Abstract

Many viruses synthesize mRNAs containing an internal ribosomal entry site (IRES) that mediates cap-independent translation. IRESs differ greatly from one another and use distinct mechanisms for initiation. Our studies of the molecular mechanisms of initiation on 4 different IRESs will provide a basis for understanding their cell type-specificity, for the design of inhibitors, and may identify underlying mechanistic similarities. IRESs often act in a cell type-specific manner that determines viral pathogenesis. The attenuated neurovirulence of poliovirus vaccine strains is due to defective IRES function, likely due to a need for cell-specific IRES trans-acting factors (ITAFs). We will identify the complete set of canonical factors/ITAFs required by this IRES using biochemical reconstitution of initiation in vitro, determine how ribosomes reach the initiation codon approximately 60 nucleotides downstream of the IRES and map the interactions of components of the translation apparatus with IRESs of attenuated and virulent PV strains. This analysis will elucidate an important aspect of viral tissue tropism. Hepatitis A virus contains a 580 nucleotide-long IRES that also determines viral growth characteristics and pathogenicity in humans. We shall use similar approaches to determine the complete set of canonical factors and ITAFs needed by this IRES and to map their interactions with the IRES. Hepatitis C virus (HCV) and Cricket paralysis virus (CrPV) IRESs use very different mechanisms of initiation. The HCV IRES binds both eIF3 and the 40S ribosomal subunit and then needs only eIF2/GTP/tRNA to form a 48S complex. We shall characterize interactions that lead to 48S complex formation in detail, examine regulation of eIF3's activity during chronic HCV infection and determine how ribosomal subunits join on the IRES. We shall also identify how and where the CrPV IRES binds the 40S subunit and whether its binding excludes eIF2/tRNA from the P site. We shall determine how This IRES induces translocation of aa-tRNA from ribosomal A to P site without concomitant peptide formation so that protein synthesis can begin. We shall reconstitute this process in vitro to determine how this occurs.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI051340-03
Application #
6738149
Study Section
Virology Study Section (VR)
Program Officer
Koshy, Rajen
Project Start
2002-05-01
Project End
2007-04-30
Budget Start
2004-05-01
Budget End
2005-04-30
Support Year
3
Fiscal Year
2004
Total Cost
$344,250
Indirect Cost

  Institution

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

  Publications

Asnani, Mukta; Pestova, Tatyana V; Hellen, Christopher U T (2016) PCBP2 enables the cadicivirus IRES to exploit the function of a conserved GRNA tetraloop to enhance ribosomal initiation complex formation. Nucleic Acids Res 44:9902-9917
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; Pestova, Tatyana V; Hellen, Christopher U T (2016) Initiation on the divergent Type I cadicivirus IRES: factor requirements and interactions with the translation apparatus. Nucleic Acids Res 44:3390-407
Abaeva, Irina S; Pestova, Tatyana V; Hellen, Christopher U T (2016) Attachment of ribosomal complexes and retrograde scanning during initiation on the Halastavi árva virus IRES. Nucleic Acids Res 44:2362-77
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 57:1059-1073
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
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
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

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