Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies

Hua, Rong-Hong; Liu, Li-Ke; Chen, Zhenshi; Li, Yenan; Bu, Zhigao

doi:10.1371/journal.pone.0067553

Cited by 18 publications

(14 citation statements)

References 35 publications

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“…The JEV SA14 strain was propagated and titrated onto BHK-21 cells. Mouse monoclonal antibodies against the JEV prM (31,32), E (33), and NS1 (34,35) proteins were generated previously. Mouse monoclonal antibody against JEV NS2B was a gift from Shengbo Cao (Huazhong Agricultural University, Wuhan, China).…”

Section: Methodsmentioning

confidence: 99%

Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B

Zhang

et al. 2018

J Virol

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Signal peptidase complex subunit 1 (SPCS1) is a newly identified host factor that regulates flavivirus replication, but the molecular mechanism is not fully understood. Here, using Japanese encephalitis virus (JEV) as a model, we investigated the mechanism through which the host factor SPCS1 regulates the replication of flaviviruses. We first validated the regulatory function of SPCS1 in JEV propagation by knocking down and knocking out endogenous SPCS1. The loss of SPCS1 function markedly reduced intracellular virion assembly and the production of infectious JEV particles but did not affect cell entry, RNA replication, or translation of the virus. SPCS1 was found to interact with nonstructural protein 2B (NS2B), which is involved in posttranslational protein processing and virus assembly. Serial deletion mutation of the JEV NS2B protein revealed that two transmembrane domains, NS2B(1-49) and NS2B(84-131), interact with SPCS1. Further mutagenesis analysis of conserved flavivirus residues in two SPCS1 interaction domains of NS2B demonstrated that G12A, G37A, and G47A in NS2B(1-49) and P112A in NS2B(84-131) weakened the interaction with SPCS1. Deletion mutation of SPCS1 revealed that SPCS1(91-169), which contains two transmembrane domains, was involved in interactions with both NS2B(1-49) and NS2B(84-131). Taken together, these results demonstrate that SPCS1 affects viral replication by interacting with NS2B, thereby influencing the posttranslational processing of JEV proteins and the assembly of virions. Understanding virus-host interactions is important for elucidating the molecular mechanisms of virus propagation and identifying potential antiviral targets. Previous reports demonstrated that SPCS1 is involved in the flavivirus life cycle, but the mechanism remains unknown. In this study, we confirmed that SPCS1 participates in the posttranslational protein processing and viral assembly stages of the JEV life cycle but not in the cell entry, genome RNA replication, or translation stages. Furthermore, we found that SPCS1 interacts with two independent transmembrane domains of the flavivirus NS2B protein. NS2B also interacts with NS2A, which is proposed to mediate virus assembly. Therefore, we propose a protein-protein interaction model showing how SPCS1 participates in the assembly of JEV particles. These findings expand our understanding of how host factors participate in the flavivirus replication life cycle and identify potential antiviral targets for combating flavivirus infection.

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Section: Methodsmentioning

confidence: 99%

Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B

Zhang

et al. 2018

J Virol

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“…The inability of dengue serological assays to specifically identify dengue serotypes is a result of cross‐reactivity reactions between serotypes and other related flaviviruses . Dengue shares similar NS1 epitope sequences with JEV, WNV, and ZIKV . Within the four serotypes of dengue, it was also previously reported that sequence homology may reach up to 70% .…”

Section: Discussionmentioning

confidence: 99%

A synthetic peptide analog of in silico‐predicted immunogenic epitope unique to dengue virus serotype 2 NS1 antigen specifically binds immunoglobulin G antibodies raised in rabbits

