A putative loop E motif and an H-H kissing loop interaction are conserved and functional features in a group C enterovirus RNA that inhibits ribonuclease L.

Abstract

A phylogenetically conserved RNA structure within the open reading frame of poliovirus and other group C enteroviruses functions as a competitive inhibitor of the antiviral endoribonuclease RNase L. Hence, we call this viral RNA the RNase L competitive inhibitor RNA (RNase L ciRNA). In this investigation we used phylogenetic information, RNA structure prediction software, site-directed mutagenesis, and RNase L activity assays to identify functionally important sequences and structures of the RNase L ciRNA. A putative loop E motif is phylogenetically conserved in the RNA structure and mutations of nucleotides within the putative loop E motif destroyed the ability of the RNA molecule to inhibit RNase L. A putative H-H kissing loop interaction is phylogenetically conserved in the RNA structure and covariant polymorphisms that maintain the Watson-Crick complementarity required for the kissing interaction provide evidence of its importance. Compensatory mutations that disrupted and then restored the putative kissing interaction confirm that it contributes to the ability of the viral RNA to inhibit RNase L. RNase L was activated late during the course of poliovirus replication in HeLa cells, as virus replication and assembly neared completion. We conclude that a putative loop E motif and an H-H kissing loop interaction are key features of the group C enterovirus RNA associated with the inhibition of RNase L.