FIG.1.

Expression and primary structure of the SARS-CoV nsp13 helicase. (A) Overview of the domain organization and (predicted) proteolytic processing of the SARS-CoV replicase polyproteins pp1a and pp1ab. Nsp13 is encoded by ORF 1b and is processed from pp1ab by the 3C-like proteinase. The processing end products of pp1a are designated nsp1 to nsp11, and those of pp1ab are designated nsp1 to nsp10 and nsp12 to nsp16. Note that nsp1 to nsp10 may be released by proteolytic processing of either pp1a or pp1ab, whereas nsp11 is processed from pp1a and nsp12 to nsp16 are processed from pp1ab. Nsp11 and nsp12 have a number of common residues at their N termini. Cleavage sites that are (predicted to be) processed by the viral main proteinase are indicated by grey arrowheads, and sites that are processed by the papain-like proteinase 2 are indicated by black arrowheads. Ac, acidic domain (92); X, X domain (21), which is predicted to have ADP-ribose 1"-phosphatase activity (65); SUD, SARS-CoV unique domain (65); PL2, papain-like cysteine proteinase 2 (72); Y, Y domain containing a transmembrane domain and a putative metal-binding domain (65, 72, 92); TM1, TM2, and TM3, putative transmembrane domains 1 to 3, respectively; 3CL, 3C-like main proteinase (3, 72); RdRp, putative RNA-dependent RNA polymerase domain (19, 29, 43, 52); HEL, superfamily 1 helicase domain (72); ExoN, putative 3′-to-5′ exonuclease (65); XendoU, putative poly(U)-specific endoribonuclease (65); MT, putative S-adenosylmethionine-dependent ribose 2′-O-methyltransferase (65, 81); C/H, domains containing conserved Cys and His residues and predicted to bind metal ions. (B) Sequence comparison of coronavirus helicases. The alignment was generated with the ClustalW program (version 1.82) (http://www.ebi.ac.uk/clustalw/) and used as the input for the ESPript program, version 2.1 (http://prodes.toulouse.inra.fr/ESPript/cgi-bin/ESPript.cgi). The nsp13 sequences of SARS-CoV (isolate Frankfurt 1; accession no. AY291315), mouse hepatitis virus (MHV, strain A59; NC_001846), bovine coronavirus (BCoV, isolate LUN; AF391542), human coronavirus 229E (HCoV-229E; X69721), porcine epidemic diarrhea virus (PEDV, strain CV777; AF353511); transmissible gastroenteritis virus (TGEV, strain Purdue 46; AJ271965), and avian infectious peritonitis virus (IBV, strain Beaudette; M95169) were derived from the replicative polyproteins of these viruses, whose sequences were obtained from the DDBJ, EMBL, and GenBank databases. Conserved helicase motifs I to VI (18) are indicated. Near the N terminus, the 12 conserved Cys and His residues predicted to form a binuclear zinc-binding cluster (77) are indicated by @. Also indicated is the conserved Lys288 residue (corresponding to Lys5589 in pp1ab), which, in the MBP-nsp13_KA control protein, was replaced with Ala. Lys288 is part of conserved helicase motif I (18), which is also called the Walker A box (82). Highlighted in grey is the C-terminal nsp13 sequence against which the rabbit antiserum, α-nsp13, used in this study was raised.