title: Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions between NS2A and NS2B Transmembrane Segments.
abstract: 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 >1,000-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 virus-induced CPE. These results expand the understanding of flavivirus replication. The knowledge may also facilitate studies of pathogenesis and novel vaccine and antiflaviviral drug development.
authors.affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China.
authors.forename: Ren-Huang
authors.initials: RH
authors.lastname: Wu
authors.affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China.
authors.forename: Ming-Han
authors.initials: MH
authors.lastname: Tsai
authors.affiliation: Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan, Republic of China.
authors.forename: Kuen-Nan
authors.initials: KN
authors.lastname: Tsai
authors.affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China.|Department of Life Sciences, National Central University, Jhongli, Taiwan, Republic of China.
authors.forename: Jia Ni
authors.initials: JN
authors.lastname: Tian
authors.affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China.
authors.forename: Jian-Sung
authors.initials: JS
authors.lastname: Wu
authors.affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China.
authors.forename: Su-Ying
authors.initials: SY
authors.lastname: Wu
authors.affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China.
authors.forename: Jyh-Haur
authors.initials: JH
authors.lastname: Chern
authors.affiliation: Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan, Republic of China chunhong@nhri.org.tw andrewyueh@nhri.org.tw.
authors.forename: Chun-Hong
authors.initials: CH
authors.lastname: Chen
authors.affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China chunhong@nhri.org.tw andrewyueh@nhri.org.tw.|Department of Life Sciences, National Central University, Jhongli, Taiwan, Republic of China.
authors.forename: Andrew
authors.initials: A
authors.lastname: Yueh
mesh_terms: D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D049109:Cell Proliferation / Q000235:genetics; D003588:Cytopathogenic Effect, Viral; D003716:Dengue Virus / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007930:Leucine / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics; D012367:RNA, Viral / Q000378:metabolism; D017421:Sequence Analysis; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019065:Virus Assembly; D014779:Virus Replication / Q000235:genetics
chemical_list: C079341:NS2A protein, Dengue virus type 2; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D010649:Phenylalanine; D007770:L-Lactate Dehydrogenase; D007930:Leucine; D000409:Alanine
keywords: RNA replication; cytopathic effect; dengue virus; intermolecular interaction; scanning mutagenesis; transmembrane segment