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 negatively charged and helical polymer heparin is able to occupy the binding sites of RNA and DNA polymerases, and can thus directly compete with RNA and DNA templates. To verify that the full-length and longer RNA products were derived from single nsp(7+8) complexes bound to the template (i.e. from a processive activity), and not from multiple binding and extension events (i.e. a distributive activity), we performed the primer extension reaction in the presence of heparin to trap any unbound nsp(7+8). We first tested the concentration required to saturate all nsp(7+8) complexes in the reaction by titrating 0-100 mM into the reaction (Supplementary Figure S2A) and observed that the incorporation levels were stable above 1 mM (Supplementary Figure S2B) , suggesting that these reactions represent single initiation-extension events. We next assessed whether the activity of nsp8 or nsp(7+8) was distributive or processive by quantifying the incorporated signal in full-length or longer products in the presence of 1 mM heparin ( Figure 4C ). As shown in Figure 4D , 66 ± 4% (mean ± standard deviation) of the nsp8 products were full length compared to 61 ± 2% of the nsp(7+8) products, suggesting that both the enzymes complexes are mostly processive and that nsp7 does not confer additional processivity to nsp8. Interestingly, both nsp8 and nsp(7+8) are able to extend the RNA primers beyond template length in the presence of heparin ( Figure 4D and Supplementary Figure S2B ), suggesting that these extensions result from terminal transferase activity and not from template switching, as was previously observed for poliovirus 3D pol (20) .