Human adenoviruses are under intense scrutiny as vehicles for gene therapy and as anti-cancer agents in humans. Nevertheless, many basic aspects of the mechanisms by which these viruses subvert normal cellular processes to ensure efficient replication remain poorly understood. These include posttranscriptional regulation of viral and cellular gene expression by the adenoviral E1B 55 kDa and E4 0rf 6 proteins, which induce selective export of viral mRNAs from the nucleus to the cytoplasm during the late phase of infection. Those two proteins also exhibit transforming activity, and can induce accelerated degradation of the cellular tumor suppressor protein p53 and block transcriptional activation by p53. The long-term objective of these studies is elucidation of the molecular mechanism(s) by which the adenoviral E1B 55 kDa protein regulates mRNA export, and its relationship to the seemingly disparate functions of the protein in modulation of p53 turnover and activity.
The specific aims of this proposal comprise complementary molecular, genetic and biochemical approaches to address these issues. Both mutations targeted to specific sequences and more systematic changes will be introduced into the E1B 55kDa protein coding sequence in the viral genome, and mRNA export and related phenotypes exhibited by the mutant viruses examined in infected HeLa cells. This information will yield a detailed description of the protein, and identification of sequences required for mRNA export regulation will facilitate molecular and biochemical investigations of the mechanism(s) by which the E1B protein induces selective export of viral mRNAs. These studies will employ a variety of methods, including inhibition of individual RNA export pathways by cell permeable, inhibitory peptides, to address such questions as which cellular RNA export pathway(s) are subverted by the viral protein, and whether its binding to RNA in vivo is necessary for selective mRNA export. The effects of the mutations on E1B 55 kDa protein functions and activities in normal, diploid human cells will also be examined. This analysis is designed to establish the contribution of regulation of mRNA export and modulation of p53 turnover (or activity) to efficient virus reproduction. It will also reveal any mechanistic relationships between these activities of the E1B protein, or previously unknown roles of the protein. ? ?
