Structural basis for the regulatory function of a complex zinc-binding domain in a replicative arterivirus helicase resembling a nonsense-mediated mRNA decay helicase

Deng, Zengqin; Lehmann, Kathleen C.; Li, Xiaorong; Feng, Chong; Wang, Guoqiang; Zhang, Qi; Qi, Xiaoxuan; Yu, Lin; Zhang, Xingliang; Feng, Wenhai; Wu, Wei; Gong, Peng; Tao, Ye; Posthuma, Clara C.; Snijder, Eric J.; Gorbalenya, Alexander E.; Chen, Zhongzhou

Previous studies identified the nsp carrying RNA helicase activity (arterivirus nsp10 and coronavirus nsp13) as one of the two most evolutionarily conserved nidovirus proteins. Biochemical studies using recombinant arterivirus and coronavirus helicases revealed similar enzymatic properties, including nucleic acid-stimulated ATPase and 5 0 -3 0 duplex unwinding activities on both RNA and DNA substrates containing 5 0 single-stranded regions (34, 35) . A unique nidovirus helicase feature is the presence of an N-terminal (predicted) complex zincbinding domain (ZBD) of 80-100 residues. ZBD includes 12 or 13 conserved Cys/His residues (36) and is a nidoviral genetic marker not found in any other RNA virus group (19) . ZBD is separated from the downstream HEL1 domain by an uncharacterized domain that varies in size and sequence between arteri-and coronaviruses (37) . For the arterivirus prototype EAV, the significance of the nsp10 ZBD was evaluated extensively using sitedirected mutagenesis in combination with biochemical assays and reverse genetics. Amino acid substitutions in ZBD or the adjacent 'spacer' that connects it to the downstream domain can profoundly affect EAV helicase activity and RNA synthesis, with most replacements of conserved Cys or His residues yielding replicationnegative virus phenotypes (36, 37) . Intriguingly, some mutations in the spacer region selectively inactivated transcription, while not affecting replication (36, 38) , strongly suggesting a specific role for nsp10 in the unique mechanism of discontinuous sg RNA synthesis.