Profiling of viral proteins expressed from the genomic RNA of Japanese encephalitis virus using a panel of 15 region-specific polyclonal rabbit antisera: implications for viral gene expression.
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is closely related to West Nile (WN), yellow fever (YF), and dengue (DEN) viruses. Its plus-strand genomic RNA carries a single open reading frame encoding a polyprotein that is cleaved into three structural (C, prM/M, and E) and at least seven nonstructural (NS1/NS1', NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins, based on previous work with WNV, YFV, and DENV. Here, we aimed to profile experimentally all the viral proteins found in JEV-infected cells. We generated a collection of 15 JEV-specific polyclonal antisera covering all parts of the viral protein-coding regions, by immunizing rabbits with 14 bacterially expressed glutathione-S-transferase fusion proteins (for all nine viral proteins except NS2B) or with a chemically synthesized oligopeptide (for NS2B). In total lysates of JEV-infected BHK-21 cells, immunoblotting with these antisera revealed: (i) three mature structural proteins (~12-kDa C, ~8-kDa M, and ~53-kDa E), a precursor of M (~24-kDa prM) and three other M-related proteins (~10-14 kDa); (ii) the predicted ~45-kDa NS1 and its frameshift product, ~58-kDa NS1', with no evidence of the predicted ~25-kDa NS2A; (iii) the predicted but hardly detectable ~14-kDa NS2B and an unexpected but predominant ~12-kDa NS2B-related protein; (iv) the predicted ~69-kDa NS3 plus two major cleavage products (~34-kDa NS3N-term and ~35-kDa NS3C-term), together with at least nine minor proteins of ~16-52 kDa; (v) the predicted ~14-kDa NS4A; (vi) two NS4B-related proteins (~27-kDa NS4B and ~25-kDa NS4B'); and (vii) the predicted ~103-kDa NS5 plus at least three other NS5-related proteins (~15 kDa, ~27 kDa, and ~90 kDa). Combining these data with confocal microscopic imaging of the proteins' intracellular localization, our study is the first to provide a solid foundation for the study of JEV gene expression, which is crucial for elucidating the regulatory mechanisms of JEV genome replication and pathobiology.
Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.
Jin-Kyoung
JK
Kim
Department of Microbiology, College of Medicine, Chungbuk National University, Cheongju, South Korea.
Jeong-Min
JM
Kim
Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.
Byung-Hak
BH
Song
Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.
Sang-Im
SI
Yun
Department of Microbiology, College of Medicine, Chungbuk National University, Cheongju, South Korea.
Gil-Nam
GN
Yun
Animal Biotechnology Division, Korea National Institute of Animal Science, Suwon, South Korea.
Sung-June
SJ
Byun
Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.
Young-Min
YM
Lee
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D004664:Encephalitis Virus, Japanese / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D007106:Immune Sera / Q000276:immunology*; D007114:Immunization; D018613:Microscopy, Confocal; D011817:Rabbits; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D007106:Immune Sera; D014764:Viral Proteins