Induction and Utilization of an ATM Signaling Pathway by Polyomavirus

Abstract

Progression from G 1 to S is essential for polyomavirus DNA replication and depends on the interaction of large T with the retinoblastoma gene product pRb. This virus-induced replication pathway is accompanied by p53 activation resembling a DNA damage response (12). We sought to determine whether this pathway depends in part on activation of the ATM (ataxia telangiectasia mutated) kinase and whether the virus gains advantages from this pathway beyond that of entry into S. We show that polyomavirus infection activates the S-and G 2 -phase checkpoints in primary as well as established mouse cells. Infected cells undergo a prolonged S phase compared to uninfected serum-stimulated cells and show no evidence of a G 2 3M transition before lytic death ensues. Infection is accompanied by increases in ATM activity in vitro and in the level of ATM-S1981-P in vivo. The incubation of infected cells with caffeine, a known ATM inhibitor, did not block entry into S but reduced the rate of viral compared to cellular DNA synthesis. Importantly, caffeine lowered the yields of viral DNA an average of 3-to 6-fold and those of infectious virus by as much as 10-fold. Virus yields were 10-fold lower in ATM ؊/؊ p53 ؊/؊ than in ATM ؉/؉ p53 ؊/؊ mouse embryo fibroblasts, indicating a p53-independent role of ATM in productive infection. Replacement of the normal SMC1 (structural maintenance of chromosomes, or cohesin) protein, a critical ATM substrate in the DNA repair pathway, with its phosphorylation mutant SMC1 S957AS966A also lowered virus yields by roughly 90%. We suggest that polyomavirus activates and utilizes a component(s) of an ATM pathway of DNA repair to prolong S phase and aid its own replication.A productive infection by polyomavirus is accompanied by activation as well as inactivation of tumor suppressor gene pathways. A critical event is the interaction of the large T antigen with pRb, the product of the retinoblastoma tumor suppressor gene. This interaction leads to an override of the G 1 checkpoint and the entry of cells into S, thus allowing viral DNA synthesis to occur (17). A productive infection by polyomavirus also results in the induction of p53 and a DNA damage-like response. Interestingly, this induction also depends on a large T-pRb interaction, based presumably on E2F activation and the assembly of initiation complexes on viral DNA with the formation of theta structures (12). The p53 response, although accompanied by the induction of p21 CIP1/WAF1 (p21), does not lead to G 1 arrest because of the downstream intervention at the pRb checkpoint by large T. The viral replication pathway leading to p53 induction may impose additional events on the infected cell beyond those of entry into S and raises questions about the possible utilization of a DNA damage response-like pathway by the virus.Previous studies have shown that cells infected by polyomavirus accumulate in S and G 2 phase. This has been investigated with various wild-type and mutant strains of virus in both permissive mouse cells and nonpermissive rat cell...

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