Transmembrane Domains of NS2B Contribute to both Viral RNA Replication and Particle Formation in Japanese Encephalitis Virus

Abstract

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

Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions between NS2A and NS2B Transmembrane Segments

“…Previously, fluorescent resonance energy transfer-and biomolecular fluorescent complementation (BiFC)-based imaging methods were applied to show that WNV NS2B could interact with itself, as well as most other NS proteins, e.g., NS2A, NS4A, and NS4B (34). Recently, the interaction between NS2A and NS2B of JEV was also shown by the BiFc-based imaging methodology (35). Clearly, our virus genetics results for the first time provided genetics results regarding the interaction between DENV NS2A and NS2B, possibly through intricate protein-protein interactions between pTMSs within DENV NS2A and NS2B.…”

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“…NS3 interacts with the ER-resident hydrophobic integral membrane protein, NS2B (4-8; reviewed in references 2 and 9) and forms an active serine protease (NS2B-NS3pro). The viral protease in conjunction with the ER resident cellular proteases is involved in the processing of the polyprotein precursor into mature proteins prior to viral replication and viral assembly (10). The C-terminal region of NS3 exhibits ATP-dependent RNA helicase and 5= RNA triphosphatase activities which are involved in viral replication (11)(12)(13) and in the first step in 5= RNA capping (14,15).…”

“…The N-terminal region of NS5 has the guanylyltransferase, the N-7 and 2=-O-methyltransferase activities (17)(18)(19) that are essential for the formation of type 1 cap at the 5= end. The C-terminal region of NS5 codes for RNA-dependent RNA polymerase activity involved in RNA replication (10,(20)(21)(22). NS2A, NS2B, NS4A, and NS4B contain transmembrane domains (TMDs) that penetrate the ER.…”

“…NS2B has three TMDs; one is located at the N terminus, and two are at the C terminus of NS2B (8,26,27; reviewed in reference 9). TMDs in NS2B contribute to both viral RNA replication and virion assembly (10). In addition, two TMDs also exist in E protein that are essential for virion assembly in the ER (28).…”

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“…Such proteins are present in a variety of RNA viruses and perform different functions. For example, in flaviviruses, small membrane proteins NS2B and NS4A (with sizes similar to the nuORF) perform multiple functions, ranging from enzyme cofactors (38,39) to participation in particle formation (40,41) and protection against the cellular antiviral response. Influenza A M2 protein acts as a proton-selective ion channel (42), whereas Coronavirus E protein acts as a viroporin and plays a role in the induction of the regulation of the unfolded protein response (43).…”