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

Abstract

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

Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis

“…In addition, a 10-fold reduction in the amount of genomic positivestrand RNA was detected for all mutant replicons (data not shown), suggesting that ExoN, NendoU, and 2Ј-O-MT are essential for efficient CoV replication and transcription. Although it was previously reported that NendoU and ExoN were essential for CoV RNA synthesis (14,19), this is the first evidence that 2Ј-O-MT activity is also essential. Further genetic and biochemical studies will be required to address the specific role of these enzymes in CoV transcription and replication.…”

“…Formally, it is not possible to exclude the possibility of a lethal effect of these deletions at the level of RNA structure or pp1ab processing, even though their N-and C-terminal borders were relatively distant from the proteolytic cleavage sites. To overcome this potential limitation, three point mutants, in which putative catalytic and highly conserved residues of ExoN (Asp-6145), NendoU (His-6678), and 2Ј-O-MT (Asp-6905) (14,19,25) were replaced by Ala (D6145A, H6678A, and D6905A, respectively), were generated. In addition, since the Ser-6744 residue has been described to be nonessential for NendoU activity (14), an additional mutant containing an Ala instead of a Ser at this position (S6744A) was generated as a control to confirm that single amino acid substitutions could be tolerated in this domain of the polyprotein (Fig.…”

How RNA viruses maintain their genome integrity

“…The nsp14 protein of these viruses possesses sequence motifs that are analogous to the active-site motifs of cellular exonuclease enzymes, and the nsp14 protein of severe acute respiratory syndrome (SARS)-related coronavirus has been shown to have 39A59 exonuclease activity in vitro (Chen et al, 2007;Minskaia et al, 2006). Evidence that this protein functions as a proofreading enzyme comes from the fact that mutant viruses that have a defective nsp14 protein are more error-prone than wild-type virus by a factor of at least 15-fold (Eckerle et al, 2007).…”

Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mRNA degradation

“…Probably most of the gene 1 proteins are important for viral RNA synthesis (5,(7)(8)(9)(10)(11). Some gene 1 proteins are predicted to have biological functions related to RNA synthesis (11), and some have demonstrated functions that appear to be involved in RNA synthesis (7,8,10,12), whereas some gene 1 proteins may have functions other than viral RNA synthesis (13)(14)(15)(16).…”