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Regulation of viral and cellular gene expression in adenovirus-infected cells

Like all viruses, human adenoviruses are molecular parasites that rely on cellular mechanisms for expression of their genetic information. They are therefore excellent model systems for investigation of fundamental cellular processes. Furthermore, several adenoviral genes can contribute to neoplastic transformation of cells in culture and certain adenovirus serotypes are tumorigenic in laboratory animals. Study of these oncogenic viral gene products has provided important insights into the mechanisms that regulate progression through the cell cycle.

The adenoviral infectious cycle can be described in terms of an orderly program of the expression of viral genes that comprise the double-stranded DNA genome, culminating in the synthesis of prodigious quantities of viral macromolecules and inhibition of cellular gene expression. Viral genes are expressed via the cellular biosynthetic machinery, yet infection induces transcriptional and post-transcriptional regulatory mechanisms controlling viral and cellular gene expression. Our work aims to elucidate such regulatory events.

Products of the adenoviral E1A gene initially activate transcription of a subset of viral genes by cellular RNA polymerase II, while transcription of other genes later in the infectious cycle requires viral DNA synthesis in the infected cell. To investigate mechanisms of DNA synthesis-dependent activation of transcription, we chose to focus on the late IVa2 promoter and demonstrated that uninfected cells contain a repressor of IVa2-transcription that binds specifically to a sequence superimposed on those of the IVa2 promoter. As adenovirus infection does not lead to inactivation of this cellular transcriptional repressor, we proposed that late phase-specific transcriptional activity of the IVa2 promoter is the result of titration of the cellular repressor following initiation of viral DNA synthesis. As the IVa2 protein is itself an activator of transcription from a second, viral late promoter, the cellular repressor may control a regulatory cascade determining the temporal program of viral gene expression. We have established the validity of the repressor titration model for regulation of expression of the IVa2 gene during the infectious cycle and are currently investigating the identity and mechanisms of action of the cellular repressor using genetic, molecular, and biochemical methods. We are applying similar methods to elucidation of the mechanism(s) by which the IVa2 protein stimulates transcription from the major late promoter.

The early E2E promoter, which controls production of viral replication proteins, was extensively characterized in early studies. More recently, we have established that an RNA polymerase III promoter active in adenovirus infected cells is superimposed on the typical E2E RNA polymerase II promoter. The properties of RNA polymerase III transcription during infection and of the RNA products of such transcription suggest that recognition of the E2E promoter by RNA polymerase III may serve to damp, or set a threshold for, RNA polymerase II transcription. Such a novel regulatory mechanism would ensure that production of replication proteins, and therefore entry into the late phase of infection, take place only when the host cell milieu has been optimized for viral replication by the action of E1A (and other) proteins that induce both entry of infected cells into S phase and efficient RNA polymerase II transcription from the E2E promoter. An important aim of our current studies is, therefore, to establish whether such a novel mechanism of regulation of transcription operates in adenovirus-infected cells.

The inhibition of cellular gene expression characteristic of the late phase of adenovirus infection is in part the result of an unusual post-transcriptional regulatory mechanism, induction of selective export of newly-synthesized viral mRNAs from the nucleus to the cytoplasm. Two viral early proteins, the E1B 55 kDa and the E4 Orf 6 proteins, are required for efficient export of viral mRNAs with concomitant inhibition of export of the majority of newly synthesized cellular mRNA species. These two proteins can also each independently inhibit the activity of the cellular p53 protein, which regulates the response of cells to genotoxic stress and induces cell-cycle arrest or apoptosis. These adenoviral E1B and E4 proteins also cooperate to increase the rate of degradation of the p53 protein. Despite such important roles, the mechanisms by which these early proteins regulate mRNA export and p53 activity and concentration are not well understood. We have demonstrated that the E1B 55 kDa protein is primarily responsible for directing viral late mRNAs for selective export and is required to protect normal diploid, human cells against adeno-virus-induced apoptosis. We are therefore investigating molecular functions of this protein using genetic and biochemical approaches.



 
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Journal of Virology

 

Virology

 

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