E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

The transition from G(1) to S phase in the cell cycle requires sequential activation of cyclin-dependent kinase 4 (cdk4) and cdk2, which phosphorylate the retinoblastoma protein, causing the release of E2F. Free E2F upregulates the transcription of genes involved in S phase and cell cycle progression. Recent studies from this and other laboratories have shown that herpes simplex virus 1 stabilizes cyclin D3 early in infection and that early events in viral replication are sensitive to inhibitors of some cdks. On the other hand cdk2 is not activated. Here we report studies on the status of members of E2F family in cycling HEp-2 and HeLa cells and quiescent serum-starved, contact-inhibited human lung fibroblasts. The results show that (i) at 8 h postinfection or thereafter, E2F-1 and E2F-5 were posttranslationally modified and/or translocated from nucleus to the cytoplasm, (ii) E2F-4 was hyperphophorylated, and (iii) overall, E2F binding to cognate DNA sites was decreased at late times after infection. These results concurrent with those cited above indicate that late in infection activation of S-phase genes is blocked both by posttranslational modification and translocation of members of E2F family to inactive compartments and by the absence of active cdk2. The observation that E2F were also posttranslationally modified in quiescent human lung fibroblasts that were not in S phase at the time of infection suggests that specific viral gene products are responsible for modification of the members of E2F family and raises the possibility that in infected cells, activation of the S phase gene is an early event in viral infection and is then shut off at late times. This is consistent with the timing of stabilization of cyclin D3 and the events blocked by inhibitors of cdks.

细胞周期中G₁期向S期的转变，需要细胞周期蛋白依赖性激酶4（cyclin-dependent kinase 4, CDK4）与CDK2依次激活；二者可磷酸化视网膜母细胞瘤蛋白（retinoblastoma protein, Rb），进而引发E2F转录因子的释放。游离的E2F可上调参与S期进程与细胞周期运转的相关基因的转录水平。本实验室及其他团队的近期研究表明，单纯疱疹病毒1型（herpes simplex virus 1, HSV-1）可在感染早期稳定细胞周期蛋白D3（cyclin D3），且病毒复制的早期事件对部分CDK抑制剂敏感，但CDK2并未被激活。本研究针对增殖状态的HEp-2细胞、HeLa细胞，以及经血清饥饿诱导静止、接触抑制的人肺成纤维细胞中E2F家族成员的表达状态展开分析，结果显示：（1）在感染后8小时及更晚时间点，E2F-1与E2F-5发生翻译后修饰，且/或从细胞核转位至细胞质；（2）E2F-4出现过度磷酸化；（3）整体而言，感染后期E2F与其同源DNA结合位点的结合活性显著降低。结合前述研究结果，上述数据表明，在感染后期，S期基因的激活受到双重阻断：一是E2F家族成员发生翻译后修饰并转位至非活性区域，二是细胞内缺乏活性CDK2。研究还观察到，在感染时未处于S期的静止人肺成纤维细胞中，E2F同样发生了翻译后修饰；这一结果提示，特定病毒基因产物介导了E2F家族成员的修饰，并提示在感染细胞中，S期基因的激活是病毒感染的早期事件，而该激活过程会在感染后期被关闭。这一结论与细胞周期蛋白D3的稳定时间进程，以及受CDK抑制剂阻断的病毒复制事件的时序高度吻合。