A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus

Abstract

In (+) RNA coronaviruses, replication and transcription of the giant approximately 30 kb genome to produce genome- and subgenome-size RNAs of both polarities are mediated by a cognate membrane-bound enzymatic complex. Its RNA-dependent RNA polymerase (RdRp) activity appears to be supplied by non-structural protein 12 (nsp12) that includes an RdRp domain conserved in all RNA viruses. Using SARS coronavirus, we now show that coronaviruses uniquely encode a second RdRp residing in nsp8. This protein strongly prefers the internal 5'-(G/U)CC-3' trinucleotides on RNA templates to initiate the synthesis of complementary oligonucleotides of <6 residues in a reaction whose fidelity is relatively low. Distant structural homology between the C-terminal domain of nsp8 and the catalytic palm subdomain of RdRps of RNA viruses suggests a common origin of the two coronavirus RdRps, which however may have evolved different sets of catalytic residues. A parallel between the nsp8 RdRp and cellular DNA-dependent RNA primases is drawn to propose that the nsp8 RdRp produces primers utilized by the primer-dependent nsp12 RdRp.

Figure 1

Sequence alignment of nsp8 proteins. The alignment of coronavirus nsp8 sequences was generated with the ClustalW program, version 1.82 (http://www.ebi.ac.uk/clustalw/). This alignment and individual nsp8 sequences were used to search sequence databases as described in Snijder et al (2003). Using results of these searches, the original alignment was extended to a distantly related (see text) torovirus sequence using the MUSCLE program. The resulting alignment was converted into this figure using the ESPript program, version 2.2 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Residues that are conserved in all or >70% sequences are boxed in red and yellow, respectively. Above the alignment, numbering and secondary structure elements (Zhai et al, 2005) for SARS-CoV nsp8 protein are depicted. National Center for Biotechnology Information (NCBI) Accession Numbers for replicase polyprotein sequences including nsp8: SARS-CoV, NP_828866; Breda torovirus (BToV-1), AY427798; Transmissible Gastroenteritis coronavirus (TGEV), NP_840006; Porcine epidemic diarrhea virus CV777 (PEDV), NP_839962; Human coronavirus NL63 (NL63), AAS58176; Human coronavirus 229E (229E), NP_835349; Human coronavirus HKU1 genotype A (HKU1), AAT98578; Human coronavirus OC43 (OC43), NP_937947; Bovine coronavirus (BCoV), NP_742135; Mouse Hepatitis virus strain A59 (MHV-A59), NP_740613; Avian Infectious Bronchitis Virus (AIBV), NP_740626.

Figure 2

Kinetics of RNA synthesis using nsp8 protein, NS5B polymerase of HCV and NS5 polymerase of Dengue virus and an oligo(rC₁₅) RNA template. 1 μM of nsp8, 1 μM HCV NS5B and 400 nM of Dengue NS5 proteins were mixed with [α-³²P]GTP (10 μM) and oligo(rC₁₅) template (10 μM) to start the reaction (as described under ‘Materials and methods'). At various times (0, 1, 15 and 30 min), reaction aliquots were quenched by the addition of EDTA/formamide, and the RNA products were resolved on 14% polyacrylamide/7 M urea gel. Due to the likely formation of poly(rG) secondary structures, product size and abundance is not visually accurate for >8-mer products. M, RNA marker synthesized using T7 RNA polymerase and an appropriate template; each band product is indicated on the left.

Figure 3

Nsp8 polymerase template requirements. (A) Time course of nucleotide incorporation using a purified 373 nt RNA template and 1 μM nsp8 (left panel) or 1 μM HCV NS5B (right panel) together with 500 μM ATP, UTP, CTP, and 10 μM [α³²P]GTP. Reaction products are indicated on the side of the gel autoradiograph. Positions of the RNA markers (M) synthesized using T7 RNA polymerase are shown on the left. (B) Time course of nucleotide incorporation using RNA template 10 (5′-UAUAGUCCCAAA-3′) together with 1 μM nsp8 and 10 μM [α³²P]GTP. Reactions were performed with all required nucleotides or lacking either ATP, UTP or CTP, as indicated.

Figure 4

Inhibition of RNA synthesis by 3′-dGTP using a poly(rC) template. (A) Effect of increasing concentrations of 3′-dGTP on the initiation and full-length RNA product formation. The nucleoside analog was added to reaction mixtures containing 1 μM poly(rC), 10 μM [α³²-P] GTP and 1 μM nsp8. Lanes 1–5 show the products of the reactions performed using the following concentrations of 3′-dGTP: 0, 100, 500 nM, 1 and 10 μM. Reactions were allowed to proceed for 60 min. M, RNA marker synthesized using T7 RNA polymerase and an appropriate template. (B) Same as in (A) using 10 μM oligo(rC₁₅) as a template in conjunction with 1 μM HCV NS5B. Each chain terminated band product is indicated on the right by an asterisk (^*). For each 3′-dGTP concentration (lanes 1–5: 0, 5, 10, 50 and 100 μM, respectively), the reaction was allowed to proceed for 30, 60 and 120 min.

Figure 5

Alanine scanning mutagenesis of the nsp8 protein. Indicated amino acids were replaced by alanine using site-directed mutagenesis. One micromolar of nsp8 wt and 1 μM of Ala mutants were tested for polymerase activity by measuring [³H]GTP incorporation using a poly(rC) template. Nsp8 polymerase specific activity is represented as follows: empty lozenge (◊); 100% activity relative to nsp8 wt; black lozenge (⧫), between 12 and 40% activity and; asterisk (*), less than 4% activity (see Supplementary Figure 3 for precise values).

Figure 6

Model of two monomers of the nsp8 protein in complex with an ssRNA template (5′-UAGC-3′) and two nucleotides (GTP and CTP). RNA template, GTP and CTP are shown by a stick model. (A) The amino-acid residues related to null mutants, Lys-58 and Arg-75, are represented in yellow. The two first NTPs incorporated (GTP in +1 and CTP in +2) are indicated. Discontinuous purple line represents distance between GTP 3′-OH (incorporated nucleotide in position +1) and α-phosphate of CTP (incorporated nucleotide in +2 position), estimated to 3.8 Å. (B) The surface is colored according to the electrostatic potential nomenclature (blue, positive charge; red, negative charge). Images were generated using PYMOL.