Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicasetranscriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3 35 direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2 -O-methylated RNA substrates and required divalent metal ions for activity. A range of 5 -labeled ssRNA substrates were processed to final products of Ϸ8 -12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5 -labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.replication ͉ ribonuclease ͉ severe acute respiratory syndrome
The three-dimensional structure of the lipase-procolipase complex, co-crystallized with mixed micelles of phosphatidylcholine and bile salt, has been determined at 3 A resolution by X-ray crystallography. The lid, a surface helix covering the catalytic triad of lipase, adopts a totally different conformation which allows phospholipid to bind to the enzyme's active site. The open lid is an essential component of the active site and interacts with procolipase. Together they form the lipid-water interface binding site. This reorganization of the lid structure provokes a second drastic conformational change in an active site loop, which in its turn creates the oxyanion hole (induced fit).
Macro domains constitute a protein module family found associated with specific histones and proteins involved in chromatin metabolism. In addition, a small number of animal RNA viruses, such as corona-and toroviruses, alphaviruses, and hepatitis E virus, encode macro domains for which, however, structural and functional information is extremely limited. Here, we characterized the macro domains from hepatitis E virus, Semliki Forest virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). The crystal structure of the SARS-CoV macro domain was determined at 1.8-Å resolution in complex with ADP-ribose. Information derived from structural, mutational, and sequence analyses suggests a close phylogenetic and, most probably, functional relationship between viral and cellular macro domain homologs. The data revealed that viral macro domains have relatively poor ADP-ribose 1؆-phosphohydrolase activities (which were previously proposed to be their biologically relevant function) but bind efficiently free and poly(ADP-ribose) polymerase 1-bound poly-(ADP-ribose) in vitro. Collectively, these results suggest to further evaluate the role of viral macro domains in host response to viral infection.
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