Guevarra

Boado

Ceñidoza

et al. 2020

Microbiology and Immunology

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Development of a serotyping-capable dengue detection test is hampered by the absence of an identified unique marker that can detect specific dengue virus (DENV) serotype. In the current commercially available antibody-capture diagnostic methods, immobilized nonstructural 1 (NS1) antigen indiscriminately binds and detects immunoglobulin M or immunoglobulin G against any serotype, thus limiting its capability to distinguish existing serotypes of dengue. Identification of dengue serotype is important because certain serotypes are associated with severe forms of dengue as well as dengue hemorrhagic fever. In this study, we aimed to identify an immunogenic epitope unique to DENV2 NS1 antigen and determine the binding specificity of its synthetic peptide mimotope to antibodies raised in animal models.Selection of a putative B-cell epitope from the reported DENV2 NS1 antigen was done using Kolaskar and Tongaonkar Antigenicity prediction, Emini surface accessibility prediction, and Parker hydrophilicity prediction available at the immune epitope database and analysis resource. Uniqueness of the B-cell epitope to DENV2 was analyzed by BLASTp. Immunogenicity of the synthetic peptide analog of the predicted immunogenic epitope was tested in rabbits. The binding specificity of the antibodies raised in animals and the synthetic peptide mimotope was tested by indirect ELISA. A synthetic peptide analog comprising the unique epitope of DENV2 located at the 170th-183rd position of DENV2 NS1 was found to be immunogenic in animal models. The antipeptide antibody produced in rabbits showed specific binding to the synthetic peptide mimotope of the predicted unique DENV2 NS1 immunogenic epitope. K E Y W O R D Sdengue detection, dengue serotype, epitope prediction, immune epitopes, mimotopeAbbreviations: BLAST, basic local alignment search tool; BSA, bovine serum albumin; DENV, dengue virus; DENV2 NS1 Ag, dengue virus serotype 2 nonstructural 1 antigen; DHF, dengue hemorrhagic fever; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; IEDB, immune epitope database; IgG, immunoglobulin G; IgM, immunoglobulin M; JEV, Japanese encephalitis; LFA, lateral flow assay; NCBI, National Center for Biotechnology Information; NS1, nonstructural 1; PBS, phosphate buffered saline; WNV, West Nile virus; ZIKV, Zika virus.

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“…(19) Thus far monoclonal antibodies against the NS1 protein of Japanese encephalitis virus have been developed, and five B-cell epitopes have been screened by using them, which lays the foundation for further study of the antigenic structure of NS1 protein and the potential clinical application of epitope-based methods. (20) The development of monoclonal antibodies against NS1 protein of dengue virus has provided an effective tool for early diagnosis and classification of dengue virus infections clinically. The function of NS1 protein and mechanism of protein-protein interaction, once ascertained, may ultimately lead to the production of effective antiviral agents.…”

Section: Discussionmentioning

confidence: 99%

Development and Characterization of Monoclonal Antibodies Against NS1 Protein of Duck Tembusu Virus

Li²,

Zhao

et al. 2016

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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To produce monoclonal antibodies (MAbs) against NS1 protein of duck Tembusu virus (TMUV), the NS1 gene sequence was cloned into the prokaryotic expression vector pET-28a(+). The prokaryotic protein NS1 was successfully expressed in BL21 (DE3) and used as the immunogen in mice. Six MAbs against NS1 protein were obtained by using the standard hybridoma technique. All MAbs can react with the denatured NS1 protein in the Western blot assay and the native NS1 protein from the TMUV-infected BHK-21 cells in the immunofluorescence assay. The ELISA titers of the cell supernatants and ascites of MAbs were at a high level. The subtypes of the MAbs were determined by the Rapid Mouse Isotyping Kit-Gold series. Six MAbs possessed higher specificity and sensitivity, which indicated that MAbs against NS1 protein of TMUV may be used as valuable tools for analysis of the protein functions and pathogenesis of TMUV.

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Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies

Cited by 18 publications

References 35 publications

Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B

Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B

A synthetic peptide analog of in silico‐predicted immunogenic epitope unique to dengue virus serotype 2 NS1 antigen specifically binds immunoglobulin G antibodies raised in rabbits

Development and Characterization of Monoclonal Antibodies Against NS1 Protein of Duck Tembusu Virus

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