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.
Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their ...affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn→ Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.
Using zone velocity sedimentation and nondenaturing polyacrylamide gel electrophoresis, we have determined that purified polyoma large tumor antigen (Py T Ag) consists of discrete forms ranging from ...more abundant monomers and dimers to several higher but clearly distinguishable oligomeric species. Addition of ATP and MgCl
2 to Py T Ag caused a dramatic increase in the appearance of Py T Ag hexames, a form that, based on its SV40 T Ag counterpart, is likely to play a crucial role in its DNA replication functions. Other nucleotides in addition to ATP, as well as a nonhydrolyzable ATP derivative, were capable of inducing hexamer formation. This approach may further elucidate the role(s) of different forms of Py T Ag in viral regulatory processes.
The products of splicing of simian virus 40 early pre mRNA in HeLa cell nuclear extracts have been characterized. Of the two alternative splicing patterns exhibited by this precursor in vivo, which ...involve the use of alternative large T and small t 5′ splice sites and a single shared 3′ splice site, only one, producing large T mRNA, was found to occur in vitro. A number of possible intermediates and byproducts of splicing of large T mRNA were observed, including free large T 5′ exon, lariat form intron joined to 3′ exon and free lariat and linear forms of large T intron. The formation of these products argues strongly for a basic similarity in the mechanism underlying large T and other, non-alternative splices. A collection of RNAs resulting from protection of early pre mRNA at specific points from an endogenous 5′ to 3′ exonuclease activity in vitro have also been observed. The regions of the precursor RNA protected map to positions immediately upstream of the 5′ splice sites of large T and small t and the lariat branchpoint, and may represent interaction of these regions with components of the splicing machinery.
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