Picornaviruses as well as other positive strand RNA viruses utilize RNA templates, viral and host proteins, and the architecture provided by cytoplasmic membranes of the infected cell to orchestrate a complex series of macromolecular events that lead to the production of progeny viral RNAs. Poliovirus, coxsackievirus, and human rhinovirus are members of the Picornaviridae that share a common RNA replication strategy during their intracellular replication cycles. This proposal outlines new experimental approaches to dissect the mechanisms that underlie this complicated process. These approaches will focus on the assembly and dynamics/maturation of viral RNA replication complexes during the course of infection. The proposed experiments build on the knowledge that viral protein 3CD is an integral component of picornavirus RNA replication complexes as are host cell proteins hnRNP C and PCBP. Dual tagged forms of poliovirus 3CD will be used to isolate viral RNA replication complexes at different stages of infection and under altered conditions of proteolytic processing. Complexes will be analyzed for viral and host proteins known to play roles in picornavirus RNA replication. In addition, an unbiased, high-resolution proteomics approach will be employed by performing mass spectrometry on such isolated complexes. The overall approach will be comprehensive in its analyses because the functional significance of newly-identified protein players will be tested both by infections of human cell lines and by cell-free RNA replication reactions that can lead to the synthesis of infectious poliovirus or human rhinovirus.
The first aim of this proposal is to determine how binding of hnRNP C to both termini of poliovirus negative-strand RNA intermediates facilitates template recognition and assembly of RNA replication complexes.
The second aim of the proposal will define the complete proteome of specific poliovirus RNA replication complexes using dual-affinity tagged polypeptide 3CD and mass spectrometry. The experiments proposed in this aim will also determine the RNA replication complex proteome for human rhinovirus, which should reveal unifying principles for which viral and host proteins comprise the core picornavirus RNA replication complex and those that are unique for human rhinovirus or poliovirus. In addition to providing new mechanistic insights for how cytoplasmic RNA viruses replicate their genomic RNAs, the proposed studies should reveal novel protein-protein and protein-RNA interfaces that will serve as important targets for the development of anti-viral therapeutic approaches.

Public Health Relevance

This project will elucidate the mechanisms of viral RNA replication complex assembly and dynamics in cells infected by human picornaviruses, including poliovirus and human rhinovirus. It will also analyze the role of host proteins in picornavirus RNA synthesis. The proposed research will enhance our knowledge of virus-host interactions and identify potential new targets for anti-viral therapeutics.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AI022693-27
Application #
8648946
Study Section
Virology - A Study Section (VIRA)
Program Officer
Park, Eun-Chung
Project Start
Project End
Budget Start
Budget End
Support Year
27
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Irvine
State
CA
Country
United States
Zip Code
92697
Flather, Dylan; Cathcart, Andrea L; Cruz, Casey et al. (2016) Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5' and 3' Noncoding Regions of Genomic RNAs. Viruses 8:
Wehbe, Michel; Huguenin, Antoine; Leveque, Nicolas et al. (2016) Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells. J Virol Methods 230:1-8
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
Ertel, Kenneth J; Brunner, Jo Ellen; Semler, Bert L (2010) Mechanistic consequences of hnRNP C binding to both RNA termini of poliovirus negative-strand RNA intermediates. J Virol 84:4229-42
Brunner, Jo Ellen; Ertel, Kenneth J; Rozovics, Janet M et al. (2010) Delayed kinetics of poliovirus RNA synthesis in a human cell line with reduced levels of hnRNP C proteins. Virology 400:240-7
Sean, P; Semler, B L (2008) Coxsackievirus B RNA replication: lessons from poliovirus. Curr Top Microbiol Immunol 323:89-121
Kuznetsov, Yurii G; Daijogo, Sarah; Zhou, Jiashu et al. (2005) Atomic force microscopy analysis of icosahedral virus RNA. J Mol Biol 347:41-52
Brunner, Jo Ellen; Nguyen, Joseph H C; Roehl, Holger H et al. (2005) Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes. J Virol 79:3254-66
Brown, David M; Cornell, Christopher T; Tran, Genevieve P et al. (2005) An authentic 3' noncoding region is necessary for efficient poliovirus replication. J Virol 79:11962-73

Showing the most recent 10 out of 59 publications