ANALYSIS OF VIRAL TRANSLATION COMPLEXES

University Of California Berkeley, Berkeley CA

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Abstract

It has been long been known that eukaryotic ribosomes are heterogeneous in nature. Ribosome[unreadable] heterogeneity is exemplified by the differential presence of certain ribosomal proteins and ribosomal[unreadable] RNAs, modification of ribosomal proteins and RNA, and association of non-ribosomal proteins with[unreadable] ribosomal particles. However, it remains unknown whether the heterogeneous pools of ribosomes reflect[unreadable] distinct activities in protein biosynthesis. More recently, it has become clear that the internal ribosome[unreadable] entry sites (IRES) located in certain viral mRNAs can bind directly and with high affinity to mammalian[unreadable] 40S subunits. The aims of this proposal are to examine whether functionally heterogeneous populations[unreadable] of ribosomes exist in mammalian cells and whether distinct ribosome populations are recruited to[unreadable] picornaviral and hepatitis C viral IRES elements in infected cells. In the first aim, the composition and[unreadable] activity of total, unbound and polysome-bound ribosomal subunits from uninfected cells will be compared[unreadable] to those isolated from picornavirus infected cells by electrospray mass spectrometry (ES-MS). In the[unreadable] second aim, the presence of altered ribosomes in viral mRNA-complexes will be examined. Specifically,[unreadable] thiouridine-containing viral RNA will be generated in HeLa cells expressing the Toxoplasma gondii uracil[unreadable] phosphoribosyltransferase (UPRT) enzyme. UPRT will convert thiouracil to thiouridine 5' monophosphate[unreadable] nucleotides whose triphosphates can be selectively incorporated into viral RNA by the viral RNAdependent[unreadable] RNA polymerase in the presence of actinomycin D. Polysomal thio-labeled RNA will be[unreadable] biotinylated, isolated by streptavidin chromatography and associated ribosomes characterized by ES-MS.[unreadable] This method will also be used to identify IRES-binding proteins that are tightly linked to the viral RNA in[unreadable] infected cells. Finally, the roles of modified ribosomes in various steps of translation initiation will be[unreadable] examined in reconstituted translation systems. The outcome from these studies will reveal roles of[unreadable] modified ribosomes in mammalian cells and will provide insights by which IRES elements recruit host cell[unreadable] ribosomes during viral infection, revealing new potential targets for antiviral therapeutics.