title: 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 β-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 β-ladder domain.
  
  
  OBJECTIVE
  The 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 β-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.
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Li-Chen
authors.initials: LC
authors.lastname: Yen
authors.affiliation: Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Jia-Teh
authors.initials: JT
authors.lastname: Liao
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Hwei-Jen
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Wei-Yuan
authors.initials: WY
authors.lastname: Chou
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Chun-Wei
authors.initials: CW
authors.lastname: Chen
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.
authors.forename: Yi-Ling
authors.initials: YL
authors.lastname: Lin
authors.affiliation: Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, Republic of China National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan, Republic of China chinglen@gmail.com.
authors.forename: Ching-Len
authors.initials: CL
authors.lastname: Liao
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D004664:Encephalitis Virus, Japanese / Q000235:genetics / Q000276:immunology* / Q000502:physiology; D004672:Encephalitis, Japanese / Q000517:prevention & control; D029821:Enterovirus A, Human / Q000235:genetics / Q000276:immunology*; D004769:Enterovirus Infections / Q000517:prevention & control; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005260:Female; D008813:Mice, Inbred ICR; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D016019:Survival Analysis; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014765:Viral Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D014779:Virus Replication
chemical_list: D000939:Epitopes; C100315:NS1 protein, Flavivirus; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D017361:Viral Nonstructural Proteins; D014765:Viral Vaccines