Small and middle T antigens contribute to lytic and abortive polyomavirus infection

Abstract

Using three different polyomavirus hr-t mutants and two polyomavirus mlT mutants, we studied induction of S-phase by mutants and wild-type virus in quiescent mouse kidney cells, mouse 3T6 cells, and FR 3T3 cells. At different times after infection, we measured the proportion of T-antigen-positive cells, the incorporation of [3H]thymidine, the proportion of DNA-synthesizing cells, and the increase in total DNA, RNA, and protein content of the cultures. In permissive mouse cells, we also determined the amount of viral DNA and the proportion of viral capsid-producing cells. In polyomavirus hr-t mutant-infected cultures, onset of host DNA replication was delayed by several hours, and a smaller proportion of T-antigen-positive cells entered S-phase than in wild-type-infected cultures. Of the two polyomavirus mlT mutants studied, dl-23 behaved similarly to wild-type virus in many, but not all, parameters tested. The poorly replicating but well-transforming mutant dl-8 was able to induce S-phase, and (in permissive cells) progeny virus production, in only about one-third of the T-antigen-positive cells. From our experiments, we conclude that mutations affecting small and middle T-antigen cause a reduction in the proportion of cells responding to virus infection and a prolongation of the early phase, i.e., the period before cells enter S-phase. In hr-t mutant-infected mouse 3T6 cells, production of viral DNA was <10% of that in wild-type-infected cultures; low hr-t progeny production in 3T6 cells was therefore largely due to poor viral DNA replication.

Polyomavirus large and small T antigens cooperate in induction of the S phase in serum-starved 3T3 mouse fibroblasts

“…ST antigen was observed to enhance the infectivity of polyomavirus in mouse cells, probably by indirectly stimulating the replication of the viral genome (2,37,40,56,58). To test for this activity in an ST-antigen-expressing cell line, the cell line was transfected with a plasmid (pPyLT1) which contains the total control region, including the origin of replication, and the sequence coding for LT antigen.…”

“…It is capable of immortalizing certain cell types (11,25,47,55; reviewed in references 34 and 54) and is reported to reduce the serum requirement of immortalized cells (47,55), thus perhaps playing an important role in growth induction. MT antigen is responsible for the transforming activity of polyomavirus (45,46), while ST appears to enhance the infectivity of the virus (37,40,56,58), possibly by allowing more-extensive replication of viral DNA (2). ST antigen may also promote cell growth and cooperate with MT antigen in cell transformation (10,41).…”

Phosphorylation of polyomavirus large T antigen: effects of viral mutations and cell growth state

“…Py large T antigen is known to be phosphorylated (24,44), and multiple forms have been detected by alterations in electrophoretic mobility (26,27,29,44,52). The relationship between the different forms and functions of large T antigen is not known, although the electrophoretic conversion has been correlated with viral and cellular replication (52,53 We found that phosphorylation is the basis for the conversion. In quiescett cells the kinase activity responsible to complete this conversion must be induced.…”

“…hrt mutants also appear to differ from the wild type under certain circumstances. For example, the hrt infections can lag behind those of the wild type (20,53). When examined at such intermediate times, the mobility shift of large T antigen of mutant NG59 was less complete than that of the wild type (data not shown), and the phosphopeptide pattern showed some decrease in the amount of phosphopeptide 7 (Fig.…”

“…Py large T antigen is required for the initiation of viral DNA replication (16), and by analogy to simian virus 40 (SV40) large T antigen, it may be involved in elongation as well (50). It is also involved in stimulating host cell DNA synthesis (47,53). Large T antigen negatively autoregulates early viral transcription and positively regulates transcription of late viral genes (7,12,13) and probably some host cell genes as well (31).…”