Simian Virus 40 Large T Antigen Disrupts Genome Integrity and Activates a DNA Damage Response via Bub1 Binding

Abstract

Simian virus 40 (SV40) large T antigen (LT) is a multifunctional protein that is important for viral replication and oncogenic transformation. Previously, infection of monkey or human cells with SV40 was shownto lead to the induction of DNA damage response signaling, which is required for efficient viral replication. However, it was not clear if LT is sufficient to induce the damage response and, if so, what the genetic requirements and functional consequences might be. Here, we show that the expression of LT alone, without a replication origin, can induce key DNA damage response markers including the accumulation of ␥-H2AX and 53BP1 in nuclear foci. Other DNA damage-signaling components downstream of ATM/ATR kinases were induced, including chk1 and chk2. LT also bound the Claspin mediator protein, which normally facilitates the ATR activation of chk1 and monitors cellular replication origins. Stimulation of the damage response by LT depends mainly on binding to Bub1 rather than to the retinoblastoma protein. LT has long been known to stabilize p53 despite functionally inactivating it. We show that the activation of a DNA damage response by LT via Bub1 appears to play a major role in p53 stabilization by promoting the phosphorylation of p53 at Ser15. Accompanying the DNA damage response, LT induces tetraploidy, which is also dependent on Bub1 binding. Taken together, our data suggest that LT, via Bub1 binding, breaches genome integrity mechanisms, leading to DNA damage responses, p53 stabilization, and tetraploidy.Simian virus 40 (SV40) is a small DNA tumor virus, belonging to the polyomavirus family, that induces a productive infection in its natural host, the rhesus macaque, but yields oncogenic transformation in nonpermissive hosts such as rodent cells. The highly multifunctional large T antigen (LT) is the key early protein essential for both driving viral replication as well as inducing cellular transformation. Two other early proteins, small t antigen and 17k T antigen (17k) may perform auxiliary functions during the viral life cycle (39, 69). LT has served as a powerful model system for understanding fundamental cellular processes such as nuclear translocation, transcriptional regulation, eukaryotic DNA replication, immortalization, and malignant transformation (reviewed in references 26 and 37).LT overrides cellular control mechanisms and reprograms the host cell to create a permissive environment for viral replication. The deregulation of cellular proliferation is dependent on LT's interaction with specific host proteins, among which the tumor suppressors p53 and the retinoblastoma protein (pRB) are the best characterized (reviewed in reference 37). Transformation in vitro and tumor induction in vivo frequently depend on LT binding and functionally inactivating these key tumor suppressors (37).

Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

“…BUB1 participates with proteins acting in DNA repair-related pathways, including principal NHEJ factors, and DSBs in BUB1-depleted cells induce ␥-H2AX phosphorylation and 53BP1 focus formation. The latter finding and the presence of Bub-phosphorylated substrates on Tel1 S/T-Q sites reinforce the finding that ATM-mediated BUB1-dependent phosphorylation is likely to activate the DDR (21) and accumulate ␥-H2AX and 53BP1 focus formation in a BUB1 binding-and ATM-dependent manner (56).…”

Bocavirus Infection Induces a DNA Damage Response That Facilitates Viral DNA Replication and Mediates Cell Death

“…8) strongly supports the notion that the MRN complex is required for replication of the MVC genome ( Fig. 10) and that its role may be to recruit DNA repair factors to the replication center (14,37), as p-Nbs1 is essential to DSB repair (29). On the other hand, the MRN complex senses ATM activation, which in turn may mediate proteasome-dependent degradation of the MRN subunit (94) at later stages of infection.…”

“…The DDR that is induced during simian virus 40 (SV40) infection has been suggested to be activated by the large T antigen via Bub1 binding (37). The large T antigen also interacts with the MRN complex (30,49,90).…”

A combination of MSH2 DNA mismatch repair deficiency and expression of the SV40 large T antigen results in cisplatin resistance of mouse embryonic fibroblasts

“…Additionally, few studies have compared the repair of cisplatin damage in primary versus transformed fibroblasts. Changes induced by transformation with simian virus 40 (SV40) have been reported to have important effects on both activation of the DNA damage response and DNA repair itself (17)(18)(19)(20).…”