Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and poses a serious health threat to immunocompromised individuals, particularly those with AIDS. This virus appears to affect the host cell metabolism in ways that mimic processes involved in cell activation, but the molecular basis of these effects and their relationship to viral replication have yet to be elucidated. We have found that the HCMV infection markedly affects key components of the cell cycle, leading to cell cycle arrest. Early after infection, HCMV induced elevated levels of cyclins E and B and their associated kinase activities, p53, and hyperphosphorylated Rb. However, the synthesis of cyclin A was inhibited, and only at very late times in the infection was there any increase in the levels of protein and kinase activity. Our preliminary studies indicate that p53, but not Rb, becomes sequestered in viral replication centers in the nucleus. We also have evidence which shows that both the enhanced expression of cyclin E and the inhibition of cyclin A are due to effects at the level of transcription, and we hypothesize that the E2F/DP family of transcription factors are involved. In contrast, the increased levels of cyclin B appear to be due to lack of cell-cycle mediated degradation. Preliminary data also indicate that during the infection there is a distinct redistribution of the proteasomes and their overall level increases. Thus, it appears that the HCMV infection has altered the transcription, subcellular localization, and stability of key cell cycle regulatory proteins. The goal of the proposed studies is to decipher the molecular basis of these cell cycle perturbations. The approach is to couple in vivo analyses with in vitro biochemical and molecular assays to achieve the following specific aims: 1) identification of the cis-acting sequences and trans-acting factors responsible for the HCMV-mediated effects on the transcription of the cyclin E and cyclin A genes; 2) determination of the role of ubiquitination and the proteasomes in mediating the effects of the HCMV infection on the stability of cell cycle regulatory proteins; and 3) analysis of the functional properties of the HCMV 1E2 86 and IE1 72 gene products with respect to dysregulation of the cell cycle. These studies are important not only to advance our knowledge of how the complex interactions of viral and host functions relate to viral replication and pathogenesis but also to help elucidate the general mechanisms that operate to control the cell cycle.
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