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Structural and molecular analyses of functional epitopes and escape mutants in Japanese encephalitis virus envelope protein domain III.

ABSTRACT: The Japanese encephalitis virus (JEV) is one of the vector borne causes of encephalitis found in southeastern Asia. This positive single-stranded RNA virus is a member of the Flaviviridae family, which notably includes dengue, tick-borne, West Nile, Zika as well as yellow fever, and transmits to humans by infected mosquitos. The main site of interactions for antibodies against this virus is the envelope protein domain III (ED3). The present report investigates the time-dependent structural and conformational changes of JEV ED3 functional epitopes and escape mutants by computer simulations. The results indicate the presence of significant structural differences between the functional epitopes and the escape mutants. Mutation-induced structural/conformational instabilities of this type can decrease the antibody neutralization activity. Among the different escape mutants studied here, Ser40Lys/Asp41Arg appear to be most unstable, while Ser40Glu/Asp41Leu exhibit the lowest structural variations. The highest level of escape mutation observed in Ser40Lys is linked to the relatively higher values of root mean square deviation/fluctuation found in the molecular dynamics simulation of this protein. Secondary-structure deviations and depletion of H bonding are other contributing factors to the protein's increased instability. Overall, the proteins with residue 41 mutations are found to be structurally more ordered than those with residue 40 mutations. The detailed time-based structural assessment of the mutant epitopes described here may contribute to the development of novel vaccines and antiviral drugs necessary to defend against future outbreaks of JEV escape mutants.

SUBMITTER: Roy U

PROVIDER: S-EPMC7243247 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Structural and molecular analyses of functional epitopes and escape mutants in Japanese encephalitis virus envelope protein domain III.

Roy Urmi U

Immunologic research 20200401 2

The Japanese encephalitis virus (JEV) is one of the vector borne causes of encephalitis found in southeastern Asia. This positive single-stranded RNA virus is a member of the Flaviviridae family, which notably includes dengue, tick-borne, West Nile, Zika as well as yellow fever, and transmits to humans by infected mosquitos. The main site of interactions for antibodies against this virus is the envelope protein domain III (ED3). The present report investigates the time-dependent structural and c ...[more]

PMID: 32445181

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Project description:Japanese encephalitis virus (JEV) is a mosquito-transmitted flavivirus that is closely related to other emerging viral pathogens, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout Southeast Asia, the Pacific Islands, and the Far East, causing around 68,000 cases of infection worldwide each year. In this report, we present a 2.1-Å-resolution crystal structure of the C-terminal ?-ladder domain of JEV nonstructural protein 1 (NS1-C). The surface charge distribution of JEV NS1-C is similar to those of WNV and ZIKV but differs from that of DENV. Analysis of the JEV NS1-C structure, with in silico molecular dynamics simulation and experimental solution small-angle X-ray scattering, indicates extensive loop flexibility on the exterior of the protein. This, together with the surface charge distribution, indicates that flexibility influences the protein-protein interactions that govern pathogenicity. These factors also affect the interaction of NS1 with the 22NS1 monoclonal antibody, which is protective against West Nile virus infection. Liposome and heparin binding assays indicate that only the N-terminal region of NS1 mediates interaction with membranes and that sulfate binding sites common to NS1 structures are not glycosaminoglycan binding interfaces. This report highlights several differences between flavivirus NS1 proteins and contributes to our understanding of their structure-pathogenic function relationships.IMPORTANCE JEV is a major cause of viral encephalitis in Asia. Despite extensive vaccination, epidemics still occur. Nonstructural protein 1 (NS1) plays a role in viral replication, and, because it is secreted, it can exhibit a wide range of interactions with host proteins. NS1 sequence and protein folds are conserved within the Flavivirus genus, but variations in NS1 protein-protein interactions among viruses likely contribute to differences in pathogenesis. Here, we compared characteristics of the C-terminal ?-ladder domain of NS1 between flaviviruses, including surface charge, loop flexibility, epitope cross-reactivity, membrane adherence, and glycosaminoglycan binding. These structural features are central to NS1 functionality and may provide insight into the development of diagnostic tests and therapeutics.

Project description:Background: Due to the similarity between the dengue (DENV) and the Japanese encephalitis virus (JEV) there is potential for immune cross-reaction. We sought to identify T cell epitopes that are specific to JEV and do not cross react with DENV. Methodology: 20mer peptides were synthesized from regions which showed >90% conservation. Using IFNγ cultured ELISpot assays, we investigated JEV-specific T cell responses in DENV- and JEV- non-immune individuals (DENV-JEV- = 21), JEV seronegative and had not received the JE vaccine, but who were DENV seropositive (DENV+JEV- = 22), JEV+(seropositive for JEV and had received the JE vaccine), but seronegative for DENV (DENV-JEV+ = 23). We further assessed the responses to these peptides by undertaking ex vivo IFNγ assays and flow cytometry. Results: None of DENV-JEV- individuals responded to any of the 20 JEV-specific peptides. High frequency of responses was seen to 6/20 peptides by individuals who were JEV+ but DENV-, where over 75% of the individuals responded to at least one peptide. P34 was the most immunogenic peptide, recognized by 20/23 (86.9%) individuals who were DENV-JEV+, followed by peptide 3 and peptide 7 recognized by 19/23 (82.6%). Peptide 34 from the NS2a region, showed <25% homology with any flaviviruses, and <20% homology with any DENV serotype. Peptide 20 and 32, which were also from the non-structural protein regions, showed <25% homology with DENV. Ex vivo responses to these peptides were less frequent, with only 40% of individuals responding to peptide 34 and 16-28% to other peptides, probably as 5/6 peptides were recognized by CD4+ T cells. Discussion: We identified six highly conserved, T cell epitopes which are highly specific for JEV, in the Sri Lankan population. Since both JEV and DENV co-circulate in the same regions and since both JE and dengue vaccines are likely to be co-administered in the same geographical regions in future, these JEV-specific T cell epitopes would be useful to study JEV-specific T cell responses, in order to further understand how DENV and JEV-specific cellular immune responses influence each other.

Dataset Information

Structural and molecular analyses of functional epitopes and escape mutants in Japanese encephalitis virus envelope protein domain III.

Publications

Structural and molecular analyses of functional epitopes and escape mutants in Japanese encephalitis virus envelope protein domain III.

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