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.

Mutagenesis of nsp8 was performed to identify residues that may contribute to the catalytic centre of the nsp(7+8) polymerase, while differently tagged nsp8 recombinant proteins were constructed to explain some striking differences with previous observations. These efforts resulted in two intriguing observations. Firstly, mutation of the conserved N-terminal D/ExD/E motif, comprising D50 Table 1 ), using the synthesis of the first dinucleotide pppGpA, as previously described by Imbert et al. (12) , as readout. Nsp8 template binding mutant K58A was used as negative control. The AMP contaminant present in the used [a-32 P]ATP label is marked as loading control and size reference. (D) Side-by-side comparison of the de novo initiation activities of nps8, His-nsp8 and nsp7-8-His. (E) Elution profile of the nsp7-8-His fusion protein relative to nsp8-His. (F) Primer-extension activities of putative cleavage intermediate nsp7-8 on the U 20 template (see Figure 4 and Table 1 ). and D52 in SARS-CoV, abolished RdRp activity, whereas mutation of the C-terminal motif, including SARS-CoV residues D161 and D163, did not affect polymerase activity ( Figure 5 ). Given the general importance of acidic residues for metal-ion coordination in polymerase active sites (3) (4) (5) 22) and the D/ExD/E consensus sequence in coronaviruses at positions 50-52, we now postulate that these residues are part of the Mg 2+ -binding active site in spite of the more conserved nature of D161 and D163 ( Figure 5) , and their position in the nsp(7+8) structure (see below for further discussion).