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J. Biol. Chem., Vol. 277, Issue 35, 31551-31562, August 30, 2002

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Structure-Function Relationships of the RNA-dependent RNA Polymerase from Poliovirus (3Dpol)
A SURFACE OF THE PRIMARY OLIGOMERIZATION DOMAIN FUNCTIONS IN CAPSID PRECURSOR PROCESSING AND VPg URIDYLYLATION^*

Harsh B. Pathak^§, Saikat Kumar B. Ghosh^§, Allan W. Roberts^¶, Suresh D. Sharma, Joshua D. Yoder, Jamie J. Arnold, David W. Gohara, David J. Barton^¶, Aniko V. Paul^**, and Craig E. Cameron

From the  Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, the ^¶ Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262, and the ^** Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York 11794

The primary oligomerization domain of poliovirus polymerase, 3Dpol, is stabilized by the interaction of the back of the thumb subdomain of one molecule with the back of the palm subdomain of a second molecule, thus permitting the head-to-tail assembly of 3Dpol monomers into long fibers. The interaction of Arg-455 and Arg-456 of the thumb with Asp-339, Ser-341, and Asp-349 of the palm is key to the stability of this interface. We show that mutations predicted to completely disrupt this interface do not produce equivalent growth phenotypes. Virus encoding a polymerase with changes of both residues of the thumb to alanine is not viable; however, virus encoding a polymerase with changes of all three residues of the palm to alanine is viable. Biochemical analysis of 3Dpol derivatives containing the thumb or palm substitutions revealed that these derivatives are both incapable of forming long fibers, suggesting that polymerase fibers are not essential for virus viability. The RNA binding activity, polymerase activity, and thermal stability of these derivatives were equivalent to that of the wild-type enzyme. The two significant differences observed for the thumb mutant were a modest reduction in the ability of the altered 3CD proteinase to process the VP0/VP3 capsid precursor and a substantial reduction in the ability of the altered 3Dpol to catalyze oriI-templated uridylylation of VPg. The defect to uridylylation was a result of the inability of 3CD to stimulate this reaction. Because 3C alone can substitute for 3CD in this reaction, we conclude that the lethal replication phenotype associated with the thumb mutant is caused, in part, by the disruption of an interaction between the back of the thumb of 3Dpol and some undefined domain of 3C. We speculate that this interaction may also be critical for assembly of other complexes required for poliovirus genome replication.

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