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Journal of Virology, December 2002, p. 12008-12022, Vol. 76, No. 23
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.23.12008-12022.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication

Brandon L. Walter,1 Todd B. Parsley,1,{dagger} Ellie Ehrenfeld,2 and Bert L. Semler1*

Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697,1 Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 208922

Received 6 June 2002/ Accepted 27 August 2002

The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5'-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, CA 92697-4025. Phone: (949) 824-7573. Fax: (949) 824-8598. E-mail: blsemler{at}uci.edu.

{dagger} Present address: University of Maryland Biotechnology Institute, Center for Agricultural Biotechnology, College Park, MD 20742-4450.

Journal of Virology, December 2002, p. 12008-12022, Vol. 76, No. 23
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.23.12008-12022.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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