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. 2006 Mar 28;103(13):5108-13.
doi: 10.1073/pnas.0508200103. Epub 2006 Mar 20.

Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis

Ekaterina Minskaia  1 , Tobias Hertzig, Alexander E Gorbalenya, Valérie Campanacci, Christian Cambillau, Bruno Canard, John Ziebuhr
Affiliations

Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis

Ekaterina Minskaia et al. Proc Natl Acad Sci U S A. .

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 replicase-transcriptase 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'-->5' 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 approximately 8-12 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.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
CoV replicase genes encode a putative ExoN. (A) Functional ORFs in the SARS-CoV genome are expressed from both genomic RNA and a set of eight sg mRNAs (17). ORFs encoding the four major structural proteins, S, E, M, and N, are shown in black. The ORF1a- and ORF1b-encoded proteins, pp1a and pp1ab, are cleaved by viral proteases to yield 16 processing end products called nsp1–16. The N-terminal part of nsp14 was predicted to harbor an ExoN domain (13). (B) Partial sequence alignment of representative CoV ExoN domains. ExoN residues (pp1ab numbering) that were targeted in this study by site-directed mutagenesis are indicated for SARS-CoV (denoted in bold type) and HCoV-229E (given in brackets). Also shown are the conserved exonuclease sequence motifs I–III (18).
Fig. 2.
Fig. 2.
Substrate specificity of the SARS-CoV ExoN. SARS-CoV nsp14-HC was incubated with different 5′-[32P]-labeled substrates, and the reaction products were analyzed by denaturing PAGE and autoradiography. Lanes 1 and 2, 5′-[32P]-labeled RNA markers with sizes indicated to the left; lanes 3 and 4, reactions with ssRNA3; lanes 5 and 6, reactions with a ribose-2′-O-methylated form of ssRNA3 (designated ssRNA9); lanes 7 and 8, reactions with ssDNA97; lanes 9 and 10, reactions with dsDNA97/98; lanes 11 and 12, reactions with ssRNAm1. Reactions were done in the presence (lanes 4, 6, 8, 10, and 12) or absence (lanes 3, 5, 7, 9, and 11) of nsp14-HC (ExoN).
Fig. 3.
Fig. 3.
Ribonucleolytic activities of SARS-CoV ExoN on ssRNA and dsRNA substrates. The various RNA substrates (for sequences, see Table 1) were incubated with nsp14-HC for the indicated periods of time.
Fig. 4.
Fig. 4.
dsRNA affects the exonucleolytic activity of the SARS-CoV ExoN. 5′-[32P]-labeled RNA11 (lanes 2–13) or a ds version of RNA11 (lanes 15–18), which was obtained by annealing 5′-[32P]-labeled RNA11 with the complementary unlabeled RNA12, were incubated with nsp14-HC for the indicated periods of time in the absence (lanes 2–4, 15, and 16) or presence (lanes 5–13, 17, and 18) of the indicated unlabeled RNAs (ssRNA3, dsRNA3/8, and ssRNA4, respectively). Positions of RNA size markers are given to the left.
Fig. 5.
Fig. 5.
Effects of ExoN active-site mutations on the intracellular accumulation of viral RNAs. (A and B) BHK-21-N cells (4 × 106) were mock-transfected or transfected with HCoV-229E full-length RNAs transcribed from purified genomic DNA of vHCoV-1ab_H5859A, vHCoV-1ab_D5834A, vHCoV-1ab_D5864A, vHCoV-1ab_D5682A/E5684A, and vHCoV-inf-1 (WT), respectively. Seventy-two hours after transfection, intracellular poly(A) RNA was isolated and analyzed by Northern hybridization, as described in Materials and Methods. The blot was exposed to x-ray film for 28 (A) or 2 days (B). Transfections were done with the following RNAs: lane 1, HCoV-1ab-D6408A RNA (negative control) (15); lane 2, HCoV-1ab-H5859A RNA; lane 3, HCoV-1ab-D5834A; lane 4, HCoV-1ab-D5864A; lane 5, HCoV-1ab-D5682A/E5684A; lane 6; no RNA (mock transfection); lane 7, HCoV-229E RNA (wild-type sequence). HCoV-229E-specific RNAs 1–7 are indicated by filled arrowheads and the position of a mutant-specific sg RNA migrating slightly faster than HCoV-229E RNA3 is indicated by an open arrowhead. (C) Analysis of ratios of viral RNA species. Quantitation of bands was done in the linear range of exposure by phosphorimaging by using advanced image data analysis software (Raytest, Straubenhardt, Germany). The relative abundance of HCoV-229E (wild-type) RNAs was determined from 10 independent transfections of full-length RNA derived from vHCoV-inf-1. Transfections of RNAs containing ExoN active-site substitutions were performed in triplicate. Because very similar results were obtained for each of the four mutants, the data are presented here as a single set of data. In each experiment, the signal obtained for RNA7 was taken to be 100%, and all other signals were normalized to this value. Mean values and standard deviations are given. Because of low abundance and aberrant size, RNA2 and -3 of the ExoN mutants could not be identified unambiguously and therefore were excluded from this analysis.
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