title: Transmembrane Domains of NS2B Contribute to both Viral RNA Replication and Particle Formation in Japanese Encephalitis Virus.
abstract: Flavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly.
  
  
  
  Many flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the flavivirus life cycle. Compensatory-mutation analysis confirmed that there was a correlation between viral assembly and NS2B-NS2A interaction. TMDs of NS2B may serve as novel antiviral targets to prevent flavivirus infection, and the structure determination of NS2B will help us to understand the functional mechanism of NS2B in viral RNA replication and assembly. The results have uncovered a new function of flavivirus NS2B in virion assembly, possibly through interaction with the NS2A protein.
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Xiao-Dan
authors.initials: XD
authors.lastname: Li
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Cheng-Lin
authors.initials: CL
authors.lastname: Deng
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Han-Qing
authors.initials: HQ
authors.lastname: Ye
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Hong-Lei
authors.initials: HL
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Qiu-Yan
authors.initials: QY
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Dong-Dong
authors.initials: DD
authors.lastname: Chen
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Pan-Tao
authors.initials: PT
authors.lastname: Zhang
authors.affiliation: Departments of Biochemistry and Molecular Biology and Phamarcology and Toxicology and Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Zhi-Ming
authors.initials: ZM
authors.lastname: Yuan
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China zhangbo@wh.iov.cn.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
mesh_terms: D004252:DNA Mutational Analysis; D004664:Encephalitis Virus, Japanese / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D016296:Mutagenesis; D000072417:Protein Domains; D012367:RNA, Viral / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: C080102:NS2B protein, flavivirus; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins