the main effect of tubules on cellular protein synthesis occurs at the level of polypeptide chain initiation.     

Phosphorylation of SV40 large T antigen in SV40 nucleoprotein complexes

Plaques produced by our P^? mutants of vesicular stomatitis virus (VSV), which are defective in the inhibition of total protein synthesis in infected cells, stop increasing in size after several days of incubation under conditions where those produced by P⁺ mutants increase linearly in size. The basis for the arrest in size of P^? plaques has been shown to be due to the induction of interferon, and the phenotype is termed PIF⁺ for “plaque interferon positive.” Thus P^? plaques can inhibit the increase in size of adjacent P⁺ plaques and the factor responsible has the biological and physical properties of interferon. Also P^? mutants, when plaqued on VERO cells which cannot be induced to produce interferon, produce plaques which increase linearly in size like P⁺ plaques. Finally, the inclusion of anti-interferon antibody in the overlay medium also causes P^? mutants to produce plaques like P⁺ mutants. VERO cells were found to be useful to separate the effects of is mutations on plaque size from the interferon effect. Using other cell types the latter effect (PIF assay) can be used as an assay for the ability of viruses to induce interferon, for the isolation of PIF⁺ mutants from PIF^? virus, and as a test for the ability of cells to respond to interferon induction by PIF⁺ viruses. The assay can be increased in sensitivity through the use of specific cell types and of cell cultures preincubated for several days in the stationary phase of growth. In its most sensitive form, the assay could detect PIF⁺ behavior in certain ts mutants of VSV at permissive temperatures and in VSV mutants emerging from persistent infection. The assay has also been used to isolate novel mutants of VSV which show alterations in the viral P function.     

The isolation of SV40 tsA/deletion, double mutants and the induction of host DNA synthesis

Simian virus 40 mutants were constructed that contain both a tsA mutation leading to temperature sensitivity of the 92K T-antigen, and deletions of 20–200 base pairs leading to a loss in the expression of the 20K t-antigen. As expected, these mutants were temperature sensitive for viral growth and viral DNA replication in lytically infected cells. At nonpermissive temperatures, the ts/deletion mutants stimulated the incorporation of nucleosides into host DNA as did the tsA mutant alone. This induction of incorporation by the tsA mutants resulted from semiconservative DNA replication, not repair synthesis. At 200 μg/ml caffeine the induction of host DNA by A209 was inhibited by 30 to 50%, whereas induction by the ts/deletion mutants was abolished.     

Inhibition of SV40 DNA synthesis by vesicular stomatitis virus in doubly infected monkey kidney cells

Vesicular stomatitis virus (VSV) inhibited SV40 DNA synthesis in doubly infected synchronized Vero cells. Gel-electrophoretic profiles demonstrated that SV40 DNA monomers accumulated in all stages of supercoiling, regardless of whether cells were superinfected with VSV early or late in the S phase. These gel profiles were indistinguishable from ones obtained from SV40-infected, cycloheximide-treated cells in the absence of VSV infection. Radiolabel in the partial supercoils could be chased into supercoils, but only by restoring protein synthesis. The relative rates of SV40 DNA chain elongation were determined in VSV-superinfected and nonsuperinfected cells. The gradients of 3H incorporation as a function of distance from the origin of replication in pulse-labeled form I DNA were unaffected by VSV. It is concluded that VSV inhibition of SV40 DNA synthesis is an indirect result of inhibition of host cell protein synthesis and it is suggested that incompletely supercoiled SV40 chromatin is not a good template for DNA synthesis.     

Human fibroblasts transformed by the early region of SV40 DNA: analysis of "free" viral DNA sequences

Human fibroblastic cells (HF) were transformed with the early region of the simian virus 40 genome (0.15 – 0.73 map units) by using the DNA-calcium phosphate coprecipitation technique of F. L. Graham and A. J. Van der Eb (1973, Virology 52, 456–467). Transformation resulted in altered morphology and ability to grow in agarose. The SV40-transformed human fibroblasts (SVHF-A) have a limited life span and reach “senescence” after 10–11 passages. Analysis of the low molecular weight DNA extracted from SVHF-A cells shows a relatively high amount of free viral DNA sequences in circular supercoiled form. These circular molecules are very heterogeneous in size and contain sequences corresponding to the early region of the SV40 genome. Part of them may contain cellular DNA sequences as well. In situ hybridization experiments indicate that a minority of the SVHF-A cells (2–3%) are spontaneously induced to synthesize free viral DNA molecules and their frequency is increased by mitomycin C treatment. Immunofluorescence staining for SV40 T antigens also indicates that the cells producing free viral DNA contain higher T-antigen levels than the rest of the population. Our data suggest that the “free” viral DNA molecules derive from integrated viral sequences following replication in a minority of the cells rather than originating from a persistent extrachromosomal replication in every cell.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). VI. Mechanism of induction of SV40 transplantation immunity in mice by purified SV40 T antigen (D2 protein)

According to the base sequence homology of the gene coding for the nonstructural (NS) protein the influenza A viruses can be divided into at least three groups. Within each group the homology is about 90% or higher. The avian influenza A viruses fall at least into two groups between which the homology is about 40%. All human strains tested belong to another group. Influenza viruses of other species might comprise their own group(s). The related regions of the NS gene among the two groups of avian influenza viruses overlap completely and they are highly conserved. The results are discussed in terms of a selection pressure concerning the function exerted by the host during the evolution of the different NS genes, which also might explain a certain species specificity concerning the NS gene of influenza A viruses.     

