Infection of quiescent cell cultures by adenoviruses and papovavirus results in the induction of cellular DNA synthesis. This induction is thought to reflect a requirement for cell cycle regulated expression or activation of either cellular or viral encoded functions critical for viral DNA replication. At least a partial mechanistic explanation for this may be found in the fact that these viruses can interfere with the ability of the tumor suppressor protein pRB to regulate the cellular transcription factor E2F. The role of pRB in regulating cell cycle events derives in part from the genetics of human retinoblastoma and also from studies which correlate release of E2F from complexes with pRB or a related protein p107 with activation of E2F-responsive genes, among which are several encoding cell cycle progression or DNA replication functions. The significance of this for virus replication is suggested by the failure of viruses to replicate if they are unable to affect pRB-E2F interactions. Representatives of the three groups of herpesviruses also induce DNA synthesis upon infection of quiescent or post-mitotic cells. This research proposal is designed to test that HSV affects cell cycle regulation by inducing an S phaselike state during the course of lytic replication. As a test of this hypothesis an using adenovirus promoter sequences as a probe, we have found that S phase-specific E2F DNA binding activities containing wither pRB or p107 as well a free E2F, are induced under the regulation of HSV gene products. We speculate that this induction process by HSV is a consequence of deregulating cell cycle check points and either leads to or reflects modifications of cellular functions required for virus replication on non-cycling cells. A more thorough understanding of the mechanism(s) of E2F induction by HSV should provide important insights into the mechanism of normal cell cycle regulation. The experiments in this proposal are designed to further explore the mechanism of induction, and the functional significance of E2F induction for the HSV replication cycle.
The specific aims i nclude the following: (i) determining whether HSV induced new expression of or modifies pre-existing components of E2F DNA- binding activities; (ii) determining how HSV infection alters the ability of E2F-pRB or p- 107 complexes to bind DNA and identifying additional E2F DNA binding activities utilizing promoter sequences of E2F-responsive promoters; and (iv) characterizing the induction of E2F complex formation in quiescent cells.
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