Abstract |
Nonstructural protein 2A (NS2A) of the Japanese encephalitis virus (JEV) contributes to viral replication and pathogenesis; however, a lack of NS2A-specific antibodies restricts studies on the underlying mechanisms. In this study, we constructed a recombinant JEV with a hemagglutinin (HA)-tagged NS2A (JEV-HA/NS2A/∆NS1') to overcome this challenge. An HA-tag was fused to the N-terminus of NS2A (HA-NS2A) at the intergenic junction between NS1 and NS2A. A peptide linker, "FNG", was added to the N-terminus of HA-tag to ensure correct cleavage between the C-terminus of NS1 and the N-terminus of HA-NS2A. To avoid the side effects of an unwanted NS1' tagged with HA (HA-NS1'), an alanine-to- proline (A30P) substitution was introduced at residue 30 of NS2A to abolish HA-NS1' production. The HA-tag insertion and A30P substitution were stably present in JEV-HA/NS2A/∆NS1' after six passages and did not exhibit any significant effects on viral replication and plaque morphology. Taking advantage of HA-NS2A, we examined the activities of NS2A during JEV infection in vitro using anti-HA antibodies. NS2A was observed to be localized to the endoplasmic reticulum and interact with viral NS2B and NS3 during virus infection. These data suggest that JEV-HA/NS2A/∆NS1' can serve as a model for the analysis of the biological characteristics and functions of NS2A in vitro during JEV infection.
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Authors | Xiaochun Ma, Chenxi Li, Qiqi Xia, Yan Zhang, Yang Yang, Abdul Wahaab, Ke Liu, Zongjie Li, Beibei Li, Yafeng Qiu, Jianchao Wei, Zhiyong Ma |
Journal | Viruses
(Viruses)
Vol. 14
Issue 4
(03 29 2022)
ISSN: 1999-4915 [Electronic] Switzerland |
PMID | 35458436
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Hemagglutinins
- Viral Nonstructural Proteins
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Topics |
- Cell Line
- Encephalitis Virus, Japanese
- Encephalitis, Japanese
- Hemagglutinins
(metabolism)
- Humans
- Viral Nonstructural Proteins
(chemistry)
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