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 journalArticle

17 Citations (Scopus)

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 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
Volume90
Issue number12
DOIs
StatePublished - Jun 1 2016

Fingerprint

Japanese Encephalitis Virus
Japanese encephalitis virus
Viral RNA
Virion
virion
Flavivirus
mutation
Mutation
Proteins
proteins
Peptide Hydrolases
proteinases
mutants
RNA
Protein Domains
RNA replication
transmembrane proteins
synthesis
mutagenesis
Mutagenesis

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

Li, X. D., Deng, C. L., Ye, H. Q., Zhang, H. L., Zhang, Q. Y., Chen, D. D., ... Zhang, B. (2016). Transmembrane domains of NS2B contribute to both viral RNA replication and particle formation in Japanese encephalitis virus. Journal of Virology, 90(12), 5735-5749. https://doi.org/10.1128/JVI.00340-16

Transmembrane domains of NS2B contribute to both viral RNA replication and particle formation in Japanese encephalitis virus. / Li, Xiao Dan; Deng, Cheng Lin; Ye, Han Qing; Zhang, Hong Lei; Zhang, Qiu Yan; Chen, Dong Dong; Zhang, Pan Tao; Shi, Pei-Yong; Yuan, Zhi Ming; Zhang, Bo.

In: Journal of Virology, Vol. 90, No. 12, 01.06.2016, p. 5735-5749.

Research output: Contribution to journalArticle

Li, XD, Deng, CL, Ye, HQ, Zhang, HL, Zhang, QY, Chen, DD, Zhang, PT, Shi, P-Y, Yuan, ZM & Zhang, B 2016, 'Transmembrane domains of NS2B contribute to both viral RNA replication and particle formation in Japanese encephalitis virus', Journal of Virology, vol. 90, no. 12, pp. 5735-5749. https://doi.org/10.1128/JVI.00340-16
Li, Xiao Dan ; Deng, Cheng Lin ; Ye, Han Qing ; Zhang, Hong Lei ; Zhang, Qiu Yan ; Chen, Dong Dong ; Zhang, Pan Tao ; Shi, Pei-Yong ; Yuan, Zhi Ming ; Zhang, Bo. / Transmembrane domains of NS2B contribute to both viral RNA replication and particle formation in Japanese encephalitis virus. In: Journal of Virology. 2016 ; Vol. 90, No. 12. pp. 5735-5749.
@article{fc3f19de5ff644c1820ae1ed4fcd9453,
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 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.",
author = "Li, {Xiao Dan} and Deng, {Cheng Lin} and Ye, {Han Qing} and Zhang, {Hong Lei} and Zhang, {Qiu Yan} and Chen, {Dong Dong} and Zhang, {Pan Tao} and Pei-Yong Shi and Yuan, {Zhi Ming} and Bo Zhang",
year = "2016",
month = "6",
day = "1",
doi = "10.1128/JVI.00340-16",
language = "English (US)",
volume = "90",
pages = "5735--5749",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "12",

}

TY - JOUR

T1 - Transmembrane domains of NS2B contribute to both viral RNA replication and particle formation in Japanese encephalitis virus

AU - Li, Xiao Dan

AU - Deng, Cheng Lin

AU - Ye, Han Qing

AU - Zhang, Hong Lei

AU - Zhang, Qiu Yan

AU - Chen, Dong Dong

AU - Zhang, Pan Tao

AU - Shi, Pei-Yong

AU - Yuan, Zhi Ming

AU - Zhang, Bo

PY - 2016/6/1

Y1 - 2016/6/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84971432720&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84971432720&partnerID=8YFLogxK

U2 - 10.1128/JVI.00340-16

DO - 10.1128/JVI.00340-16

M3 - Article

C2 - 27053551

AN - SCOPUS:84971432720

VL - 90

SP - 5735

EP - 5749

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 12

ER -