Olgiate J, Ehmann G L, Vidyarthi S, Hilton M J, Bachenheimer S L
Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, 27599-7290, USA.
Virology. 1999 Jun 5;258(2):257-70. doi: 10.1006/viro.1999.9755.
Accumulation of E2F-p107 and E2F-pRB DNA binding complexes occurred after herpes simplex virus infection of U2-OS cells. Accumulation of E2F-p107 also occurred by 4 h p.i. in C33 cells. This corresponded to a time when host DNA synthesis was reduced by 50%, and lagged by >/=1 h, the onset of viral DNA synthesis. To determine the basis for increased nuclear E2F complexes, we investigated the effects of virus infection on the intracellular distribution of the E2F-dependent DNA binding complexes and their protein constituents. Western blot analyses of whole cell extracts revealed that amounts of E2F4, E2F1, DP1, and p107 remained unchanged after infection of C33 cells. Analysis of cytoplasmic and nuclear fractions, however, revealed that cytoplasmic E2F4 decreased and nuclear E2F4 increased. This correlated with a loss of cytoplasmic E2F DNA-binding activity and a corresponding increase in nuclear DNA-binding activity. Concomitant with its redistribution, the apparent molecular weight of total and p107-associated E2F4 increased, at least partially as a result of protein phosphorylation. Increased nuclear E2F-pRB in U2-OS cells was accompanied by the conversion of pRB from a hyper- to a hypophosphorylated state. Infection of U2-OS cells with viral mutants indicated that viral protein IE ICP4 was necessary for the decrease in cytoplasmic E2F-p107, and that viral protein DE ICP8 was required for nuclear accumulation of p107-E2F. In contrast, ICP8 was not required for accumulation of E2F-pRB. These results indicate that the increase in E2F-p107 may be explained by the redistribution and modification of E2F4 and the increase in E2F-pRB by modification of pRB.
单纯疱疹病毒感染U2-OS细胞后，E2F-p107和E2F-pRB DNA结合复合物出现积累。在C33细胞中，感染后4小时也出现了E2F-p107的积累。这与宿主DNA合成减少50%的时间相对应，且比病毒DNA合成开始时间滞后≥1小时。为了确定核内E2F复合物增加的基础，我们研究了病毒感染对E2F依赖性DNA结合复合物及其蛋白质成分细胞内分布的影响。对全细胞提取物的蛋白质印迹分析显示，C33细胞感染后，E2F4、E2F1、DP1和p107的量保持不变。然而，对细胞质和细胞核组分的分析显示，细胞质中的E2F4减少，而细胞核中的E2F4增加。这与细胞质E2F DNA结合活性的丧失以及核DNA结合活性的相应增加相关。伴随着其重新分布，总E2F4和与p107相关的E2F4的表观分子量增加，至少部分是蛋白质磷酸化的结果。U2-OS细胞中核内E2F-pRB的增加伴随着pRB从高磷酸化状态向低磷酸化状态的转变。用病毒突变体感染U2-OS细胞表明，病毒蛋白IE ICP4是细胞质E2F-p107减少所必需的，而病毒蛋白DE ICP8是p107-E2F核内积累所必需的。相反，E2F-pRB的积累不需要ICP8。这些结果表明，E2F-p107的增加可能是由于E2F4的重新分布和修饰，以及pRB的修饰导致E2F-pRB增加所致。
