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 proteincoding regions, by immunizing rabbits with 14 bacterially expressed glutathione-Stransferase 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 (similar to 12-kDa C, similar to 8-kDa M, and similar to 53-kDa E), a precursor of M (similar to 24-kDa prM) and three other M-related proteins (similar to 10-14 kDa); (ii) the predicted similar to 45-kDa NS1 and its frameshift product, similar to 58-kDa NS1', with no evidence of the predicted similar to 25-kDa NS2A; (iii) the predicted but hardly detectable similar to 14-kDa NS2B and an unexpected but predominant similar to 12-kDa NS2B-related protein; (iv) the predicted similar to 69-kDa NS3 plus two major cleavage products (similar to 34-kDa NS3(N-term) and similar to 35-kDa NS3(C-term)), together with at least nine minor proteins of similar to 016-52 kDa; (v) the predicted similar to 14kDa NS4A; (vi) two NS4B-related proteins (similar to 27-kDa NS4B and similar to 25-kDa NS4B'); and (vii) the predicted similar to 103-kDa NS5 plus at least three other NS5-related proteins (similar to 15 kDa, similar to 27 kDa, and similar to 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.