The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension

Uniquely among RNA viruses, replication of the ∼30-kb SARS-coronavirus genome is believed to involve two RNA-dependent RNA polymerase (RdRp) activities. The first is primer-dependent and associated with the 106-kDa non-structural protein 12 (nsp12), whereas the second is catalysed by the 22-kDa nsp8. This latter enzyme is capable of de novo initiation and has been proposed to operate as a primase. Interestingly, this protein has only been crystallized together with the 10-kDa nsp7, forming a hexadecameric, dsRNA-encircling ring structure [i.e. nsp(7+8), consisting of 8 copies of both nsps]. To better understand the implications of these structural characteristics for nsp8-driven RNA synthesis, we studied the prerequisites for the formation of the nsp(7+8) complex and its polymerase activity. We found that in particular the exposure of nsp8's natural N-terminal residue was paramount for both the protein's ability to associate with nsp7 and for boosting its RdRp activity. Moreover, this ‘improved’ recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far. The latter activity is, however, ∼20-fold weaker than that of the primer-dependent nsp12-RdRp at equal monomer concentrations. Finally, site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity.

[1]  Zihe Rao,et al.  Insights into SARS-CoV transcription and replication from the structure of the nsp7–nsp8 hexadecamer , 2005, Nature Structural &Molecular Biology.

[2]  Detlef D. Leipe,et al.  Origin and evolution of the archaeo-eukaryotic primase superfamily and related palm-domain proteins: structural insights and new members , 2005, Nucleic acids research.

[3]  Y. Guan,et al.  Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage , 2003, Journal of Molecular Biology.

[4]  J. Arnold,et al.  Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases , 2007, Proceedings of the National Academy of Sciences.

[5]  S. Goebel,et al.  Genetic Interactions between an Essential 3′ cis-Acting RNA Pseudoknot, Replicase Gene Products, and the Extreme 3′ End of the Mouse Coronavirus Genome , 2007, Journal of Virology.

[6]  R. Baric,et al.  Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture , 2010, PLoS pathogens.

[7]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[8]  Paul Ahlquist,et al.  Host Factors in Positive-Strand RNA Virus Genome Replication , 2003, Journal of Virology.

[9]  J. Arnold,et al.  The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent , 2009, Nucleic acids research.

[10]  C. Morrow,et al.  Mutation of the aspartic acid residues of the GDD sequence motif of poliovirus RNA-dependent RNA polymerase results in enzymes with altered metal ion requirements for activity , 1995, Journal of virology.

[11]  Wentao Fu,et al.  A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication , 2008, Proceedings of the National Academy of Sciences.

[12]  Bruno Canard,et al.  In Vitro Reconstitution of SARS-Coronavirus mRNA Cap Methylation , 2010, PLoS pathogens.

[13]  T. Kunkel,et al.  Fidelity of DNA synthesis catalyzed by human DNA polymerase alpha and HIV-1 reverse transcriptase: effect of reaction pH. , 1993, Nucleic acids research.

[14]  J. Arnold,et al.  The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen , 2000, Nature Medicine.

[15]  C. Cameron,et al.  Mechanisms of action of ribavirin against distinct viruses , 2005, Reviews in medical virology.

[16]  E. Snijder,et al.  Nidovirus transcription: how to make sense...? , 2006, The Journal of general virology.

[17]  P. Lécine,et al.  The SARS-Coronavirus PLnc domain of nsp3 as a replication/transcription scaffolding protein , 2008, Virus Research.

[18]  Ralph S. Baric,et al.  Processing of Open Reading Frame 1a Replicase Proteins nsp7 to nsp10 in Murine Hepatitis Virus Strain A59 Replication , 2007, Journal of Virology.

[19]  S. Perlman,et al.  Coronaviruses post-SARS: update on replication and pathogenesis , 2009, Nature Reviews Microbiology.

[20]  Xiaolu Lu,et al.  Genome-Wide Analysis of Protein-Protein Interactions and Involvement of Viral Proteins in SARS-CoV Replication , 2008, PloS one.

[21]  Shannon L. Taylor,et al.  Ovarian Tumor Domain-Containing Viral Proteases Evade Ubiquitin- and ISG15-Dependent Innate Immune Responses , 2007, Cell Host & Microbe.

[22]  Thomas J. Wisniewski,et al.  Production of "authentic" poliovirus RNA-dependent RNA polymerase (3D(pol)) by ubiquitin-protease-mediated cleavage in Escherichia coli. , 1999, Protein expression and purification.

[23]  Jindrich Cinatl,et al.  Ribavirin and interferon-β synergistically inhibit SARS-associated coronavirus replication in animal and human cell lines , 2004, Biochemical and Biophysical Research Communications.

[24]  V. Blinov,et al.  Coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis. , 1989, Nucleic acids research.

[25]  B. Coutard,et al.  A second, non-canonical RNA-dependent RNA polymerase in SARS Coronavirus , 2006, The EMBO journal.

[26]  J. Ziebuhr,et al.  Virus-encoded proteinases and proteolytic processing in the Nidovirales. , 2000, The Journal of general virology.

[27]  J. Arnold,et al.  Poliovirus RNA-dependent RNA Polymerase (3Dpol) Is Sufficient for Template Switchingin Vitro * , 1999, The Journal of Biological Chemistry.

[28]  T. Mizutani,et al.  Inhibitory effect of mizoribine and ribavirin on the replication of severe acute respiratory syndrome (SARS)-associated coronavirus , 2005, Antiviral Research.

[29]  Eric J. Snijder,et al.  Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase , 2004, Journal of Virology.

[30]  R. Baric,et al.  SARS-Coronavirus Replication/Transcription Complexes Are Membrane-Protected and Need a Host Factor for Activity In Vitro , 2008, PLoS pathogens.

[31]  A. Thompson,et al.  Structural basis for proteolysis‐dependent activation of the poliovirus RNA‐dependent RNA polymerase , 2004, The EMBO journal.

[32]  J. Ziebuhr.  The Coronavirus Replicase: Insights into a Sophisticated Enzyme Machinery , 2006, Advances in experimental medicine and biology.

[33]  M. Chan-yeung,et al.  SARS: epidemiology , 2003, Respirology.

[34]  N. Stiefl,et al.  A new lead for nonpeptidic active-site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease discovered by a combination of screening and docking methods. , 2005, Journal of medicinal chemistry.

[35]  J. Arnold,et al.  Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase , 2007, Journal of Virology.

[36]  Yunqing Liu,et al.  Molecular model of SARS coronavirus polymerase: implications for biochemical functions and drug design , 2003, Nucleic acids research.

[37]  W. Miller,et al.  Synthesis of subgenomic RNAs by positive-strand RNA viruses. , 2000, Virology.