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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI051340-09
Application #
8197408
Study Section
Special Emphasis Panel (ZRG1-IDM-R (03))
Program Officer
Park, Eun-Chung
Project Start
2002-05-01
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
9
Fiscal Year
2012
Total Cost
$381,177
Indirect Cost
$136,833
Name
Suny Downstate Medical Center
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
040796328
City
Brooklyn
State
NY
Country
United States
Zip Code
11203
Imai, Shunsuke; Kumar, Parimal; Hellen, Christopher U T et al. (2016) An accurately preorganized IRES RNA structure enables eIF4G capture for initiation of viral translation. Nat Struct Mol Biol 23:859-64
Asnani, Mukta; Kumar, Parimal; Hellen, Christopher U T (2015) Widespread distribution and structural diversity of Type IV IRESs in members of Picornaviridae. Virology 478:61-74
Zinoviev, Alexandra; Hellen, Christopher U T; Pestova, Tatyana V (2015) Multiple mechanisms of reinitiation on bicistronic calicivirus mRNAs. Mol Cell 57:1059-73
Sweeney, Trevor R; Abaeva, Irina S; Pestova, Tatyana V et al. (2014) The mechanism of translation initiation on Type 1 picornavirus IRESs. EMBO J 33:76-92
Kumar, Parimal; Sweeney, Trevor R; Skabkin, Maxim A et al. (2014) Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5'-terminal regions of cap0-, cap1- and 5'ppp- mRNAs. Nucleic Acids Res 42:3228-45
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
Sweeney, Trevor R; Dhote, Vidya; Yu, Yingpu et al. (2012) A distinct class of internal ribosomal entry site in members of the Kobuvirus and proposed Salivirus and Paraturdivirus genera of the Picornaviridae. J Virol 86:1468-86
Jackson, Richard J; Hellen, Christopher U T; Pestova, Tatyana V (2012) Termination and post-termination events in eukaryotic translation. Adv Protein Chem Struct Biol 86:45-93
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; Sweeney, Trevor R; Kafasla, Panagiota et al. (2011) The mechanism of translation initiation on Aichivirus RNA mediated by a novel type of picornavirus IRES. EMBO J 30:4423-36

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