Abstract
Nonstructural protein 2A (NS2A) of the Japanese encephalitis virus (JEV) contributes to viral replication and pathogenesis; however, a lack of NS2A-specific antibodies restricts studies on the underlying mechanisms. In this study, we constructed a recombinant JEV with a hemagglutinin (HA)-tagged NS2A (JEV-HA/NS2A/∆NS1') to overcome this challenge. An HA-tag was fused to the N-terminus of NS2A (HA-NS2A) at the intergenic junction between NS1 and NS2A. A peptide linker, "FNG", was added to the N-terminus of HA-tag to ensure correct cleavage between the C-terminus of NS1 and the N-terminus of HA-NS2A. To avoid the side effects of an unwanted NS1' tagged with HA (HA-NS1'), an alanine-to-proline (A30P) substitution was introduced at residue 30 of NS2A to abolish HA-NS1' production. The HA-tag insertion and A30P substitution were stably present in JEV-HA/NS2A/∆NS1' after six passages and did not exhibit any significant effects on viral replication and plaque morphology. Taking advantage of HA-NS2A, we examined the activities of NS2A during JEV infection in vitro using anti-HA antibodies. NS2A was observed to be localized to the endoplasmic reticulum and interact with viral NS2B and NS3 during virus infection. These data suggest that JEV-HA/NS2A/∆NS1' can serve as a model for the analysis of the biological characteristics and functions of NS2A in vitro during JEV infection.