Wu Ren-Huang, Tsai Ming-Han, Chao Day-Yu, Yueh Andrew
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China.
J Virol. 2015 Apr;89(8):4281-95. doi: 10.1128/JVI.03011-14. Epub 2015 Feb 4.
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.
The 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.
登革病毒（DENV）的NS2A蛋白有八个预测的跨膜区段（pTMSs；pTMS1至pTMS8）。NS2A已被证明参与RNA复制、病毒粒子组装和宿主抗病毒反应。然而，在病毒生命周期中，NS2A的pTMS区域内氨基酸残基的作用仍知之甚少。在本文所述的研究中，我们通过在DENV感染性克隆或亚基因组复制子的背景下，在NS2A的C端一半（pTMS4至pTMS8）引入一系列双丙氨酸或三丙氨酸取代，来探究DENV NS2A的功能。35个NS2A突变体中有14个（pTMS8内有8个）由于复制子分析显示RNA复制受损而表现出致死表型。三个在pTMS7内有突变的NS2A突变体，即CM20、CM25和CM27突变体，表现出相似的表型，病毒产量低（>100倍降低）、野生型样复制子活性以及通过瞬时转包装实验得到的感染性病毒样颗粒产量低，这表明在病毒组装和分泌方面存在缺陷。从CM20、CM25和CM27突变病毒衍生的回复病毒测序显示，在pTMS7内的第181密码子处存在一个一致的回复突变，即亮氨酸（L）突变为苯丙氨酸（F）。将L181F突变引入全长NS2A突变体，即CM20、CM25和CM27构建体中，完全恢复了野生型感染性。值得注意的是，L181F也显著挽救了其他在pTMS4、pTMS6和pTMS8内有突变的严重RNA复制缺陷突变体，即CM2、CM3、CM13、CM31和CM32突变体。总之，结果揭示了NS2A的pTMS4至pTMS8在RNA复制和/或病毒组装及分泌中的重要作用。NS2A蛋白的pTMS7与pTMS4、pTMS6或pTMS8之间的分子内相互作用也被涉及。
所报道的登革病毒NS2A C端一半的特征是第一项全面的诱变研究，旨在探究黄病毒NS2A在病毒生命周期各步骤中的功能。特别是，对NS2A预测跨膜区段（pTMSs）内参与RNA复制和/或病毒组装及分泌的氨基酸残基进行了详细定位。一项回复遗传学研究还表明，pTMS7内的L181F是一个一致的回复突变，可挽救NS2A其他三个pTMS内有突变的RNA复制缺陷型以及病毒组装和分泌缺陷型突变体。总体而言，这些发现阐明了NS2A在病毒生命周期中所起的作用，可能是通过NS2A蛋白内pTMS7与其他pTMS之间复杂的分子内相互作用。
