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PMID:16549795

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Citation

Minskaia, E, Hertzig, T, Gorbalenya, AE, Campanacci, V, Cambillau, C, Canard, B and Ziebuhr, J (2006) Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 103:5108-13

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.

Links

PubMed PMC1458802 Online version:10.1073/pnas.0508200103

Keywords

Amino Acid Sequence; Cations, Divalent/chemistry; Cell Nucleus/enzymology; Cell Nucleus/genetics; Conserved Sequence; Coronavirus/enzymology; Coronavirus/genetics; Coronavirus/physiology; Exoribonucleases/chemistry; Exoribonucleases/genetics; Exoribonucleases/metabolism; Metals/chemistry; Metals/pharmacology; Molecular Sequence Data; RNA, Double-Stranded/metabolism; RNA, Viral/biosynthesis; RNA, Viral/genetics; Sequence Alignment; Substrate Specificity; Transcription, Genetic/genetics; Virus Replication

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

CVHSA:R1AB

GO:0000175: 3'-5'-exoribonuclease activity

ECO:0000314:

F

Fig. 3 and Fig. 4

complete
CACAO 2448

CVHSA:R1AB

GO:0019083: viral transcription

ECO:0000315:

P

Fig. 5

complete
CACAO 2449

CVHSA:R1AB

enables

GO:0000175: 3'-5'-exoribonuclease activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

CVHSA:R1AB

involved_in

GO:0019083: viral transcription

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

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