Mechanism of interferon action: stability and translation of simian virus-40 early mRNA coding for T-antigen is comparable in untreated and interferon-treated monkey cells

The effect of interferon treatment on the translation and the stability of simian virus 40 (SV40) early mRNA coding for T-antigen was examined in tsA-infected monkey kidney BSC-1 cells at 40°. Neither the translation nor the stability of SV40 early mRNA was altered by interferon under cellular conditions where the synthesis of reovirus polypeptides was significantly inhibited by interferon. SV40 early mRNA decayed with a half-life of about 3 hours as measured by T-antigen synthesis; the decay rate was indistinguishable between untreated and interferon-treated cells.     

Existence of a form of SV40 T antigen incapable of interacting with DNA

The interaction of SV40 T-antigen and viral DNA was studied by using adsorption of DNA-protein complexes on nitrocellulose filters. The T-antigen purification procedure included ion-exchange chromatography on DEAE-cellulose, selective adsorption of cellular proteins on single-stranded DNA-cellulose, chromatography on heparin-Sepharose and removal of cell proteins by an immunosorbent. Only the latter step allowed to remove the contamination of cellular DNA-binding proteins, judging from the reaction of T-antigen neutralization by specific antibodies. It was shown that T-antigen and cellular DNA-binding proteins interact with SV40 DNA at different values of pH, namely ah 6,0-6,4 and 7,9, respectively. The T-antigen obtained was passed through a column with native DNA-cellulose at pH and ionic strength values optimal for interaction with DNA. The bulk of T-antigen (30-40%) did not bind to native thymus DNA and did not interact with SV40 DNA. It is assumed that this fraction is a form of T-antigen, which undergoes structural or functional changes during specific interaction with viral or cellular DNAs.     

pZ402, an improved SV40-based shuttle vector containing a T-antigen mutant unable to interact with wild-type p53

Shuttle vectors are useful tools for studying DNA replication and mutagenesis. SV40-based shuttle vectors are popular because of their ease of use and quick results. However, one complication with the use of SV40-based shuttle vectors is the interaction of cellular p53 protein with the T-antigen of SV40. Wild-type, but not mutant, p53 has been shown to be involved in DNA replication and DNA repair. To address this concern, we have modified an SV40-based shuttle vector, pZ189, by exchanging the wt T-antigen for a mutant SV40 T-antigen, which is unable to bind with p53. This shuttle vector, pZ402, provides us with a tool to study DNA replication and genomic instability in cells with varying genetic backgrounds without interference from the interaction of T-antigen with p53.     

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.     

Interaction of SV40 T-antigen with homologous and heterologous DNA

Using the DNA filter binding assay, the effects of ionic strength and pH on SV40 T-antigen interaction with viral DNA were studied. The apparent association constants for T-antigen binding to SV40 DNA in Scatchard coordinates in the presence of 40 mM NaCl are equal to 0.67 . 10(6) M-1 (pH 6.0) and 0.86 x 10(7) M-1 (pH 7.4). These data indicate that the interaction between T-antigen and SV40 DNA is more specific at pH 7.4. The coincident values of association constants for T-antigen binding to viral and cellular DNAs (Ka = 0.9 x 10(7) M-1 for cellular DNA) at pH 7.4 and the absence of competition between the two DNA species upon binding with T-antigen suggest that viral and cellular DNAs possess similar sites for T-antigen binding. Denatured DNA competes with viral DNA only at pH 6.0, when the T-antigen--SV40 DNA interaction is less specific.     

