West Nile virus NS2A protein facilitates virus-induced apoptosis independently of interferon response

Melian, Ezequiel Balmori; Edmonds, Judith H.; Nagasaki, Tomoko; Hinzman, Edward; Floden, Nadia; Khromykh, Alexander A.

doi:10.1099/vir.0.047076-0

Cited by 38 publications

(32 citation statements)

References 30 publications

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“…Interestingly, no apparent CPE was found in HEK-293 cells transiently transfected with a plasmid harboring a WT DENV NS2A gene driven by the CMV promoter alone. A similar observation was also reported in the overexpression of WNV KUN NS2A, suggesting the other viral proteins in the replication complex are possibly required for NS2A protein to cause CPE of cells (28). Thus, we believe that the DENV NS2A protein may facilitates virus-induced cell death by inducing apoptosis through intrinsic and/or extrinsic apoptotic signaling cascades.…”

Section: Discussionsupporting

confidence: 55%

“…1B). The CPE-defective phenotypes of the NM5, NM7, NM9, NM17, NM18, and NM19 mutants are similar to that of the A30P WNV KUN NS2A CPE-defective mutant, which failed to induce apoptosis in Vero cells (28). The WNV KUN A30P mutant virus was able to form plaques in BHK21 cells and displayed no apparent reduction in virus yield compared to WT WNV KUN .…”

Section: Discussionmentioning

confidence: 59%

“…An A30P mutation in the N-terminal half of WNV KUN attenuated virus virulence in mice and inhibited the ability of the virus to downregulate type I interferon production (21). Although this mutation did not appear to affect virus yields, the WNV KUN A30P mutant virus failed to cause a virus-induced CPE independently of interferon, which is a novel and mysterious phenotype (28). Despite the fact that several studies have evaluated the NS2A protein, as discussed above, the regions within the N-terminal half of the flavivirus NS2A protein that are essential for the various stages of the viral life cycle remain poorly defined.…”

Section: Importancementioning

confidence: 97%

See 2 more Smart Citations

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: 59%

Section: Importancementioning

confidence: 97%

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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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“…A second-site reversion mutation at position 149 (from threonine to proline [T149P]) in KUNV NS2A could rescue the defect in virus assembly and secretion and virus production of virus mutants bearing the NS2A-I59N mutation, suggesting a potential interaction between codons 59 and 149. Several lines of evidence have shown that flavivirus NS2A is also involved in the interferon response (15,(23)(24)(25)(26) and virus-induced cytopathic effects (CPEs) (27,28). Recently, the topological study of DENV NS2A suggested that NS2A has eight predicted transmembrane segments (pTMSs; pTMS1 to pTMS8) and five integral transmembrane segments (pTMS3, pTMS4, pTMS6 to pTMS8) that span the lipid bilayer of the endoplasmic reticulum (ER) membrane.…”

mentioning

confidence: 99%

Scanning Mutagenesis Studies Reveal a Potential Intramolecular Interaction within the C-Terminal Half of Dengue Virus NS2A Involved in Viral RNA Replication and Virus Assembly and Secretion

Tsai

Chao³

et al. 2015

J Virol

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The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMSs; pTMS1 to pTMS8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. In the study described here, we explored the function of DENV NS2A by introducing a series of double or triple alanine substitutions into the C-terminal half (pTMS4 to pTMS8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (8 within pTMS8) of 35 NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by a replicon assay. Three NS2A mutants with mutations within pTMS7, the CM20, CM25, and CM27 mutants, displayed similar phenotypes, low virus yields (>100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transient trans-packaging experiments, suggesting a defect in virus assembly and secretion. The sequencing of revertant viruses derived from CM20, CM25, and CM27 mutant viruses revealed a consensus reversion mutation, leucine (L) to phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, CM25, and CM27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants with mutations within pTMS4, pTMS6, and pTMS8, i.e., the CM2, CM3, CM13, CM31, and CM32 mutants. In conclusion, the results revealed the essential roles of pTMS4 to pTMS8 of NS2A in RNA replication and/or virus assembly and secretion. The intramolecular interaction between pTMS7 and pTMS4, pTMS6, or pTMS8 of the NS2A protein was also implicated. IMPORTANCEThe reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly and secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication-defective and virus assembly-and secretion-defective mutants with mutations within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein.M ost flaviviruses are transmitted by mosquito or tick vectors and cause serious human and animal diseases (1, 2). Certain members of the genus Flavivirus are reemerging global health threats, especially in urban areas of developing countries (3). Flaviviruses include dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yello...

