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

te Velthuis, Aartjan J.W.; van den Worm, Sjoerd H. E.; Snijder, Eric J.

eight copies of nsp7 (13,17) ( Figure 1B ). Based on the large diameter, positive charge of the hexadecamer's channel and in silico docking, it was proposed to be able to encircle dsRNA ( Figure 1B) . However, the functional significance of the compound interactions between nsp7 and nsp8 is poorly understood, as are the polymerase activities associated with monomeric nsp8 or nsp8-containing multimers. So far, strategies for the purification of recombinant nsp8 have involved the use of affinity tags [e.g. His 6 or glutathione-S-transferase (GST) (12, 13) ] that were fused to one terminus to facilitate protein recovery. Inadvertently though, such tags or other exogenous sequences may significantly impede the correct folding of enzymes and thus alter their stability or activity, as exemplified by studies of the poliovirus (3D pol ) and SARS-CoV (nsp12) RdRp subunits (10, 14, 18) . To circumvent this issue, we developed a protocol in which SARS-CoV nsp8 was expressed as a ubiquitin (ub) fusion protein carrying a C-terminal His 6 -tag (ub-nsp8-His), which was subsequently processed at both termini in two steps. The first step was co-translational and involved the release of the N-terminal ub fusion partner by the co-expressed ubiquitin carboxyl-terminal hydrolase 1 (Upb1, Figure 2A ) (10, 14) . The second proteolytic step, catalysed by a recombinant form of the SARS-CoV nsp5 main protease (15) , removed the C-terminal His 6 -tag and was performed either in solution (Figure 2A and B) or when nsp8-His was immobilised to Talon beads. This procedure yielded SARS-CoV nsp8 with its exact natural Nand C-terminus (replicase residues Ala-3920 and Gln-4117, respectively; Figure 2A ), the product that is normally liberated by the nsp5-driven autoprocessing of the SARS-CoV replicase polyproteins (19) .