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 proposed signalling network can now be used to rationalize, in a structural context, the previously reported phenotypes of EAV ZBD mutants carrying replacements of residues not directly involved in Zn-binding. For instance, a replication-negative phenotype was described for mutant D45A (36) . It is now clear that Asp45 forms two hydrogen bonds with the main and side chain of Thr35 and electrostatically interacts with the side chain of His34, which both belong to the RING-like zinc finger ( Figure 4D ). Replacement of Asp45 may thus greatly reduce these interactions and disrupt ZBD integrity, potentially affecting the structural integrity of the HEL1. Another residue, Ser59, was probed extensively by mutagenesis after the finding that a virus mutant (EAV030F) carrying a S59P mutation replicates its genomic RNA with wild-type efficiency, while being completely defective in sg mRNA synthesis (38) . This transcription-negative phenotype was attributed to the severe structural constraints exerted by Pro residues on the local conformation of the proposed hinge region, as various substitutions of Ser59 alone (to Ala, Cys, Gly, His, Leu or Thr) yielded virus mutants with a wild-type phenotype, while combining the neutral S59G mutation with a P60G substitution reproduced the specific defect in sg mRNA synthesis (36) . This interpretation is now further supported by the nsp10Á structure in which Ser59 and Pro60 are located in the hinge connecting the treble-clef zinc finger and a4 of ZBD. The main chain of Ser59 forms three hydrogen bonds with the treble-clef Thr54, which is also connected to the Pro60 side chain and Lys61 main chain ( Figure 4D ). Owing to the unique properties of the Pro residue, the Ser59-to-Thr54 bonds are likely disrupted by the S59P mutation, but are not affected by the alternative replacements tested. Consequently, also owing to the main chain rigidity associated with the introduction of a Pro residue, the orientation of a4 relative to 1A and/or the main body of ZBD is likely affected in mutant S59P, which carries adjacent Pro residues at positions 59 and 60. Likewise, the introduction of two Gly residues at these positions [double mutant S59G/P60G; (36) ] probably gives rise to excessive flexibility of the hinge region, thus compromising nsp10 function in a similar manner.