SV40 T-antigen is a histocompatibility antigen of SV40-transgenic mice

Although the extensive family of non-H-2 histocompatibility (H) antigens provides a formidable barrier to transplantation, the origin of their encoding genes are unknown. Recent studies have demonstrated both the linkage between H genes and retroviral sequences and the ability of integrated Moloney-murine leukemia virus to encode what is operationally defined as a non-H-2 H antigen. The experiments described in this communication reveal that skin grafts from an SV40 T-antigen transgenic C57BL/6 mouse strain are rejected by coisogenic C57BL/6 recipients with a median survival time of 49 days, which is comparable to those of many previously defined non-H-2 H antigens. The specificity of this response for SV40 T-antigen was demonstrated by the identification of SV40 T-antigen-specific cytolytic T lymphocytes and antibodies in multiply-grafted recipients. Although these cytolytic T lymphocytes could detect SV40 T-antigen on syngeneic SV40-transformed fibroblasts, they neither could be stimulated by splenic lymphocytes from T-antigen transgenics nor could they lyse lymphoblast targets from T-antigen transgenics. These observations suggest a limited tissue distribution of SV40 T-antigen in these transgenics. These results confirm the role of viral genes in the determination of non-H-2 histocompatibility antigenes by the strict criteria that such antigenes stimulate (1) tissue graft rejection and (2) generation of cytolytic T lymphocytes. Furthermore, they suggest that the SV40 enhancer and promoter region can target expression of SV-40 T-antigen to skin cells of transgenic animals.     

Regulation of gene amplification and expression in cells that constitutively express a temperature sensitive SV40 T-antigen.

Simian cells have been transformed with SV40 origin-defective recombinant plasmids containing the tsA209 T-antigen gene. These plasmids contain deletions of either 5 or 52 nucleotides that include the BglI site at the SV40 ori, are defective for replication in COS-1 cells but retain a functional SV40 early promoter. Two cell lines transformed with these plasmids, U4 and S7, and their respective clonal derivatives E5 and F11, contain the tsA209 T-antigen gene integrated into the cell DNA and express T-antigen as detected by immunoprecipitation and immunofluorescence. These cells behave as ts-COS cells, since they complement in a temperature dependent manner the replication of an SV40 derived recombinant plasmid. When transfected with recombinant plasmids containing the chloramphenicol acetyl transferase (CAT) gene cloned into SV40 replicons, ts-COS cells were able to regulate the induction of the CAT activity by temperature. The ratios of CAT activity observed at permissive versus restrictive temperature were in the range of 20-400. Thus, these ts-COS cells are useful systems for the regulated expression of cloned genes in simian cells.     

Transgenic mice harboring SV40 t-antigen genes develop characteristic brain tumors

A high percentage of transgenic mice developing from eggs microinjected with plasmids containing the SV40 early region genes and a metallothionein fusion gene develop tumors within the choroid plexus. A line of mice has been established in which nearly every affected animal succumbs to this brain tumor. Thymic hypertrophy and kidney pathology are also observed in some mice. SV40 T-antigen mRNA and protein are readily detected in affected tissues; however, SV40 T-antigen gene expression is barely detectable in unaffected tissues or in susceptible tissues prior to overt pathology, suggesting that tumorigenesis depends upon activation of the SV40 genes. Comparison of DNA from tumor tissue (or cell lines derived from tumors) with DNA from unaffected tissues reveals structural rearrangements as well as changes in DNA methylation of the foreign DNA. The SV40 genes are frequently amplified in tumor tissue, which further indicates that their expression is intimately involved in tumorigenesis in transgenic mice.     

Simian virus 40 cRNA is processed into functional mRNA in microinjected monkey cells.

Monkey cells, microinjected with simian virus 40 (SV40) in vitro synthesized cRNA produce full-size tumor (T)-antigen. This was verified by analyzing immunoprecipitates of microinjected cells by polyacrylamide gel electrophoresis. Early SV40 DNA contains an intron within the large T-antigen coding sequences. Therefore, cRNA copied in vitro from the early DNA strand requires removal of the intron in order to become a functional mRNA. Polyadenylation of the cRNA in vitro by Escherichia coli poly(A)-polymerase increased the biological activity of the RNA. Detection of T-antigen by gel electrophoresis required as little as 50 poly(A)-cRNA injected cells. Splicing of the microinjected cRNA appears to be a nuclear process. Cells enucleated by cytochalasin B prior to injection do not synthesize large T-antigen. However, small t-antigen, a protein with a continuous sequence, is synthesized in these cells. Finally, it is shown that the process of splicing is not required for the transport of mRNA from the nucleus into the cytoplasm. Authentic T-antigen mRNA, isolated from virus infected cells, induced T-antigen synthesis with similar efficiency after either nuclear or cytoplasmic injection.     

