Abstract
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.
Highlights
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, permitting the head-to-tail assembly of 3Dpol monomers into long fibers
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 be critical for assembly of other complexes required for poliovirus genome replication
The RNA-dependent RNA polymerase (RdRP)1 is the key component of the replication machinery of RNA viruses
Summary
RdRP, RNA-dependent RNA polymerase; DMEM, Dulbecco’s modified Eagle’s medium; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; oligo, oligonucleotide; sym/sub, symmetrical substrate; PEI, polyethyleneimine; PBS, phosphate-buffered saline; nt, nucleotide(s); GdnHCl, guanidine HCl. Given the role of these residues in the stability of interface I (Fig. 1C) [1], these data provided the first evidence that oligomerization of 3Dpol might play a significant role in some step of the virus multiplication cycle. Mutation of the residue that interacts with Arg-455, Asp-349, does not produce a lethal phenotype [4]. We test the hypothesis that the lethal phenotype associated with mutations on the back of the thumb of 3Dpol arises from a requirement for this subdomain that is independent of oligomerization. We find that 1) oligomerization via interface I may not be essential for virus multiplication and 2) the back of the thumb of 3Dpol may interact with host and viral factors to modulate capsid protein processing and initiation of protein-primed RNA synthesis, respectively. The implications of heteromeric interactions between 3Dpol and another viral factor on the mechanism for negative-strand RNA synthesis will be discussed
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