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

Abstract

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 NS3 N-term and ∼35-kDa NS3 C-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.