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.

Our comparative study revealed that the N-terminal His 6 -tag of His-nsp8 greatly influences the primerextension activity of nsp8 ( Figure 7A ), its multimerization profile and its association with nsp7 ( Figure 2 ). To test if this inhibitory effect was His 6 -tag specific, we assessed the activity of a ub-nsp8-His fusion protein. At the same time, control reactions were performed in which we (i) followed the activity of this protein as it was being processed by a recombinant form of the ubiquitin-cleaving nsp2 protease of equine arteritis virus (28) or (ii) monitored the activity of nsp8-His. As shown in Supplementary Figure S3 , the presence of the ub-tag decreased nsp8 activity to a level that was comparable to that of N-terminally His 6 -tagged nsp8. Upon cleavage by EAV nsp2, however, a partial recovery of the primer extension activity was observed (Supplementary Figure S3) . Unfortunately, we were not able to perform the same experiment with purified ub-nsp8, since our recombinant nsp5 removed the N-terminal ub-tag with similar efficiency as the C-terminal His 6 -tag (Supplementary Figure S4) .