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.

The expression of SARS-CoV nsp7 with a C-terminal His 6 -tag (nsp7-His) was achieved from plasmid pDEST14-nsp7-His 6 according to the protocol previously described for EAV nsp9 (11) . SARS-CoV nsp5-His 6 (nsp5-His) was expressed as a self-cleaving maltose binding protein (MBP)-fusion protein and was purified via its C-terminal His 6 -tag (15) . The pASK3-His-nsp8 plasmid for expression of the N-terminally His 6 -tagged The coronavirus genome contains two large 5 0 -proximal ORFs (ORF1a and 1 b) that encode the two replicase polyproteins, whose mature products assemble into the viral replication and transcription complex. Both polyproteins are cleaved (cleavage sites indicated with arrow heads) by the proteinase activities of nsp3 (black lines) and nsp5 (red lines), which releases the mature nsps. Also indicated are the 5 0 cap structure and the 3 0 polyA tail (A n ). (B) The SARS-CoV nsp8 crystal structure (pdb 2AHM) resembles a 'golf club-like' shape, as presented by the yellow ribbon structure. This nsp8 conformation connects to a much larger, hexadecameric structure that is composed of seven additional nsp8 subunits (grey) and eight nsp7 subunits (green). The hollow hexadecameric ring structure has a positively charged channel (blue background shading) that was proposed to mediate RNA binding. The outside of the structure is predominantly negatively charged (red background shading). nsp8 was kindly provided by Dr Imbert and Dr Canard (University of Marseille, France).