The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion

Abstract

NS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using a trans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core ␤-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core ␤-ladder domain. IMPORTANCEThe positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core ␤-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice, despite having retained the brain replication ability observed in wild-type JEV. Mother dams immunized with recombinant JEV expressing EV71 epitope-NS1 fused proteins elicited neutralizing antibodies that protected the newborn mice against lethal EV71 challenge. Together, our results implied a potential application of JEV NS1 as a viral carrier protein to express a heterologous epitope to stimulate dual/multiple protective immunity concurrently against several pathogens. M osquito-borne flaviviruses comprise several medically important viruses, including Japanese encephalitis virus (JEV), West Nile virus (WNV), dengue virus (DENV), and yellow fever virus (YFV). Flaviviruses have a single-stranded positive-sense RNA genome with only a single open reading frame (ORF), from which polyprotein precursors are translated and processed by cellular signalase and/or viral protease in the cytoplasm of infected cells, resulting in three structural proteins, capsid (C), membrane (prM/M), and envelope (E), and seven nonstructural proteins (NS), NS1, NS2A, NS2B, NS3, NS4A, NS4...

Japanese Encephalitis Virus: An Update on the Potential Antivirals and Vaccines

“…However, none of the siRNA were carried forward for clinical development. Furthermore, a study by Yen et al, has indicated that NS1 acts as a heterologous epitope and induces a cross-reactive immune response against various pathogens [24]. The NS2A protein has direct involvement in the viral RNA genome synthesis and its assembly and NS2B forms the complex with NS3 and helps in the viral protease activity.…”

Japanese Encephalitis Virus: An Emerging Threat in the Indo-Pacific Region

“…However, none of the siRNA was carried forward for clinical development. Also, a study by Yen et al, 2015, indicated that NS1 acts as a heterologous epitope and induces a cross-reactive immune response against various pathogens [25]. However, the NS2A protein has direct involvement in the viral RNA genome synthesis and its assembly.…”

Flavivirus NS1: a multifaceted enigmatic viral protein

“…The association of NS1 with the membrane and replication complex is mediated through these three distinct structural domains. Yen et al [ 14 ] reported the flexible nature of core β-ladder NS1 protein. JEV NS1 has been reported as a natural viral protein carrier for expressing the heterologous epitopes to stimulate the immune response against various pathogens [ 14 ].…”