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“…Although the precise mechanisms by which wnv induces apoptosis are not completely understood, it has been shown that viral proteins like ns3 and Capsid c induce apoptosis by activating caspases 8 and 3 [41,42]. Also, Balmori et al [43] have suggested a possible role of the protein ns2a in the ifn-independent apoptotic cell death. The authors have shown how a single amino acid (alanine or lysine) might control the cytopathic effect and apoptosis in the absence of ifn-α/β response.…”

Section: Immunopathogenesismentioning

confidence: 99%

Immunopathogenesis of West Nile Virus infection

Diosa-Toro

Hernández-López

2015

Revista Curare

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Abstract. The reappearance of West Nile virus (wnv) infection in the last years has highlighted that arthropod-borne diseases are not circumscribed to tropical regions of the world. wnv is maintained in enzootic cycles involving Culex spp. mosquitoes and avian hosts, with epizootic spread to mammals, including humans. Human infection results in mild symptomatic illness in 25% of cases or neurological disease in less than 1% of infected persons. Additional understandings on how wnv interacts with its hosts are recently emerging; the virus exploits immune system, both at the peripheral tissues and the central nervous system, which could explain the differences in virulence, progression and severity of wnv infection. The continuing spread of wnv, combined with the lack of specific therapeutics or vaccines to combat or prevent infection, imparts a pressing need to identify the viral and host processes that control the outcome and immunity to wnv infection. Here, we provide an overview of a subset of information regarding the immune-pathological response generated during wnv infection. Keywords: cytokines, viral infection, inflammation, T cells. Inmunopatogénesis de la infección por el virus del Nilo OccidentalResumen. La reaparición de la infección por el virus del Nilo Occidental (vno) en los últimos años ha puesto de manifiesto que las enfermedades transmitidas por artrópodos no se circunscriben a las regiones tropicales del mundo. El vno se mantiene en ciclos enzoóticos que involucran mosquitos del género Culex spp. y hospederos aviares, con distribución epizoótica en los mamíferos, incluidos los seres humanos. La infección en seres humanos ocasiona enfermedad sintomática leve en el 25% de los casos o enfermedad neurológica en menos del 1% de las personas infectadas. Recientemente, han surgido otros entendimientos sobre la forma en que el vno interactúa con sus hospederos; el virus se aprovecha del sistema inmune, tanto en los tejidos periféricos y el sistema nervioso central, lo que podría explicar las diferencias en la virulencia, evolución y gravedad de la infección por el vno. La continua propagación del vno, junto con la falta de terapias o vacunas específicas para combatir o prevenir la infección, genera la necesidad de identificar los procesos virales y del hospedero que determinan el pronóstico y la inmunidad a la infección por el vno. En el presente artículo, ofrecemos una visión general de información con respecto a la respuesta inmunopatológica generada durante la infección por el vno.Palabras clave: citoquinas, infección viral, inflamación, linfocitos T. Imunopatogênese da infeção pelo vírus do Nilo OcidentalResumo. O ressurgimento da infecção pelo Vírus do Nilo Ocidental (vno) nos últimos anos tem demonstrado que as doenças transmitidas por artrópodes não estão vinculadas às regiões tropicais do mundo. O vno mantém-se em ciclos enzoóticos que envolvem mosquitos do gêne-ro Culex spp. e hospedeiros aviares, com distribuição epizoótica nos mamíferos, incluídos os seres humanos. A infecção nestes ocasion...

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West Nile virus NS2A protein facilitates virus-induced apoptosis independently of interferon response

Cited by 38 publications

References 30 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

Scanning Mutagenesis Studies Reveal a Potential Intramolecular Interaction within the C-Terminal Half of Dengue Virus NS2A Involved in Viral RNA Replication and Virus Assembly and Secretion

Immunopathogenesis of West Nile Virus infection

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