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標題: 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
作者: Wu, Ren-Huang
Tsai, Ming-Han
Chao, Day-Yu
Yueh, Andrew
關鍵字: Base Sequence;Blotting, Western;DNA Primers;Dengue Virus;Enzyme-Linked Immunosorbent Assay;Escherichia coli;HEK293 Cells;Humans;Molecular Sequence Data;Mutagenesis;Saccharomyces cerevisiae;Sequence Analysis, DNA;Viral Nonstructural Proteins;Viral Plaque Assay;Virus Assembly;Virus Replication
Project: Journal of virology, Volume 89, Issue 8, Page(s) 4281-95.
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.
DOI: 10.1128/JVI.03011-14
Appears in Collections:微生物暨公共衛生學研究所

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