The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

Tan, Jinzhi; Vonrhein, Clemens; Smart, Oliver S.; Bricogne, Gerard; Bollati, Michela; Kusov, Yuri; Hansen, Guido; Mesters, Jeroen R.; Schmidt, Christian L.; Hilgenfeld, Rolf

Full-length SUD (Nsp3 residues 389-726) and the fragment SUD core (Nsp3 residues 389-652, previously called ''SUDc5b'') of SARS-CoV strain TOR2 (acc. no. AY274119) were produced recombinantly in E. coli as described [31] . The coding regions for the SUD-N subdomain (Nsp3 residues 389-524) and the SUD-M subdomain (Nsp3 residues 525-652) were constructed by introducing an appropriate deletion into the previously described plasmid pQE30-Xa-c5b [31] using site-directed mutagenesis. Plasmids encoding SUD-N and SUD-M were prepared using primers listed in Table S1 . The coding regions for four sets of mutations of SUD core , M1 (K505A+K506A), M2 (K476A+K477A), M3 (K563A+K565A+K568A), and M4 (K565A+K568A+E571A), were constructed by introducing appropriate mutations into plasmid pQE30-Xa-c5b [31] using site-directed mutagenesis. Plasmids encoding these mutants were prepared using primers also listed in Table S1 . All plasmids provided an N-terminal His-tag and a short linker sequence encoding a factor-Xa cleavage site. The coding regions of the expression plasmids were verified by DNA sequencing. E. coli M15 (pRep4) was used as expression host for these constructs. SUD-N, SUD-M, and the mutated proteins were purified according to the same protocol as for SUD core [31] .