The genomes of many positive sense RNA viruses contain an internal ribosomal entry site (IRES) that mediates end-independent initiation of translation. IRESs belong to different structural classes and use distinct mechanisms for initiation. Our proposed studies of the process of initiation on representatives of four classes of IRES that use distinct mechanisms will identify important cis-acting elements and provide detailed mechanistic insights into the actions of canonical initiation factors and cellular IRES trans-acting factors (ITAFs) in promoting internal ribosomal entry. Our studies will address mechanisms of increasing complexity. The ~180 nt-long intergenomic region (IGR) IRESs of dicistroviruses mediate initiation without initiator tRNA or initiation factors and the first elongation cycle consequently occurs without deacylated tRNA in the ribosomal P site. We will characterize whether elements in the IRES play an analogous role during elongation to deacylated tRNA when it has been translocated to the E site. The IRES of classical swine fever virus, a pestivirus, is substantially resistant to inhibition by eIF2 phosphorylation, which can be accounted for by the use of two exceptional eIF2-independent mechanisms of initiation. We will determine whether these mechanisms account for the relaxation on this IRES of the ribosome's normally strict avoidance of initiation at non-AUG codons. Type 2 picornavirus IRESs such as the encephalomyocarditis virus IRES bind specifically to eIF4G/eIF4A: we will investigate how these factors promote recruitment of 43S complexes to the initiation codon, how ITAFs induce conformational changes in these IRESs and why only a subset of type 2 IRESs require their activity. The mechanism of initiation on type 1 picornaviruses (e.g. poliovirus) is not known, but also involves specific interaction with eIF4G. We shall characterize this interaction in detail, validate previously identified candidate ITAFs and if necessary isolate additional ITAFs to reconstitute the entire initiation process on this IRES in vitro. We will then characterize interactions between factors and the IRES using chemical/enzymatic footprinting and directed hydroxyl radical cleavage. The studies will provide a framework for understanding of mechanistic details of IRES-mediated initiation, for understanding the cell-type specificity of IRES function and for the design of inhibitors.

Public Health Relevance

The genomes of a number of viruses, including poliovirus, hepatitis C virus and foot-and-mouth disease virus contain structured RNA elements known as `internal ribosomal entry sites'that promote viral protein synthesis by mechanisms that are distinct from those used by the cell. The size and complexity of IRESs varies, but they all coordinate the activities of protein factors in order to recruit ribosomes to translate viral proteins and are consequently essential for virus viability. We are investigating how IRESs work, which may give insights that, will eventually be used in the development of antiviral drugs.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1-IDM-R (03))
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Park, Eun-Chung
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Suny Downstate Medical Center
Schools of Medicine
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Hashem, Yaser; des Georges, Amedee; Dhote, Vidya et al. (2013) Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit. Nature 503:539-43
Abaeva, Irina S; Marintchev, Assen; Pisareva, Vera P et al. (2011) Bypassing of stems versus linear base-by-base inspection of mammalian mRNAs during ribosomal scanning. EMBO J 30:115-29
Yu, Yingpu; Abaeva, Irina S; Marintchev, Assen et al. (2011) Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors. Nucleic Acids Res 39:4851-65
Skabkin, Maxim A; Skabkina, Olga V; Dhote, Vidya et al. (2010) Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling. Genes Dev 24:1787-801
Jackson, Richard J; Hellen, Christopher U T; Pestova, Tatyana V (2010) The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11:113-27
Hellen, Christopher U T (2009) IRES-induced conformational changes in the ribosome and the mechanism of translation initiation by internal ribosomal entry. Biochim Biophys Acta 1789:558-70
Pisareva, Vera P; Pisarev, Andrey V; Komar, Anton A et al. (2008) Translation initiation on mammalian mRNAs with structured 5'UTRs requires DExH-box protein DHX29. Cell 135:1237-50
Galkin, Oleksandr; Bentley, Amber A; Gupta, Sujatha et al. (2007) Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5. RNA 13:2116-28
Hellen, Christopher U T; de Breyne, Sylvain (2007) A distinct group of hepacivirus/pestivirus-like internal ribosomal entry sites in members of diverse picornavirus genera: evidence for modular exchange of functional noncoding RNA elements by recombination. J Virol 81:5850-63
Pisareva, Vera P; Hellen, Christopher U T; Pestova, Tatyana V (2007) Kinetic analysis of the interaction of guanine nucleotides with eukaryotic translation initiation factor eIF5B. Biochemistry 46:2622-9

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