Adenovirus-infected cells provide an ideal opportunity to investigate post-transcriptional regulation of gene expression: during the late phase of infection, cellular gene expression is completely inhibited as a result of selective transport of mature, viral mRNA species form the nucleus to the cytoplasm and selective translation of cytoplasmic, viral mRNA mRNAs. The experiments described in this proposal are designed to provide a better understanding of the mechanisms whereby such post- transcriptional discrimination among mRNA species is mediated and thus improve our knowledge not only of the repertoire of regulatory circuits that operate in eukaryotic cells, but also of the ways in which virus infection can disrupt cellular metabolism. The roles of such parameters as location in the viral genome, activation of transcription and specific signals carried by viral mRNA in selection of mRNA species for transport form adenovirus-infected cell nuclei to the cytoplasm will be investigated, using both a series of non- adenoviral genes expressed from sites within the viral genome, the cellular genome or from episomal plasmids, and mutated, viral, late genes. In each case, all steps in production of mRNA species from such genes, as well as cytoplasmic mRNA stability, will be examined during adenovirus infection so that the effects of such parameters on the transport step itself can be assessed. The viral E1B 55kd and E4 early proteins have been implicated in selective transport of mRNA during the late phase of productive infection. Viruses carrying mutations in these coding sequences will therefore be used to investigate the role of these protein in recognition of any parameter that appear essential to selective transport. We have recently established, by screening of temperature-sensitive adenovirus mutants, that the late, non-structural 100kd protein plays an important role in efficient translation of viral, late mRNA species, by a mechanism that appears to operate at the level of initiation of translation. This molecular function of the 100kd protein will be investigated in more detail, both by analysis of the interactions of this protei with mRNA and translational components, and by establishment of in vitro system in which its translational function is reproduced.
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