Herpes Simplex Virus Type 1 Infection Imposes a G1/S Block in Asynchronously Growing Cells and Prevents G1 Entry in Quiescent Cells

Herpes simplex virus type 1 (HSV-1) infection disrupted cell cycle regulation in at least two ways. First, infection of quiescent human embryonic lung cells simultaneously with readdition of serum caused inhibition of cyclin D/cyclin-dependent kinase (CDK) 4,6-specific and cyclin E/CDK2-specific phosphorylation of the retinoblastoma protein pRb. The inhibition of cyclin D/CDK4,6 kinase activity corresponded to a loss of cyclin D1 protein and a failure of CDK4 and CDK6 to translocate to the nucleus. Failure to detect cyclin E/CDK2 kinase activity was accompanied by a loss of cyclin E protein and a failure of CDK2 to translocate to the nucleus. Levels of pocket protein p130 persisted, whereas p107 did not accumulate. As a result of these effects on cyclin kinase, G0-infected cells failed to reenter the cell cycle. The second type of HSV-induced cell cycle dysregulation was observed in asynchronously dividing cell cultures. A rapid inhibition of preexisting cyclin E/CDK2 and cyclin A/CDK2 activities was observed in human embryonic lung cells, as well as two other human cell lines: C33 and U2OS. HSV-1 immediate-early gene expression was necessary for the inhibition of CDK2 kinase activity. Cyclin and CDK subunit protein levels, intracellular localization, and complex stability were unaffected by infection. In addition, levels of cyclin-dependent kinase inhibitors, p27 and p21, were not affected by HSV-1. Previous experiments demonstrated that in asynchronous infected cells, hypophosphorylated pRb and pocket protein–E2F complexes accumulated, and cellular DNA synthesis was rapidly inhibited. Coupled with the present results, this indicates that HSV-1 has evolved mechanisms for preventing cells in G1 from proceeding through the restriction point and for cells in S from completing a round of DNA replication

Herpes Simplex Virus Induces Intracellular Redistribution of E2F4 and Accumulation of E2F Pocket Protein Complexes

AbstractAccumulation 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