Flavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly. ) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (1). The structural proteins form the virus particle, while the nonstructural proteins function in viral RNA replication, virion assembly, and evasion of the host antiviral immune responses (2-5). Among them, NS3 is a multifunctional protein with an N-terminal protease domain and a C-terminal RNA helicase/NTPase domain. The NS3 protease activity requires NS2B as a cofactor (NS2B-NS3 pro ) and is responsible for cleaving the C terminus of mature capsid protein, as well as the junctions of NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 (6).
IMPORTANCE
Many flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is es-NS2B is a small integral membrane protein (approximately 130 amino acids) with a molecular mass of 14 kDa. It contains a conserved central hydrophilic region (amino acids 51 to 95 in JEV) and three hydrophobic regions that are predicted to be transmembrane domains (TMDs) (7,8). It has been demonstrated that the central hydrophilic region of NS2B is necessary and sufficient for the activation of NS3 protease, and mutations in this region could affect protease activities and NS3 stability, resulting in defects of viral replication (1, 7, 9-13). Its hydrophobic TMDs are generally considered to help the NS2B-NS3 pro complex anchor into the host endoplasmic reticulum (ER) membranes for efficient
