Picornaviruses as well as other positive strand RNA viruses utilize sequences in the 5'noncoding regions (5'NCRs) of their genomic RNAs to bind host cell and viral proteins to carry out important functions during their intracellular replication cycles. Poliovirus, coxsackievirus, and human rhinovirus are members of the Picornaviridae that share a common RNA secondary structure in their 5'NCRs required for internal ribosome entry during translation initiation. These structures (i.e., IRES elements) bind several host RNA binding proteins, including poly(rC) binding protein 2 (PCBP2). Binding of PCBP2 is required for poliovirus translation initiation, a process that also requires the function of a nucleo-cytoplasmic shuttling protein, SRp20.
The first aim of this proposal is to identify components of translation initiation complexes assembled with PCBP2-bound poliovirus IRES sequences. Genetic, biochemical, and confocal microscopy experiments will test the hypothesis that SRp20 acts as a molecular bridge between PCBP2 bound to the poliovirus IRES and specific elements of the cellular translation initiation apparatus. In addition to its role in poliovirus translation, cellular protein PCBP2 is required for negative-strand viral RNA synthesis. During poliovirus infection of HeLa cells, PCBP2 is cleaved by a viral proteinase to generate a truncated protein that is unable to function in translation but retains its role in RNA replication.
The second aim of the proposal will utilize cell culture and in vitro translation/RNA replication approaches to determine if this cleavage is responsible for clearing poliovirus genomic RNAs of translating ribosomes prior to the onset of viral RNA synthesis, providing a mechanistic switch for these two competing functions utilizing positive- strand RNA templates. Results from these proposed studies will provide new mechanistic insights into the interplay between translation functions and picornavirus RNA replication. In a new direction for this project, IRES elements have been identified in the long 5'NCRs of two cellular mRNA molecules, one that encodes a voltage-gated potassium channel (Kv1.4) and one that encodes a transcription factor (LEF-1). The experiments proposed in the third aim will examine the nature of RNP complexes formed with the 5'NCRs of these two cellular mRNAs to define the determinants of IRES functions for translation of mRNAs that, unlike picornavirus RNAs, are synthesized in the nucleus of cells and must be transported to the cytoplasm prior to their association with 40S ribosomal subunits. Results from these latter studies will provide important side-by-side comparisons with picornavirus IRES functions and new insights into how eukaryotic cells initiate translation from mRNAs with long, highly-structured 5'NCRs.

Public Health Relevance

This project will elucidate the mechanisms of gene expression and viral RNA replication in cells infected by human picornaviruses. It will also analyze a specialized class of mammalian cell protein synthesis that is important under certain physiological and cell- specific conditions. The proposed research will enhance our knowledge of virus-host interactions as well as normal cell mechanisms of gene expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI026765-23
Application #
8497565
Study Section
Virology - B Study Section (VIRB)
Program Officer
Park, Eun-Chung
Project Start
1988-07-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
23
Fiscal Year
2013
Total Cost
$338,053
Indirect Cost
$107,729
Name
University of California Irvine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Chase, Amanda J; Semler, Bert L (2014) Differential cleavage of IRES trans-acting factors (ITAFs) in cells infected by human rhinovirus. Virology 449:35-44
Tsai, Becky Pinjou; Jimenez, Judith; Lim, Sharon et al. (2014) A novel Bcr-Abl-mTOR-eIF4A axis regulates IRES-mediated translation of LEF-1. Open Biol 4:140180
Fitzgerald, Kerry D; Semler, Bert L (2013) Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein. Virus Res 176:223-31
Huang, Cheng; Lokugamage, Kumari G; Rozovics, Janet M et al. (2011) Alphacoronavirus transmissible gastroenteritis virus nsp1 protein suppresses protein translation in mammalian cells and in cell-free HeLa cell extracts but not in rabbit reticulocyte lysate. J Virol 85:638-43
Fitzgerald, Kerry D; Semler, Bert L (2009) Bridging IRES elements in mRNAs to the eukaryotic translation apparatus. Biochim Biophys Acta 1789:518-28
Sean, Polen; Nguyen, Joseph H C; Semler, Bert L (2009) Altered interactions between stem-loop IV within the 5'noncoding region of coxsackievirus RNA and poly(rC) binding protein 2: effects on IRES-mediated translation and viral infectivity. Virology 389:45-58
Jang, Gwendolyn M; Tanaka, Brian S; Gutman, George A et al. (2008) Alternative polyadenylation signals in the 3'non-coding region of a voltage-gated potassium channel gene are major determinants of mRNA isoform expression. Gene 408:133-45
Sean, Polen; Nguyen, Joseph H C; Semler, Bert L (2008) The linker domain of poly(rC) binding protein 2 is a major determinant in poliovirus cap-independent translation. Virology 378:243-53
Bedard, Kristin M; Daijogo, Sarah; Semler, Bert L (2007) A nucleo-cytoplasmic SR protein functions in viral IRES-mediated translation initiation. EMBO J 26:459-67
Perera, Rushika; Daijogo, Sarah; Walter, Brandon L et al. (2007) Cellular protein modification by poliovirus: the two faces of poly(rC)-binding protein. J Virol 81:8919-32

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