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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 less than 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.
Insights
Polyomavirus mutants affecting small and middle T-antigen delay host DNA replication and reduce the number of cells entering S-phase. These mutations also decrease viral DNA replication and progeny production in infected cells.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Polyomaviruses are small DNA viruses that can induce cell proliferation.
- Viral T-antigens play crucial roles in regulating the host cell cycle and viral replication.
Purpose of the Study:
- To investigate the role of polyomavirus small and middle T-antigens (T-antigens) in inducing S-phase entry.
- To analyze the impact of hr-t and mlT mutations on host DNA replication and viral production.
Main Methods:
- Infection of quiescent mouse kidney, mouse 3T6, and FR 3T3 cells with wild-type and mutant polyomaviruses.
- Measurement of T-antigen expression, [3H]thymidine incorporation, and DNA synthesis.
- Quantification of viral DNA, RNA, protein content, and progeny virus production.
Main Results:
- Polyomavirus hr-t mutants delayed host DNA replication and reduced S-phase entry compared to wild-type.
- Polyomavirus mlT mutants showed varied effects; dl-23 was similar to wild-type, while dl-8 induced S-phase and progeny in only a third of T-antigen-positive cells.
- Viral DNA replication in hr-t mutant-infected cells was less than 10% of wild-type levels, primarily limiting progeny production.
Conclusions:
- Mutations in small and middle T-antigen reduce the proportion of cells responding to infection and prolong the pre-S-phase period.
- Impaired viral DNA replication is a major factor limiting hr-t mutant progeny production in mouse 3T6 cells.