Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
J Virol. 2011 May;85(10):5159-71. doi: 10.1128/JVI.02099-10. Epub 2011 Mar 2.
The envelope (E) of dengue virus (DENV) is a determinant of tropism and virulence. At the C terminus of E protein, there is a stem region containing two amphipathic α-helical domains (EH1 and EH2) and a stretch of conserved sequences in between. The crystal structure of E protein at the postfusion state suggested the involvement of the stem during the fusion; however, the critical domains or residues involved remain unknown. Site-directed mutagenesis was carried out to replace each of the stem residues at the hydrophobic face with an alanine or proline in a DENV serotype 4 (DENV4) precursor membrane (prM)/E expression construct. Most of the 15 proline mutations at either EH1 or EH2 severely affected the assembly of virus-like particles (VLPs). Radioimmunoprecipitation and membrane flotation assays revealed that EH1 mutations primarily affect prM-E heterodimerization and EH2 mutations affect the membrane binding of the stem. Introducing four proline mutations at either EH1 or EH2 into a DENV2 replicon packaging system greatly affects assembly and entry. Moreover, introducing these mutations into a DENV2 infectious clone confirmed the impairment in assembly and infectivity. Sequencing analysis of adaptive mutations in passage 5 viruses revealed a change to a leucine or wild-type residue at the original site, suggesting the importance of maintaining the helical structure. Collectively, these findings suggest that the EH1 and EH2 domains are involved in both assembly and entry steps of the DENV replication cycle; this feature, together with the high degree of sequence conservation, suggests that the stem region is a potential target of antiviral strategies.
登革病毒（DENV）的包膜（E）是决定亲嗜性和毒力的因素。在 E 蛋白的 C 末端，有一个茎区，包含两个两亲性α螺旋结构域（EH1 和 EH2）和其间的一段保守序列。融合后 E 蛋白的晶体结构表明，茎区在融合过程中起作用；然而，涉及的关键结构域或残基仍不清楚。通过定点突变，用丙氨酸或脯氨酸替换 DENV 血清型 4（DENV4）前膜（prM）/E 表达构建体中茎区疏水面的每个残基。EH1 或 EH2 中的 15 个脯氨酸突变中的大多数严重影响病毒样颗粒（VLPs）的组装。放射免疫沉淀和膜漂浮分析表明，EH1 突变主要影响 prM-E 异二聚体化，EH2 突变影响茎的膜结合。在 DENV2 复制子包装系统中引入 EH1 或 EH2 中的四个脯氨酸突变会极大地影响组装和进入。此外，将这些突变引入 DENV2 感染性克隆中证实了组装和感染性受损。传代 5 病毒中的适应性突变的测序分析显示，原始位点处的亮氨酸或野生型残基发生了变化，这表明保持螺旋结构的重要性。总之，这些发现表明 EH1 和 EH2 结构域参与 DENV 复制周期的组装和进入步骤；这种特征以及高度的序列保守性表明，茎区是抗病毒策略的潜在靶点。
