Abstract

The proposed project will utilize picornavirus molecular recombinants and mutants to assay the functional domains of the 5'-non-coding region of poliovirus RNA. We will determine the effects of genetic alteration of the 5'-untranslated region of the polio genome on RNA replication and ribosome binding/viral- specific protein synthesis. We will carry out in vitro manipulation of cloned cDNAs to construct a number of different site-specific recombinant and mutant genomes within the 5'-noncoding regions of polio-virus and coxsackievirus RNAs. The 5'-noncoding cDNA chimeras and mutants will then be ligated to the coding region from an infectious cDNA clone of poliovirus RNA. Plasmids containing the complete recombinant/mutant genomes will be assayed for infectivity by transfection of cultured primate cells. Infectious recombinant and mutant viruses will be recovered and analyzed for altered or temperature-sensitive expression of (i) RNA replication functions,(ii) viral protein synthesis functions, and (iii) host cell shut off functions. Finally, any of the recombinant plasmids that do not produce infectious virus in the transfection assay will be analyzed for in vitro translation efficiency using in vitro transcripts derived from a bacteriophage T7 promoter and RNA polymerase system. Our overall goal in this project is to understand the role of specific blocks of noncoding nucleotides within a viral genome in the regulation of expression of the single translational unit of polio gene products. The health-relatedness of the proposed project is based upon previous studies that implicate the 5'-noncoding region of poliovirus RNA in both the replicative fitness of the virus and the full expression of viral neurovirulence determinants. The information gained from the proposed project should provide a rational scheme for the genetic modification of vaccine strains of picornaviruses by recombinant DNA methodology. Such noncoding region modifications have the potential advantage of reducing vaccine strain cytopathogenicity and instability without perturbing the virion structure that is critical for eliciting a fully- protective immune response.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI026765-03
Application #
3140699
Study Section
Virology Study Section (VR)
Project Start
1988-07-01
Project End
1992-06-30
Budget Start
1990-07-01
Budget End
1992-06-30
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost

  Institution

Name
University of California Irvine
Department
Type
Schools of Medicine
DUNS #
161202122
City
Irvine
State
CA
Country
United States
Zip Code
92697

  Publications

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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