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 oligoribonucleotide substrates used for polymerase assays are listed in Table 1 and were prepared as described previously (10) . Primer-extension assays for nsp8, the nsp7-8 polyprotein, and the nsp(7+8) complex were essentially performed as described previously for SARS-CoV nsp12 (10, 11) . In each primer-extension reaction, typically 1 mM wild-type or mutant nsp8 was incubated with 4 mM MgCl 2 , 50 mM GTP, 50 mM ATP, 0.17 mM [a-32 P]ATP, 1 mM DTT, 0.1% Triton X-100, 10 mM KCl and 20 mM Tris (pH 9.5). At most, 10 mM NaCl and 5% glycerol were introduced with the nsp8 storage buffer. Gels were run and analysed as described previously (10) . To convert the phosphorimager signal into the amount of [a-32 P]AMP incorporated, a 10 À2 to 10 À5 dilution series of the [a-32 P]ATP stock was spotted in triplicate on Whatman filter paper and exposed alongside the PAGE gel. The amount of incorporated label was ultimately corrected for the concentration of competing, unlabelled nucleotides present in the reaction mixture.