Viral DNA synthesis in nonpermissive rat F-111 cells and its role in neoplastic transformation by polyomavirus.

Abstract

We have investigated the occurrence and role of polyomavirus DNA synthesis in neoplastic transformation by this virus. We show that after infection of Fischer rat F-111 cells at 37 degrees C, there is two- to threefold increase in the level of viral DNA as compared with the input signal, with a peak observed between 5 and 7 days postinfection. Viral DNA synthesis is about 10 times higher at 33 degrees C and increases up to 15 days postinfection. Most of the viral DNA produced is supercoiled (form I DNA). On the basis of in situ hybridization, it appears that viral replication is restricted to a small fraction of the population. At the lower temperature, more cells are permissive for viral DNA synthesis and the level of synthesis per permissive cell is higher. The DNA synthesis observed is large T-antigen dependent, and the increase in viral DNA synthesis at 33 degrees C is paralleled by an increase in the expression of this viral protein. When large T antigen is inactivated, the half-life of de novo-synthesized viral DNA is less than 12 h, suggesting that large T antigen may be responsible for the stability of the viral genomes as well as their synthesis. Surprisingly, at early times postinfection (0 to 48 h), when the essential function of large T antigen in transformation is expressed (as demonstrated in shift-up experiments with tsa mutants), the level of large T antigen is below the detection level and is at least 10-fold lower than the levels observed in permissive infections at the start of viral DNA synthesis. The difference in viral DNA at 37 and 33 degrees C allowed us to study its effect on transformation. Although an increase in transformation frequency is observed in wild-type A2 infections carried at 33 degrees C (frequencies two to three times higher than at 37 degrees C), this increase appears to be unrelated to the increase in viral DNA synthesis. Furthermore, the overall level of viral DNA and large T antigen in F-111 cells may not affect the integration of the viral genome, since the patterns of integration in cells transformed by wild-type A2 at 33 and 37 degrees C appear similar. The results are compatible with a role for large T antigen in integration-transformation which is not simply to amplify the viral genome to enhance the probability of its integration.