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.

Interestingly, the primer extension activity of nsp(7+8) did not require manganese ions as was previously reported for the His-nsp8 homomer (12) . In fact, similar to the SARS-CoV nsp12-RdRp (10), the addition of Mn 2+ was found to reduce the fidelity of nsp(7+8) and induce both transversional and transitional misincorporations in a pulse-chase experiment ( Figure 6E and F) . Interestingly, the assay also revealed a discrimination against the widely Figure 5 . Mutagenesis of SARS-CoV nsp8. (A) Alignment of nsp8 sequences from representative alpha-, beta-and gammacoronaviruses. Fully conserved residues are shaded red, while partially conserved residues are boxed. The residues targeted by mutagenesis are indicated with asterisks. Please see 'Material and Methods' section for the Genbank accession numbers associated with the presented sequences. (B) To verify that the observed extension activity was nsp8-dependent, we tested the incorporation of AMP into the primed U 20 template by 1, 5 or 10 mM of wild-type nsp8 or template-binding mutant K58A. Mutation of K58 resulted in a $95% reduction of AMP incorporation. (C) To assess the importance of the two D/ExD/E motifs in nsp8, we engineered alanine substitution mutants of these residues and tested their primer extension activity on the primed UC 10 template (see Figure 4 ). Reactions were stopped after 60 min and compared to the activity of the wild-type nsp(7+8) complex on a 20% PAGE/ used ATP and GTP analogue ribavirin triphosphate (RTP) (24, 25) . Whether this may offer an explanation for SARS-CoV's relative resistance to this antiviral drug (26, 27) remains an open question for future research.