The proposed project will extend our molecular genetic analysis of the 5' noncoding regions (5'NCRs) of picornavirus RNAs to include biochemical assays as functional tests for RNA-protein interactions. We will utilize point mutations, deletions, and linker scanning mutations within predicted stem-loop structures in the poliovirus 5' NCR to address the role of sequence versus structure in the formation of functional complexes required for cap-independent translation. Our functional analysis for viable viruses containing 5' NCR lesions will include assays for viral-specific protein and RNA synthesis, complementation with genetically-marked viruses, determination of growth characteristics in human cells of neuronal origin, and RNA sequence analysis of pseudo-revertants. The need by picornavirus RNAs for specific complexes of cellular proteins distinct from those employed by cap-dependent translation mechanisms will be explored by binding and translation assays in the presence of fractionated cell extracts enriched in known and perhaps novel translation components. We propose a series of experiments employing U.V. crosslinking, primer extension footprinting, and chemical modification to define the specific nucleotide sequences in the 5'NCR of poliovirus RNA that are involved in the formation RNA-protein complexes. In addition, we will identify and isolate cellular proteins that bind to specific stem-loop structures in the poliovirus 5' NCR. The overall goal of the proposed studies is to determine the viral genetic and cellular polypeptide determinants in RNA-proteins interactions within the 5' NCR of genomic RNAs, information that ultimately will help to unravel the molecular mechanisms of cap-independent translation initiation utilized by picornaviruses. The health relatedness of this project stems from uncovering genetic and biochemical mechanisms employed by viral pathogens to subvert the cellular translation apparatus for dedicated synthesis of their own viral gene products. On a more general level, our studies will contribute to a basic understanding of how RNA-protein interactions are involved in translational regulation in eukaryotic cells.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Experimental Virology Study Section (EVR)
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University of California Irvine
Schools of Arts and Sciences
United States
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Ullmer, Wendy; Semler, Bert L (2018) Direct and Indirect Effects on Viral Translation and RNA Replication Are Required for AUF1 Restriction of Enterovirus Infections in Human Cells. MBio 9:
Lévêque, Nicolas; Garcia, Magali; Bouin, Alexis et al. (2017) Functional Consequences of RNA 5'-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation. J Virol 91:
Ullmer, Wendy; Semler, Bert L (2016) Diverse Strategies Used by Picornaviruses to Escape Host RNA Decay Pathways. Viruses 8:
Lévêque, Nicolas; Semler, Bert L (2015) A 21st century perspective of poliovirus replication. PLoS Pathog 11:e1004825
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
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
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
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
Cathcart, Andrea L; Semler, Bert L (2014) Differential restriction patterns of mRNA decay factor AUF1 during picornavirus infections. J Gen Virol 95:1488-92
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

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