SV40 early mutants that are defective for viral DNA synthesis but competent for transformation of cultured rat and simian cells

An early SV40 mutant (C6/SV40) was isolated by rescuing the viral DNA integrated into the genome of the C6 cell line which is a CV1 cell line transformed with uv-irradiated SV40. The DNA of C6/SV40 mutant does not replicate detectably in permissive, simian cells, but it transforms them with high efficiency. It also transforms established rat cells with an efficiency equal to that of wild-type DNA, although the phenotype of the mutant-transformed cells is somewhat different from that of the wild-type transformants. Genetic analysis shows that C6/SV40 carries multiple mutations. These mutations were partially segregated into separate viral genomes, C6-1 and C6-2. Nucleotide sequencing showed that the mutations in both C6-1 and C6-2 were transversions (G to T at nucleotide 5074 and A to T at nucleotide 5011 in C6-1, and A to C at nucleotide 4360 in C6-2); all nucleotide numbers are in SV system (J. Tooze (ed.) (1982), “Molecular Biology of Tumor Viruses,” Part 2, “DNA Tumor Viruses,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.). The predicted amino acid changes are Met-Ile and Lys-Asn in C6-1 and Asn-Thr in C6-2. In the case of C6-1, these substitutions would occur in the N-terminal portion of large T antigen that is held in common with small t antigen (amino acids 30 and 51). The mutated amino acid in C6-2 resides in the unique portion of large T antigen at position 153. Both C6-1 and C6-2 transform established rat cells as efficiently as wild-type SV40. However, no replication of C6-2 DNA is detectable in permissive CV1 cells, while replication of C6-1 DNA occurs with significantly reduced efficiency. A revertant of C6-1 was isolated and found to have retained the two original mutations and to have acquired a new mutation (G to A at nucleotide 4977), predicting an amino acid change of Glu-Lys at amino acid position 63, which is common to both large and small T antigens.     

Monoclonal antibody to SV40 T-antigen blocks lysis of cloned cytotoxic T-cell line specific for SV40 TASA

The lysis of SV40-transformed target cells by a cloned cytotoxic T cell line (CTB6) that is SV40 TASA-specific and H-2Kb-restricted is blocked by a monoclonal antibody reactive with the SV40 T-antigen. The blocking is maximized at high antibody concentrations and low effector-to-target cell ratios. These data indicate that at least one antigenic determinant of the SV40 T-antigen molecule is expressed at the cell surface and is either recognized by this cytotoxic T lymphocyte or is proximate to that determinant recognized by this cytotoxic T lymphocyte.     

Macromolecular synthesis in cells infected by frog virus 3. X. Inhibition of cellular protein synthesis by heat-inactivated virus

Heat-inactivated frog virus 3 (ΔFV3) inhibits host cell protein synthesis without interfering with protein synthesis directed by active FV3. An examination of various parameters of cellular protein synthesis after exposure of cells to ΔFV3 revealed the following. (i) The rate of inhibition was multiplicity dependent. (ii) There was a rapid disaggregation of both free and membrane-bound polysomes. (iii) Rates of protein chain elongation remained unaltered. (iv) There was neither a detectable breakdown nor modification of cellular mRNAs; the mRNAs isolated from ΔFV3-treated cells could be translated in vitro and the pattern of the translational products appeared identical to that of mRNAs from untreated cells. These data are consistent with the conclusion that inhibition of host cell protein synthesis by ΔFV3 is the result of a selective effect on a step(s) essential for the initiation of translation of cellular mRNAs.     

Characterization of measles polypeptides by monoclonal antibodies

Five stable mouse spleen cell myeloma hybrids (hybridomas) producing monoclonal antibodies to measles virus proteins were produced. The specificity of these monoclonal antibodies was established by immunoprecipitation and PAGE analysis and by immunoflourescence. The antibodies from three clones (F/20, H-1, E-1) neutralize infectious virus and are specific for the hemagglutinin (Pl) polypeptide. Two clones produce antibodies reacting with the nucleocapsid protein (P3). The monoclonal antibodies were then used to study the antigenic relationships of polypeptides of measles virus and canine distemper virus. It is shown that the glycoproteins of two strains of wild type measles virus and three strains of SSPE all share common antigenic determinants as detected by two of the neutralizing hybridomas while canine distemper was not recognized by those sera. The monoclonal antibody to nucleocapsid supports these findings since it immunoprecipitates wild type virus and SSPE nucleoproteins but not that from canine distemper. The monoclonal antibodies against P3 recognized a number of polypeptides with molecular weights smaller than that of P3 in certain measles-infected cellular extracts. Several of these are in the molecular weight range of the previously reported P4 polypeptide. Evidence is presented that these polypeptides are degradation products of the nucleocapsid protein, and not randomly produced or artifacts of cell extraction procedure. They probably represent selective proteolysic cleavage products of host proteolysis by certain specific enzymes.