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
Flather, Dylan; Semler, Bert L (2015) Picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus. Front Microbiol 6:594
Lévêque, Nicolas; Semler, Bert L (2015) A 21st century perspective of poliovirus replication. PLoS Pathog 11:e1004825
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
Chase, Amanda J; Daijogo, Sarah; Semler, Bert L (2014) Inhibition of poliovirus-induced cleavage of cellular protein PCBP2 reduces the levels of viral RNA replication. J Virol 88:3192-201
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
Langereis, Martijn A; Feng, Qian; Nelissen, Frank H T et al. (2014) Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA. Nucleic Acids Res 42:2473-82
Cathcart, Andrea L; Semler, Bert L (2014) Differential restriction patterns of mRNA decay factor AUF1 during picornavirus infections. J Gen Virol 95:1488-92
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
Cathcart, Andrea L; Rozovics, Janet M; Semler, Bert L (2013) Cellular mRNA decay protein AUF1 negatively regulates enterovirus and human rhinovirus infections. J Virol 87:10423-34
Fitzgerald, Kerry D; Chase, Amanda J; Cathcart, Andrea L et al. (2013) Viral proteinase requirements for the nucleocytoplasmic relocalization of cellular splicing factor SRp20 during picornavirus infections. J Virol 87:2390-400

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