Biochemical Characterization of a Recombinant SARS Coronavirus Nsp12 RNA-dependent RNA Polymerase Capable of Copying Viral RNA Templates

Overview

Journal Arch Virol

Specialty Microbiology

Date 2012 Jul 14

PMID 22791111

Citations 93

Authors

Dae-Gyun Ahn

Jin-Kyu Choi

Deborah R Taylor

Jong-Won Oh

Affiliations

Soon will be listed here.

Abstract

The severe acute respiratory syndrome coronavirus (SARS-CoV) RNA genome is replicated by a virus-encoded RNA replicase, the key component of which is the nonstructural protein 12 (nsp12). In this report, we describe the biochemical properties of a full-length recombinant SARS-CoV nsp12 RNA-dependent RNA polymerase (RdRp) capable of copying viral RNA templates. The purified SARS-CoV nsp12 showed both primer-dependent and primer-independent RNA synthesis activities using homopolymeric RNA templates. The RdRp activity was strictly dependent on Mn(2+). The nsp12 preferentially copied homopolymeric pyrimidine RNA templates in the absence of an added oligonucleotide primer. It was also able to initiate de novo RNA synthesis from the 3'-ends of both the plus- and minus-strand genome of SARS-CoV, using the 3'-terminal 36- and 37-nt RNA, respectively. The in vitro RdRp assay system established with a full-length nsp12 will be useful for understanding the mechanisms of coronavirus replication and for the development of anti-SARS-CoV agents.

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References

Hong Z, Cameron C, Walker M, Castro C, Yao N, Lau J . A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B polymerase. Virology. 2001; 285(1):6-11. DOI: 10.1006/viro.2001.0948. View

Tsai C, Cheng C, Peng C, Lin B, Lin N, Hsu Y . Sufficient length of a poly(A) tail for the formation of a potential pseudoknot is required for efficient replication of bamboo mosaic potexvirus RNA. J Virol. 1999; 73(4):2703-9. PMC: 104026. DOI: 10.1128/JVI.73.4.2703-2709.1999. View

Spagnolo J, Hogue B . Requirement of the poly(A) tail in coronavirus genome replication. Adv Exp Med Biol. 2002; 494:467-74. DOI: 10.1007/978-1-4615-1325-4_68. View

Luo G, Hamatake R, Mathis D, Racela J, Rigat K, LEMM J . De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus. J Virol. 2000; 74(2):851-63. PMC: 111606. DOI: 10.1128/jvi.74.2.851-863.2000. View

Knoops K, Kikkert M, van den Worm S, Zevenhoven-Dobbe J, van der Meer Y, Koster A . SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol. 2008; 6(9):e226. PMC: 2535663. DOI: 10.1371/journal.pbio.0060226. View

van Dijk A, Makeyev E, Bamford D . Initiation of viral RNA-dependent RNA polymerization. J Gen Virol. 2004; 85(Pt 5):1077-1093. DOI: 10.1099/vir.0.19731-0. View

Imbert I, Guillemot J, Bourhis J, Bussetta C, Coutard B, Egloff M . A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus. EMBO J. 2006; 25(20):4933-42. PMC: 1618104. DOI: 10.1038/sj.emboj.7601368. View

Ahn D, Lee W, Choi J, Kim S, Plant E, Almazan F . Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication. Antiviral Res. 2011; 91(1):1-10. PMC: 4728714. DOI: 10.1016/j.antiviral.2011.04.009. View

Shevchenko A, Wilm M, Vorm O, Mann M . Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996; 68(5):850-8. DOI: 10.1021/ac950914h. View

10.

Paul A, Rieder E, Kim D, VAN Boom J, Wimmer E . Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg. J Virol. 2000; 74(22):10359-70. PMC: 110910. DOI: 10.1128/jvi.74.22.10359-10370.2000. View

11.

Cazenave C, Uhlenbeck O . RNA template-directed RNA synthesis by T7 RNA polymerase. Proc Natl Acad Sci U S A. 1994; 91(15):6972-6. PMC: 44320. DOI: 10.1073/pnas.91.15.6972. View

12.

Marra M, Jones S, Astell C, Holt R, Brooks-Wilson A, Butterfield Y . The Genome sequence of the SARS-associated coronavirus. Science. 2003; 300(5624):1399-404. DOI: 10.1126/science.1085953. View

13.

Te Velthuis A, Arnold J, Cameron C, van den Worm S, Snijder E . The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent. Nucleic Acids Res. 2009; 38(1):203-14. PMC: 2800238. DOI: 10.1093/nar/gkp904. View

14.

Larsen G, Semler B, Wimmer E . Stable hairpin structure within the 5'-terminal 85 nucleotides of poliovirus RNA. J Virol. 1981; 37(1):328-35. PMC: 171010. DOI: 10.1128/JVI.37.1.328-335.1981. View

15.

Beerens N, Selisko B, Ricagno S, Imbert I, van der Zanden L, Snijder E . De novo initiation of RNA synthesis by the arterivirus RNA-dependent RNA polymerase. J Virol. 2007; 81(16):8384-95. PMC: 1951334. DOI: 10.1128/JVI.00564-07. View

16.

Jacobson A, Zuker M . Structural analysis by energy dot plot of a large mRNA. J Mol Biol. 1993; 233(2):261-9. DOI: 10.1006/jmbi.1993.1504. View

17.

Snijder E, Bredenbeek P, Dobbe J, Thiel V, Ziebuhr J, Poon L . Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol. 2003; 331(5):991-1004. PMC: 7159028. DOI: 10.1016/s0022-2836(03)00865-9. View

18.

Selisko B, Dutartre H, Guillemot J, Debarnot C, Benarroch D, Khromykh A . Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases. Virology. 2006; 351(1):145-58. DOI: 10.1016/j.virol.2006.03.026. View

19.

Svitkin Y, Costa-Mattioli M, Herdy B, Perreault S, Sonenberg N . Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP). RNA. 2007; 13(12):2330-40. PMC: 2080607. DOI: 10.1261/rna.606407. View

20.

Reigadas S, Ventura M, Castroviejo M, Litvak S, Astier-Gin T . HCV RNA-dependent RNA polymerase replicates in vitro the 3' terminal region of the minus-strand viral RNA more efficiently than the 3' terminal region of the plus RNA. Eur J Biochem. 2001; 268(22):5857-67. DOI: 10.1046/j.0014-2956.2001.02532.x. View