Hein Jennifer, Boichuk Sergei, Wu Jiaping, Cheng Yuan, Freire Raimundo, Jat Parmjit S, Roberts Thomas M, Gjoerup Ole V
Hillman Cancer Center, Research Pavilion Suite 1.8, 5117 Centre Avenue, Pittsburgh, PA 15213, USA.
J Virol. 2009 Jan;83(1):117-27. doi: 10.1128/JVI.01515-08. Epub 2008 Oct 15.
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 shown to 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 gamma-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.
猿猴病毒40（SV40）大T抗原（LT）是一种多功能蛋白，对病毒复制和致癌转化至关重要。此前研究表明，SV40感染猴细胞或人细胞会导致DNA损伤反应信号的诱导，而这是高效病毒复制所必需的。然而，尚不清楚LT是否足以诱导损伤反应，如果是，其遗传需求和功能后果可能是什么。在此，我们表明，在没有复制起点的情况下，单独表达LT就能诱导关键的DNA损伤反应标志物，包括γ-H2AX和53BP1在核灶中的积累。ATM/ATR激酶下游的其他DNA损伤信号成分也被诱导，包括chk1和chk2。LT还与Claspin介导蛋白结合，该蛋白通常促进chk1的ATR激活并监测细胞复制起点。LT对损伤反应的刺激主要取决于与Bub1的结合，而不是与视网膜母细胞瘤蛋白的结合。长期以来已知LT能稳定p53，尽管在功能上使其失活。我们表明，LT通过Bub1激活DNA损伤反应似乎在通过促进p53丝氨酸15位点的磷酸化来稳定p53方面发挥主要作用。伴随DNA损伤反应，LT诱导四倍体，这也依赖于Bub1结合。综上所述，我们的数据表明，LT通过与Bub1结合破坏基因组完整性机制，导致DNA损伤反应、p53稳定和四倍体形成。
