Characterization of duck tembusu virus NS2A membrane topology and functional residues in transmembrane domain-3 on viral proliferation

Flavivirus nonstructural protein 2A (NS2A) is a small endoplasmic reticulum (ER)-resident, hydrophobic transmembrane protein that function in viral replication, virion assembly and evasion of the host immune response. Despite previous studies on the role of duck Tembusu virus (DTMUV) NS2A in inhibiting the host immune response, its membrane topology has not been clearly addressed (Zhang et al., 2020; Zhang et al., 2022). Here, we present the first report on the membrane topology model and functional characterization of DTMUV NS2A. Our findings demonstrate that DTMUV NS2A localizes to the endoplasmic reticulum (ER) and associates with viral double-stranded RNA, with a single segment (TMD3, amino acids 72 to 95) spanning the ER membrane. To better delineate the residues in NS2A-TMD3 related to viral properties, specific mutations were introduced to generate DTMUV replicons and infectious cDNA clones. Functional analysis indicates that L77, Q86 and L89 of NS2A are crucial for viral RNA synthesis, while residues M79 and F83 are crucial for the assembly or release of viral particles. Moreover, these mutations attenuated the virulence of DTMUV in vivo. Collectively, our results shed light on the relationship between the transmembrane of DTMUV NS2A and its functions in virus proliferation, providing insights for further understanding the molecular mechanisms of NS2A in the virus life cycle.