Poliovirus 2C Protein Forms Homo-oligomeric Structures Required for ATPase Activity

Peter Adams,Eaazhisai Kandiah,Grégory Effantin,Alasdair C Steven,Ellie Ehrenfeld

doi:10.1074/jbc.m109.031807

Abstract

The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily. A feature common to AAA+ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5-8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect oligomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked oligomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain.

Highlights

There was some variability in the relative amounts of different sized aggregates of MBP-2C among different preparations of this protein, perhaps indicating a greater sensitivity to misfolding and/or proteolysis
Similar to what was observed with the full-length MBP-2C, only a specific oligomeric form of ⌬19 2C displayed any ATPase activity, and the specific activity was reduced
There was some evidence for oligomerization of MBP-2C proteins with C-terminal deletions, the ATPase activity was very low

Summary

Introduction

There was some evidence for oligomerization of MBP-2C proteins with C-terminal deletions, the ATPase activity was very low (data not shown). Both profiles were similar to that of wild-type polio MBP-2C (Fig. 1B), immunoblots of fractions eluting from the gel-filtration column detected increased levels of 2C-containing material in the eluate corresponding to monomeric or dimeric MBP fusions with the chimeric proteins (data not shown), which did not occur in the wild-type 2C preparations (Fig. 1B).

Results

Conclusion