title: A Dimerization-Dependent Mechanism Drives the Endoribonuclease Function of Porcine Reproductive and Respiratory Syndrome Virus nsp11.
abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) RNA endoribonuclease nsp11 belongs to the XendoU superfamily and plays a crucial role in arterivirus replication. Here, we report the first crystal structure of the arterivirus nsp11 protein from PRRSV, which exhibits a unique structure and assembles into an asymmetric dimer whose structure is completely different from the hexameric structure of coronavirus nsp15. However, the structures of the PRRSV nsp11 and coronavirus nsp15 catalytic domains were perfectly superimposed, especially in the "active site loop" (His129 to His144) and "supporting loop" (Val162 to Thr179) regions. Importantly, our biochemical data demonstrated that PRRSV nsp11 exists mainly as a dimer in solution. Mutations of the major dimerization site determinants (Ser74 and Phe76) in the dimerization interface destabilized the dimer in solution and severely diminished endoribonuclease activity, indicating that the dimer is the biologically functional unit. In the dimeric structure, the active site loop and supporting loop are packed against one another and stabilized by monomer-monomer interactions. These findings may help elucidate the mechanism underlying arterivirus replication and may represent great potential for the development of antiviral drugs.
  
  
  OBJECTIVE
  Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirales PRRSV is a major agent of respiratory diseases in pigs, causing tremendous economic losses to the swine industry worldwide. The PRRSV nsp11 endoribonuclease plays a vital role in arterivirus replication, but its precise roles and mechanisms of action are poorly understood. Here, we report the first dimeric structure of the arterivirus nsp11 from PRRSV at 2.75-Å resolution. Structural and biochemical experiments demonstrated that nsp11 exists mainly as a dimer in solution and that nsp11 may be fully active as a dimer. Mutagenesis and structural analysis revealed NendoU active site residues, which are conserved throughout the order Nidovirales(families Arteriviridae and Coronaviridae) and the major determinants of dimerization (Ser74 and Phe76) in Arteriviridae Importantly, these findings may provide a new structural basis for antiviral drug development.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
authors.forename: Yuejun
authors.initials: Y
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
authors.forename: Youwen
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Lei
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Ye
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Shen
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
authors.forename: Limeng
authors.initials: L
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Luo
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
authors.forename: Dang
authors.initials: D
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.|Pathology Department, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA.
authors.forename: Zhen F
authors.initials: ZF
authors.lastname: Fu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
authors.forename: Shaobo
authors.initials: S
authors.lastname: Xiao
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China penggq@mail.hzau.edu.cn.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.|The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: Guiqing
authors.initials: G
authors.lastname: Peng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002460:Cell Line; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D019143:Evolution, Molecular; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D019316:Porcine respiratory and reproductive syndrome virus / Q000145:classification / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013552:Swine; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D004722:Endoribonucleases