Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis

• DOI: 10.1073/pnas.0508200103
• Publication Date: 2006-03-21T01:44:26Z
• Authors: Ekaterina Minskaia; Tobias Hertzig; Alexander E. Gorb...; Valérie Campanacci; Christian Cambillau; Bruno Canard; John Ziebuhr
• Keywords: Exoribonuclease; Coronavirus; RNA Silencing; Subgenomic mRNA; Five-prime cap; Endoribonuclease

Replication of the giant RNA genome severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis as many eight subgenomic (sg) mRNAs are mediated by a viral replicase-transcriptase outstanding complexity that includes an essential endoribonuclease activity. Here, we show CoV replicative machinery, unlike other viruses, also uses exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms SARS-CoV nsp14 were shown to act on both ssRNAs dsRNAs in 3′→5′ direction. The activity depended residues conserved DEDD exonuclease superfamily. did not hydrolyze DNA or ribose-2′- O -methylated substrates required divalent metal ions for A range 5′-labeled ssRNA processed final products ≈8–12 nucleotides. When part dsRNA presence nonlabeled dsRNA, significantly smaller products, indicating binding cis trans modulates exonucleolytic nsp14. Characterization human 229E ExoN active-site mutants revealed defects synthesis, no viable virus could be recovered. Besides strongly reduced replication, specific sg such aberrant sizes RNAs changes molar ratios between individual species, observed. Taken together, study identifies involved multiple from exceptionally large genomic templates CoVs.