Helpers for efficient encapsidation of SV40 pseudovirions

Plasmid DNA that carries the simian virus 40 (SV40) ori can be packaged as SV40 pseudovirions. The pseudovirions are very efficient in gene transmission into a variety of cell types, including human hemopoietic cells. They are routinely prepared with wild-type (wt) SV40 as a helper. In the present study, several parameters required for the helper function were investigated. Plasmids that carry pBR322 sequences in addition to the late genes of SV40 were inefficient in providing helper functions, presumably because the prokaryotic sequences interfered with expression of the SV40 late genes. Efficient helpers were plasmid pSVPiC [Villarreal and Soo, Mol. Appl. Genet. 3 (1985) 62-71] and an SV40 defective virus SLT3 (presently constructed). Plasmid pSVPiC carries a duplication of the SV40 ori and enhancer regions, and pi AN7 sequences. Because of its large size it was not packaged into virion particles. However, it underwent extensive recombination generating infective SV40 particles. Almost no prokaryotic sequences are included in SLT3, that carries the SV40 late gene. In spite of its small size (3.5 kb) it was packaged efficiently, creating defective (T-antigen-negative) SV40 virions. The availability of T-antigen positive and negative pseudovirion mixtures enabled us to suggest that T-antigen drives gene amplification in the target human hemopoietic cells.     

Control of ribonucleic acid function by oligonucleoside methylphosphonates

Oligodeoxyribonucleoside methylphosphonates contain nonionic 3'-5' linked methylphosphonate internucleotide bonds in place of the normal charged phosphodiester linkage of natural nucleic acids. These oligomers are resistant to nuclease hydrolysis, can pass through the membranes of mammalian cells in culture and can form stable hydrogen-bonded complexes with complementary nucleotide sequences of cellular RNAs such as mRNA. The oligomers are readily synthesized on insoluble polymer supports. Their chainlength and nucleotide sequence can be determined by chemical sequencing procedures. Oligonucleoside methylphosphonates which are complementary to the 5'-end, initiation codon region, or coding region of rabbit globin mRNA inhibit translation of the mRNA in rabbit reticulocyte lysates and globin synthesis in rabbit reticulocytes. This inhibition is due to the interaction of the oligomers with mRNA and the extent of inhibition is influenced by the secondary structure of the mRNA and the location of oligomer binding site on the mRNA. Oligomers complementary to the initiation codon regions of N, NS and G protein mRNAs of Vesicular stomatitis virus (VSV) inhibit virus protein synthesis in VSV-infected Mouse L-cells. These oligomers do not affect L-cell protein synthesis or growth. Virus protein synthesis and growth can also be selectively inhibited by oligonucleoside methylphosphonates which are complementary to the donor or acceptor splice junctions of virus pre mRNA. An oligomer complementary to the donor splice junction of SV40 large T antigen mRNA inhibits T-antigen synthesis in SV40-infected African green monkey kidney cells but does not inhibit overall cellular protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Post-translational regulation of the 54K cellular tumor antigen in normal and transformed cells.

The 54K cellular tumor antigen has been translated in vitro, using messenger ribonucleic acids from simian virus 40 (SV40)-transformed cells or 3T3 cells. The in vitro 54K product could be immunoprecipitated with SV40 tumor serum and had a peptide map that was similar, but not identical, to the in vivo product. The levels of this 54K protein in SV3T3 cells were significantly higher than those detected in 3T3 cells (D. I. H. Linzer, W. Maltzman, and A. J. Levine, Virology 98:308-318, 1979). In spite of this, the levels of translatable 54K messenger ribonucleic acid from 3T3 and SV3T3 cells were roughly equivalent or often greater in 3T3 cells. Pulse-chase experiments with the 54K protein from 3T3 or SV3T3 cells demonstrated that this protein, once synthesized, was rapidly degraded in 3T3 cells but was extremely stable in SV3T3 cells. Similarly, in an SV40 tsA-transformed cell line, temperature sensitive for the SV40 T-antigen, the 54K protein was rapidly turned over at the nonpermissive temperature and stable at the permissive temperature, whereas the levels of translatable 54K messenger ribonucleic acid at each temperature were roughly equal. These results demonstrate a post-translational regulation of the 54K cellular tumor antigen and suggest that this control is mediated by the SV40 large T-antigen.