Experiments are proposed to identify cell proteins required for function of the internal ribosome entry site (IRES) of hepatitis C virus (HCV), poliovirus (PV), encephalomyocarditis virus (EMCV), and the S. cerevisiae TFIID gene. IRESes are highly ordered RNA sequences at the 5' end of viral and cellular mRNAs that permit ribosomes to initiate translation independent of the 5'-end of the IIIRNA. IRES function depends on the canonical translation initiation factors as well as other cell proteins. The yeast Saccharomyces cerevisiae will be used to identify cell proteins that are required for function of IRESes.
Aim 1 : Functional expression of IRESes in S. cerevisiae. DNA encoding a bicistronic mRNA will be constructed that consists of the ADE3 gene followed by the IRES and the lacZ gene. The Ade3p protein will be produced by 5'-end dependent translation, and serves as a marker for the presence of the mRNA. B-galactosidase will be produced by internal ribosome binding, and will serve as a marker for function of the IRES. S. cerevisiae containing this plasmid will be mutagenized, and colonies that produce reduced levels of B-galactosidase will be identified. These yeast strains potentially contain mutations in genes that are important for the function of the IRES.
Aim 2 : Identification of S. cerevisiae genes required for IRES function. Mutant yeast strains identified in aim 1 presumably have a mutation in a gene that is important for IRES function. To identify the mutant genes, we will introduce a plasmid-based genomic DNA library of S. cerevisiae into the mutant strains to complement the defect in function of the IRES. Nucleotide sequence analysis will be used to identify the complementing gene from the S. cerevisiae genome database.
Aim 3 : Determination of the roles of cell proteins in IRES function. Yeast genes identified in aim 2 will be disrupted to formally prove that the encoded proteins are required for IRES function. If the yeast proteins bind to the different IRESes, the binding site on the IRES will be identified by mutagenesis. Mammalian sequence homologs of the S. cerevisiae genes important for function of the viral IRESes will be identified, and their role in IRES function will be assessed by depletion of the protein from cell-free translation extracts and from living cells. These studies will contribute to our understanding of the function of IRESes, important regulators of cell and viral growth.
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| Rosenfeld, Amy B; Racaniello, Vincent R (2005) Hepatitis C virus internal ribosome entry site-dependent translation in Saccharomyces cerevisiae is independent of polypyrimidine tract-binding protein, poly(rC)-binding protein 2, and La protein. J Virol 79:10126-37 |
