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

The nsp10 structure: defining a complex ZBD Our characterization of the EAV nsp10 structure verified and revised a model of the N-terminal ZBD based on prior studies (36, 37, 58) . It resolved the uncertainty about the number of zinc ions bound (now established to be three) and the fold of this domain (a unique structure combining a RING-like module fused with a treble-clef zinc finger). Furthermore, it redefined the C-terminal border of ZBD and placed it 13 residues downstream to include a third hitherto unrecognized structural element (helix a4). Previously, we analysed a variety of EAV nsp10 ZBD mutants in which putative zinc-binding residues were replaced in a manner (Cys!His or His!Cys) that could preserve zinc binding (36, 37) . From the solved structure, it is now apparent that the replication-negative phenotypes of these virus mutants can likely be attributed to the detrimental impact of the respective mutations on ZBD integrity and, through the extensive interaction network, HEL1 domains. It presently remains unclear why the replacement of His44 by Cys in the treble-clef zinc finger was partially tolerated. On the other hand, structural superposition of the RINGlike modules of nsp10 and hUpf1 ( Figure 4E ) reveals how the only other similarly tolerated replacement (36, 37) , that of the Zn1-coordinating Cys25 by His (found in the equivalent position in hUpf1), could be accommodated by nsp10. The RING-like module 1 of Upf1 also shares structural similarity with RING-box domains of E3 ubiquitin ligases (59) and the involvement of this module in self-ubiquitination of Upf1 was indeed demonstrated (60) . It would be interesting to see whether these results are relevant for nsp10 and its ZBD. Recently, arterivirus papain-like protease 2 was found to have deubiquitinase activity, which suppresses the innate immune response in infected host cells (61, 62) .