Membrane topology of NS2B of dengue virus revealed by NMR spectroscopy

Li, Yan; Li, Qingxin; Wong, Yiik Diew; Liew, Lynette; Kang, CongBao

doi:10.1016/j.bbamem.2015.06.010

Cited by 67 publications

(64 citation statements)

References 56 publications

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“…These well-conserved residues have been proposed to be essential for RNA replication. In agreement with this observation, sequence alignment of the amino acid residues of the nonstructural proteins among DENV2, WNV KUN , YFV, and JEV revealed that NS1, NS2B, NS3, NS4A, NS4B, and NS5 were variably conserved (33,14,40,22,15, and 51% identity, respectively), while NS2A was the least conserved (8% identity) (data not shown). Similarly, strong conservation was observed in the NS1, NS2B, NS3, NS4A, NS4B, and NS5 (61, 42, 67, 53, 73, and 67% identity, respectively) proteins among the four dengue virus serotypes, while NS2A was the least conserved (18% identity).…”

Section: Discussionsupporting

confidence: 55%

“…10. Interestingly, the second dominant reversion mutation, NS2B-I114T, is located in the fourth pTMS close to the ER lumen side (33). Several defective NS2A mutants located in pTMS1, pTMS2, pTMS3, and pTMS4 were rescued by NS2B-I114T, implicating the intricate protein complex formed by the pTMSs within NS2A and NS2B.…”

Section: Discussionmentioning

confidence: 98%

“…The results clearly indicated that the NS2A-L181F and NS2B-I114T reversion mutations have different abilities to restore the virus infectivity of defective NS2A mutant viruses. A recent nuclear magnetic resonance study revealed that the membrane topology of DENV NS2B consists of four short helical transmembrane segments (33). The NS2B-I114T reversion mutation is located at the fourth pTMS close to the ER lumen side.…”

Section: Discussionmentioning

confidence: 99%

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

Tsai

et al. 2017

J Virol

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The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMS1 to -8) and participates in RNA replication, virion assembly, and host antiviral response. However, the roles of specific amino acid residues within the pTMS regions of NS2A during the viral life cycle are not clear. Here, we explore the function of DENV NS2A by introducing a series of alanine substitutions into the N-terminal half (pTMS1 to -4) of the protein in the context of a DENV infectious clone or subgenomic replicon. Six NS2A mutants (NM5, -7, -9, and -17 to -19) around pTMS1 and -2 displayed a novel phenotype showing a Ͼ1,000-fold reduction in virus yield, an absence of plaque formation despite wild-type-like replicon activity, and infectious-virus-like particle yields. HEK-293 cells infected with the six NS2A mutant viruses failed to cause a virus-induced cytopathic effect (CPE) by MitoCapture staining, cell proliferation, and lactate dehydrogenase release assays. Sequencing analyses of pseudorevertant viruses derived from lethal-mutant viruses revealed two consensus reversion mutations, leucine to phenylalanine at codon 181 (L181F) within pTMS7 of NS2A and isoleucine to threonine at codon 114 (I114T) within NS2B. The introduction of an NS2A-L181F mutation into the lethal (NM15, -16, -25, and -33) and CPE-defective (NM7, -9, and -19) mutants substantially rescued virus infectivity and virus-induced CPE, respectively, whereas the NS2B-L114T mutation rescued the NM16, -25, and -33 mutants. In conclusion, the results revealed the essential roles of the N-terminal half of NS2A in RNA replication and virus-induced CPE. Intramolecular interactions between pTMSs of NS2A and intermolecular interactions between the NS2A and NS2B proteins were also implicated. IMPORTANCEThe characterization of the N-terminal (current study) and C-terminal halves of DENV NS2A is the most comprehensive mutagenesis study to date to investigate the function of NS2A during the flaviviral life cycle. A novel region responsible for virus-induced cytopathic effect (CPE) within pTMS1 and -2 of DENV NS2A was identified. Revertant genetics studies implied unexpected relationships between various pTMSs of DENV NS2A and NS2B. These results provide comprehensive information regarding the functions of DENV NS2A and the specific amino acids and transmembrane segments responsible for these functions. The positions and properties of the rescuing mutations were also revealed, providing important clues regarding the manner in which intramolecular or intermolecular interactions between the pTMSs of NS2A and NS2B regulate virus replication, assembly/secretion, and virusinduced CPE. These results expand the understanding of flavivirus replication. The

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Section: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

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

Tsai

et al. 2017

J Virol

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“…Notably, each of the four TMD helices contains a small-XXX-small motif ("small" represents amino acids with small side chains [Gly, Ala, and Ser], and "X" represents any amino acid) that is important for TMD-TMD interactions. Such structural characteristics of NS2B might confer more freedom to induce conformational changes in membranes under different conditions, facilitating interaction with other viral membrane proteins through its TMDs (15). Indeed, using fluorescent resonance energy transfer (FRET)-and biomolecular fluorescent complementation (BiFC)-based imaging methods, Yu et al showed that WNV NS2B could interact with itself, as well as most other NS proteins, including the other three membrane proteins, NS2A, NS4A, and NS4B (16).…”

mentioning

confidence: 99%

“…It is currently unclear how the NS2B TMDs regulate flaviviral replication. A recent nuclear magnetic resonance (NMR) study revealed the membrane topology of DENV4 NS2B (15). The hydrophilic cofactor region forms a ␤-strand structure, while the TMDs contains four short helical segments.…”

mentioning

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

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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

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Structural characterization of the linked NS2B‐NS3 protease of Zika virus

Phoo

Loh

et al. 2017

FEBS Letters

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The Zika virus (ZIKV) NS2B-NS3 protease is an important drug target. The conventional flaviviral protease constructs used for structural studies contain the NS2B cofactor region linked to the NS3 protease domain via a glycine-rich flexible linker. Here, we examined the structural dynamics of this conventional Zika protease (gZiPro) using NMR spectroscopy. Although the glycine-rich linker in gZiPro does not alter the overall folding of the protease in solution, gZiPro is not homogenous in ion exchange chromatography. Compared to the unlinked protease construct, the artificial linker affects the chemical environment of many residues including H51 in the catalytic triad. Our study provides a direct comparison of ZIKV protease constructs with and without an artificial linker.

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Membrane topology of NS2B of dengue virus revealed by NMR spectroscopy

Cited by 67 publications

References 56 publications

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

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

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

Structural characterization of the linked NS2B‐NS3 protease of Zika virus

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