Mechanism of Nucleic Acid Unwinding by SARS-CoV Helicase

Adedeji, Adeyemi O.; Marchand, Bruno; te Velthuis, Aartjan J. W.; Snijder, Eric J.; Weiss, Susan; Eoff, Robert L.; Singh, Kamalendra; Sarafianos, Stefan G.

Because nsp13 has 26 cysteine residues, 14 of which are highly conserved [34] and likely (at least for some of them) to participate in disulfide bonds, we hypothesized that if we expressed the protein in a eukaryotic expression system it would be better folded and more active than that expressed in bacteria. Hence, we prepared GST-nsp13 using a baculovirus expression system and this construct was primarily used to characterize in detail the kinetics of nucleic acid unwinding by SARS-CoV helicase, but we also carried out similar analyses with MBP-nsp13 and H 6 -nsp13 and compared them with GST-nsp13 to ensure that we were not measuring a GST-related artifact. We found that GST-nsp13 was significantly more active than the other variants and had activity comparable to other helicases [39] . In our enzyme characterization, we used both RNA and DNA substrates, as the biological role of SARS-CoV nsp13 is expected to be unwinding of RNA substrate. Moreover, we determined the effect of nsp12 on the mechanism of nsp13. Our data demonstrate that regardless of the type of fusion tag or substrate use, the helicase activity of nsp13 is enhanced in the presence of nsp12, suggesting that these proteins also interact in a functional replication complex and that their interaction contributes to the efficiency of viral replication. Our results allow mechanistic comparisons with other viral or eukaryotic helicases and set the stage for studying the intriguing evolutionary relations between helicases from nidoviruses and other groups of positive sense RNA viruses.