The major goal of the project is to understand how the human translation initiation factor elF3 contributes to the initiation mechanism and to the regulation of viral mRNAs, especially Hepatitis C Virus (HCV) mRNA that initiates translation by ribosome binding to an internal entry site (IRES). The structure of elF3, a factor comprising 12 non-identical subunits, will be probed by isolating a variety of sub-complexes formed in baculovirus-infected insect cells. The binding of elF3 and its sub-complexes to the HCV IRES will be defined quantitatively. A highly active partially fractionated mammalian cell lysate system dependent on purified elF3 will be constructed with (5-globin mRNA. This system will be used to define the role of elF3 and its subcomplexes in the translation of HCV mRNA. An in vitro translation system also will be constructed that utilizes all of the purified initiation factors, in order to define which factors are required for the translation of HCV and Dengue virus mRNAs. These studies will determine to what extent the viral mRNAs differ from capped/polyadenylated mRNAs in their initiation mechanisms, and may identify targets for therapeutic intervention of infections by these viruses. Considerable evidence indicates that elF3 plays an important role in regulating protein synthesis and cell proliferation. elF3, like other initiation factors, is phosphorylated on numerous subunit proteins. The sites of phosphorylation will be identified by mass spectroscopy, and the effects of phosphorylation on function will be tested in the translation assays. Mutant forms will be constructed and tested, with phosphorylation sites substituted with alanine to prevent phosphorylation, and with glutamate or aspartate to mimic phosphorylation. The experiments are designed to elucidate the mechanism of action of elF3 and the role of phosphorylation in regulating its activity during initiation of capped and IRES-driven mRNAs. The results may explain how disfunction or disregulation of elF3 may affect the control of cell proliferation, resulting in celt malignancy.
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