Crystal Structure of the C-Terminal Cytoplasmic Domain of Non-Structural Protein 4 from Mouse Hepatitis Virus A59

Xu, Xiaoling; Lou, Zhiyong; Ma, Yanlin; Chen, Xuehui; Yang, Zhangsheng; Tong, Xiaohang; Zhao, Qi; Xu, Yuanyuan; Deng, Hongyu; Bartlam, Mark; Rao, Zihe

MHV nsp4 was first biochemically identified as an integral membrane protein by the evidence that it is pelleted with detergent Triton_X114 extraction fraction of cell lysates, it is processed by PLP2 at amino terminal and nsp5 at carboxy terminal from a p150 precursor [21] . MHV nsp4 was localized to the endoplasmic reticulum (ER) membrane when expressed alone and recruited to the replication complex in infected cells, its amino and carboxy termini are exposed to the cytosol, while the Nglycosylated region is located between the first and second TM regions and faces the ER luminal side [25] . Substitution of the MHV nsp4 glycosylation site N237 to alanine is lethal for virus replication, while the temperature sensitive mutant N258T virus leads to a dramatic reduction in DMVs assembly at nonpermissive temperature, indicating a critical role for nsp4 in coronavirus DMVs assembly [32] . Further virus recovery analysis of several deletion mutants of MHV nsp4 revealed that it is required for viral replication: the putative TMs1-3 and specific charged residues are essential for productive virus infection, while TM4 and the carboxy terminal amino acids K398-T492 are dispensable [33] . It is evident that nsp4 plays important role in coronavirus replication and DMVs formation, and that the TM regions of nsp4 are involved in association of the coronavirus replication complex with cellular membranes. However, our understanding of the functional role of nsp4 is still at an early stage, and there has been no structural characterization of the coronavirus nsp4 to date.