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Nucleic Acids Research, 1988, Vol. 16, No. 21 10339-10352
© 1988

ENZYMOLOGY

Measurement of poliovirus RNA polymerase binding to poliovirion and nonviral RNAs using a filter-binding assay

M. Steven Oberste and James B. Flanegan

Department of Immunology and Medical Microbiology, University of Florida College of Medicine Gainesville, FL 32610, USA

Received May 17, 1988. Revised August 27, 1988. Accepted August 27, 1988.

The binding of the purified poliovirus RNA-dependent RNA polymerase to viral and nonviral RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A) -binding protein was found in viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. Optimal conditions for the binding of purified polymerase (fraction 5-PAS) to 32P-labeled oliovirion RNA were determined. The binding of purified polymerase to 32P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> >>> poly(U)> poly(C) > poly(A). In competitive binding studies, the polymerase bound with equal efficiency to virion RNA and to a subgenomic transcript which contained the 3' end of the genome. The polymerase bound to 18S ribosomal RNA and to globin mRNA equally well, but with a five-fold lower affinity than to virus-specific RNAs. The results suggest that the polymerase exhibits sequence specificity in binding and that polymerase binding sites in poliovirus RNA may contain (G- and/or U)-rich sequences.


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