Induction and Bypass of p53 during Productive Infection by Polyomavirus

Abstract

Lytic infection by polyomavirus leads to elevated levels of p53 and induction of p53 target genes p21Cip1/ WAF1 (p21) and BAX. This is seen both in polyomavirus-infected primary mouse cell cultures and in kidney tissue of infected mice. Stabilization of p53 and induction of a p53 response are accompanied by phosphorylation of p53 on serine 18, mimicking a DNA damage response. Stabilization of p53 does not depend on p19Arf interaction with mdm2. Cells infected by a mutant virus defective in binding pRb and in inducing G 1 -to-S progression show a greatly diminished p53 response. However, cells infected by wild-type virus and blocked from entering S phase by addition of mimosine still show a p53 response. These results suggest a role of E2F target genes in inducing a p53 response. Polyomavirus large T antigen coprecipitates with p53 phosphorylated on serine 18 and also with p21Cip1/WAF1. Implications of these and other findings on possible mechanisms of induction and override of p53 functions during productive infection by polyomavirus are discussed.The tumor suppressor p53 plays a pivotal role in carcinogenesis and is the most frequently mutated gene in human cancers (31,34). Most DNA tumor viruses have mechanisms to inactivate p53. The large T antigen (large T) of simian virus 40 (SV40) binds p53 and inactivates at least some of its functions (44). The E6 proteins of the highly oncogenic human papillomavirus type 16 (HPV-16) and HPV-18 promote the rapid degradation of p53 through a ubiquitin-dependent proteolytic pathway (47). The oncogenic human adenoviruses act to block p53 through dual functions of the E1B proteins acting directly on p53 and on its downstream targets (16).Thus far, none of these mechanisms have been ascribed to the mouse polyomavirus. This is surprising in view of the efficiency and rapidity with which this virus induces tumors. p53 is not stably upregulated in polyomavirus tumors, and most tumor cell lines examined show a normal p53 response to DNA damage (18). Regulation of p53 occurs through a variety of mechanisms that may operate differently in different cell types. The possibility that polyomavirus may have some way of counteracting p53 in various target cells was tested by determining the effect of the absence of p53 on tumor induction by the virus. Tumors arose significantly more rapidly in p53 Ϫ/Ϫ than in p53 ϩ/Ϫ or p53 ϩ/ϩ mice, supporting the generally held view that this virus has no effective way of blocking p53 functions during the course of tumor development (18). This result contrasts with those in the SV40 system, where the large T antigen clearly binds p53 and where the absence of p53 in the host can retard rather than accelerate tumor development (28).A single cycle of polyomavirus growth in mouse cells requires roughly 48 h and is dependent on cell cycle progression from G 0 /G 1 into S. In the absence of a counteracting mechanism(s) by the virus, induction of a p53 response leading either to cell cycle arrest or apoptosis would be expected to block virus growth. Expressio...

Induction and Utilization of an ATM Signaling Pathway by Polyomavirus

“…Lytic infection by polyomavirus was previously shown to be accompanied by induction of a p53 response with phosphorylation of p53 on serine 18 (12), indicative of an upstream activation of ATM. To gain further support for ATM activation by virus infection, the effects of caffeine on the levels of ␥-H2AX and phospho-p53 were examined.…”

“…No evidence was found for M-phase events. The initiating signal leading to ATM activation and to a DNA damage-like response to polyomavirus infection depends on the activation of E2F by a polyomavirus large T interaction with pRb (12). Among the target genes of E2F are several of the factors needed for formation of the viral initiation complex and for replication itself, including RPA, DNA polymerase ␣/primase, PCNA, and topoisomerase II (4,46).…”

“…Cells were harvested by trypsinization and frozen in 250 mM sucrose-40 mM sodium citrate-5% dimethyl sulfoxide. For analysis, nuclei were prepared and stained with propidium iodide as described previously (6,12). Total DNA was analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using the programs Cell Quest and ModFit.…”

“…BMK and A31 cells were synchronized by serum starvation for 24 to 72 h in DMEM containing 0.5% or 0.2% calf serum, respectively. The percentage of infected cells was determined as described previously (12). Viruses were titrated on UC1B cells.…”

“…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.…”