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 primer-extension and terminal transferase activity documented in Figure 4 for the complex containing the untagged nsp8 was not observed by Imbert et al. (12) when they first purified and analysed His-nsp8. To investigate whether this difference could be attributed to complex formation with nsp7 or the removal of the affinity tag, we performed the primer extension assay with three different recombinant nsp8 versions of which the gel filtration analysis is documented in Figure 2 . Interestingly, for all three variants primer-extension activity was observed ( Figure 7A ), but the activity was most pronounced for nsp8-His and the untagged nsp8 ( Figure 7A ). To estimate the effect of nsp7 on the nsp8-driven primer extension activity, we performed a direct comparison of the two enzyme complexes and found that the activity of nsp8 alone was >2-fold lower than when nsp7 and nsp8 were present at equal molarity in the reaction (Figures 4D and 7B) . A similar comparison was performed for the de novo activity of nsp8, using the assay published by Imbert et al. (12) and taking the first dinucleotide (pppGpA) product as readout. Interestingly, both nsp8 and nsp(7+8) synthesized equal amounts of the pppGpA dinucleotide ( Figure 7C) , suggesting that the effect of nsp8 is limited to the primer-extension activity of nsp8. In addition, we observed that the de novo initiation activity of nsp8 was $2-fold higher than that of His-nsp8 ( Figure 7D ).