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 mechanisms of increasing complexity 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 ~180nt-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 within the IRES play an analogous role during elongation to deacylated tRNA when it has been translocated to the E site. The IRES of the pestivirus classical swine fever virus 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 characterize whether these mechanisms account for relaxation on this IRES of the ribosome?s normally strict avoidance of initiation at non-AUG codons. Type 2 picornavirus IRESs such the encephalomyocarditis virus IRES bind specifically to eIF4G/eIF4A: we will investigate how these factors act to promote recruitment of 43S complexes to the initiation codon, how ITAFs promote binding of eIF4G/eIF4A and why only a subset of type IRESs require their activity. The mechanism of initiation on type 1 picornavirus IRESs, such as that of poliovirus, is not known. We shall characterize it by purifying and identifying all necessary factors in order to reconstitute the entire initiation process in vitro. We will then characterize interactions between factors and the ribosome 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
High Priority, Short Term Project Award (R56)
Project #
2R56AI051340-06
Application #
7668340
Study Section
Special Emphasis Panel (ZRG1-IDM-B (02))
Program Officer
Park, Eun-Chung
Project Start
2002-05-01
Project End
2008-12-14
Budget Start
2008-08-15
Budget End
2008-12-14
Support Year
6
Fiscal Year
2008
Total Cost
$390,641
Indirect Cost
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
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

Showing the most recent 10 out of 18 publications