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Utilization of a mammalian cell-based RNA binding assay to characterize the RNA binding properties of picornavirus 3C proteinases

Published online by Cambridge University Press:  01 February 1998

WADE S. BLAIR ,
TODD B. PARSLEY ,
HAL P. BOGERD ,
JONATHAN S. TOWNER ,
BERT L. SEMLER  and
BRYAN R. CULLEN
WADE S. BLAIR
Affiliation:
Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA Present address: Bristol-Myers Squibb Pharmaceuticals, Department of Virology, 5 Research Parkway, Bldg. 1, Rm. 335D, Wallingford, Connecticut 06492, USA.
TODD B. PARSLEY
Affiliation:
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697, USA
HAL P. BOGERD
Affiliation:
Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA
JONATHAN S. TOWNER
Affiliation:
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697, USA
BERT L. SEMLER
Affiliation:
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697, USA
BRYAN R. CULLEN
Affiliation:
Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA
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Abstract

Using an assay capable of detecting sequence-specific RNA/protein interactions in mammalian cells, we demonstrate that the poliovirus and rhinovirus 3C proteinases are able to bind structured target RNA sequences derived from their respective 5′ noncoding regions in vivo. Specific RNA binding by poliovirus 3C was found to be dependent on the integrity of stem-loop d of the RNA cloverleaf structure located at the 5′ end of poliovirus genomic RNA. In contrast, mutation of stem-loop b did not prevent this in vivo interaction. However, mutation of stem-loop b, which serves as the RNA binding site for a cellular co-factor important for efficient poliovirus replication, did significantly attenuate the efficiency of 3C RNA binding in vivo and 3CD RNA binding in vitro. This in vivo protein:RNA binding assay was also used to identify several residues in 3C that are critical for RNA binding, but dispensable for 3C proteinase activity. The mammalian cell-based RNA binding assay described in this study may have considerable potential utility in the future detection or analysis of in vivo RNA/protein interactions unrelated to the 3C/RNA interaction described here.

Keywords

HIV-1 Tatin vivo RNA/protein binding5′noncoding regionpoliovirusRNA structure
Type
Research Article
Information
RNA , Volume 4 , Issue 2 , February 1998 , pp. 215 - 225
Copyright
© 1998 RNA Society

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