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.

Intrigued by the primer extension activity of the SARS-CoV nsp(7+8) complex described above, we next designed a set of mutations to verify that the activity indeed was nsp(7+8) derived and to identify the most critical residues for activity in the complex. We first tested RNA-binding mutant K58A (Figure 2 ) at varying concentrations and observed a $95% loss of nucleotide incorporation activity compared to the wild-type protein ( Figure 5 ). Other likely candidates for a direct role in RdRp catalysis generally are Mg 2+ -coordinating aspartate residues and lysine or histidine residues that can function as general acid (3). In canonical RNA polymerases, the aspartates commonly reside in motifs A and C (3, 21) , while in DNA-dependent RNA primases they are usually found in a central D/ExD/E motif (22) . Given the absence of classical RdRp A and C motifs in the nsp8 sequence (12), we screened an alignment of CoV nsp8 sequences for conserved D/ExD/E motifs. Interestingly, we found such a motif in both the N-terminal and the C-terminal domain ( Figure 5A ). Subsequent alanine substitution of the N-terminal D/ ExD/E motif, composed of D50 and D52 in SARS-CoV, greatly affected primer extension activity on the CU 10 template as shown in Figure 5C . Mutation of the downstream domain (residues D161 and D163 in SARS-CoV), however, had a much smaller effect on polymerase activity, suggesting that this C-terminal D/ExD/E motif is not critical for catalysis. Controls included mutant K58A and a mutant carrying a lysine-to-alanine substitution of the non-conserved residue 127. In line with the observation of the U 20 template and its conservation in CoVs, the loss of a lysine at position 58 resulted in a near complete loss of RdRp activity, whereas mutation of K127 positively influenced RNA synthesis ( Figure 5) .