Genetic interaction between NS4A and NS4B for replication of Japanese encephalitis virus

Li, Xiao Dan; Ye, Han-Qing; Deng, Cheng; Liu, Si Qing; Zhang, Hong Lei; Shang, Bao Di; Shi, Pei–Yong; Yuan, Zhi Ming; Zhang, Bo

doi:10.1099/vir.0.000044

Cited by 28 publications

(30 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The identification of this genetic interaction between NS4A and NS4B implies that the two proteins physically associate and that NS4B-K8 and NS4A-T82, -I110, and -I116 are critical determinants for this interaction. A similar genetic interaction between the NS4A C-terminal region and the NS4B N-terminal region was recently reported for JEV (18). Interestingly, replication of an NS4A K79R mutant was impaired but could be partially rescued by the compensatory mutation Y3N in NS4B.…”

Section: ϫ2mentioning

confidence: 61%

“…Concordantly, DENV NS4B was shown to associate with viral proteins NS3 and NS4A (14,16,17). Interestingly, genetic interactions of NS4B with NS3 and NS4A were reported in West Nile virus (WNV) and Japanese encephalitis virus (JEV) infections, respectively (18,19), suggesting that these interaction are conserved among flaviviruses. At least in vitro, DENV NS4B stimulates single-stranded RNA(ssRNA) dissociation from NS3, as well as its helicase activity (16).…”

mentioning

confidence: 83%

See 1 more Smart Citation

A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

Chatel‐Chaix

Fischl

Scaturro

et al. 2015

J Virol

View full text Add to dashboard Cite

Dengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loopmore precisely, amino acid residue Q134 -as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies. IMPORTANCEWith no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target. D engue virus (DENV) is an enveloped plus-strand RNA virus belonging to the genus Flavivirus of the family Flaviviridae. DENV infection causes the most prevalent arthropod-borne viral disease worldwide, with approximately 100 million symptomatic cases per year. In 0.5 to 1% of the cases, the infection manifests with severe symptoms that can deve...

show abstract

Section: ϫ2mentioning

confidence: 61%

mentioning

confidence: 83%

A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

Chatel‐Chaix

Fischl

Scaturro

et al. 2015

J Virol

View full text Add to dashboard Cite

show abstract

“…Numerous studies have examined inhibitors of the flaviviral NS4B protein 143,144 , a highly hydrophobic integral membrane protein 145 . NS4B mediates several interactions with other non-structural viral proteins and host proteins to modulate viral replication 146,147 . Compounds targeting NS4B lacked broad-spectrum antiviral activity, and inhibitory effects were limited to a single virus or even specific serotypes 143,144,148,149 .…”

Section: Ns4b Inhibitorsmentioning

confidence: 99%

Broad-spectrum agents for flaviviral infections: dengue, Zika and beyond

Boldescu

Behnam

Vasilakis³

et al. 2017

Nat Rev Drug Discov

256

217

View full text Add to dashboard Cite

Infections with flaviviruses, such as dengue, West Nile virus, and the recently re-emerging Zika virus are an increasing and probably lasting global risk. This review summarizes and comments on the opportunities for broad-spectrum agents that are active against a range of flaviviruses. Broad-spectrum activity would be particularly desirable as preparatory measure for the next flaviviral epidemic that could emerge from as-yet-unknown or neglected viruses. Potential target sites for broad-spectrum anti-flaviviral compounds include viral proteins and host mechanisms that are exploited by these viruses during entry and replication. A variety of compounds with broad-spectrum antiviral activity have already been identified by target-specific or phenotypic assays. For some other compound classes, broad-spectrum activity can be anticipated because of their mode of action and molecular target(s).

show abstract

“…For the NS2B subcellular localization assay, the plasmids encoding NS2B-eGFP fused proteins were transfected into 293T cells using Lipofectamine 2000 (Invitrogen). After 24 hpt, the cells were fixed with cold (Ϫ20°C) 5% acetone in methanol for 10 min at room temperature and then subjected to IFA using the anti-eGFP rabbit antibody as described previously (22).…”

Section: Methodsmentioning

confidence: 99%

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

Deng

et al. 2016

J Virol

Self Cite

View full text Add to dashboard Cite

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

show abstract

Genetic interaction between NS4A and NS4B for replication of Japanese encephalitis virus

Cited by 28 publications

References 42 publications

A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

Broad-spectrum agents for flaviviral infections: dengue, Zika and beyond

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

Contact Info

Product

Resources

About