Transmembrane domains of NS2B contribute to both viral RNA replication and particle formation in Japanese encephalitis virus

Xiao Dan Li, Cheng Lin Deng, Han Qing Ye, Hong Lei Zhang, Qiu Yan Zhang, Dong Dong Chen, Pan Tao Zhang, Pei Yong Shi, Zhi Ming Yuan, Bo Zhang

Research output: Contribution to journalArticlepeer-review

44 Scopus citations


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 ofKat 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 viralRNAreplication, as well as virion assembly.

Original languageEnglish (US)
Pages (from-to)5735-5749
Number of pages15
JournalJournal of virology
Issue number12
StatePublished - Jun 1 2016

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology


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