Complete Title of Thesis:
"A Phylogenetically Conserved RNA Structure within the Poliovirus 3C ORF Competitively Inhibits the Antiviral Ribonuclease L"
Prepared under the direction of: David J. Barton, Ph.D.
Ribonuclease L (RNase L) is part of an interferon-regulated and dsRNA-activated antiviral pathway. RNase L can inhibit virus replication via several mechanisms including: direct cleavage of viral RNAs, triggering apoptosis of virus infected cells, and potentiating Type I interferon expression. This pathway has been shown to have antiviral activity against DNA and RNA viruses, including picornaviruses. Viruses, as expected, have evolved to antagonize this pathway.
This thesis describes a previously undiscovered 143 nucleotide RNA structure located in the 3C ORF of poliovirus (PV) RNA that inhibits RNase L. This RNA structure is phylogenetically conserved among group C enteroviruses including PV1, PV2, PV3, and Coxsackie A viruses 11, 13, 15, 17, 18, 20, 21, and 24. It was the scope of this thesis to:
1) identify key structural features of the viral RNA,I used purified RNase L, purified 2-5A, an RNA substrate with a 5' fluorophore and 3' quencher and the PV RNA inhibitor of RNase L in FRET assays to characterize enzyme kinetics. I determined that the viral RNA was a competitive inhibitor of RNase L (Ki of 34 nM for PV RNA versus Km of 860 nM for FRET probe RNA substrate). Single point mutations at key wobble position residues within the conserved RNA abrogated the ability to inhibit RNase L. PV replication was not inhibited by RNase L in cells, however, rRNA cleavage characteristic of RNase L activity was detected late during the course of PV infection, after assembly of intracellular virus. Mutations in the RNA structure associated with the inhibition of RNase L did not inhibit the magnitude of PV replication, consistent with the absence of RNase L activity until after virus assembly. Rather than inhibiting PV replication, RNase L activity was associated with larger plaques. I conclude that group C enteroviruses have evolved mechanisms to evade RNase L antiviral activity, and perhaps even co-opt RNase L activity to facilitate CPE associated with virus release. This is the first example of a viral RNA inhibiting an endoribonuclease and the first time a natural inhibitor of RNase L has been described.
2) determine the mechanism by which the viral RNA inhibits RNase L, and
3) examine the biological significance of the viral RNA and RNase L during picornavirus infection of cells