Université de La Réunion, INSERM U1187, CNRS UMR 9192, IRD UMR 249, Unité Mixte Processus Infectieux en Milieu Insulaire Tropical, Plateforme Technologique CYROI, 97491 Sainte-Clotilde, La Réunion, France.
Int J Mol Sci. 2019 Mar 29;20(7):1574. doi: 10.3390/ijms20071574.
Interferon-induced viperin (VP) was identified as playing an important role in the innate immune response against Zika virus (ZIKV). The 361 amino acid long human VP protein comprises of a highly conserved C-terminal region, which has been associated with VP antiviral properties against ZIKV. In the present study, we sought to determine whether the very last C-terminal amino-acid residues of VP might play a role in VP-mediated ZIKV inhibition. To address this issue, a recombinant human viperin (rVP) was overexpressed by transfection in human epithelial A549 cells. We confirmed that transient overexpression of rVP prior to ZIKV infection dramatically reduced viral replication in A549 cells. Deletion of the last 17 C-terminal amino acids of VP resulted in a higher expression level of mutant protein compared to wild-type VP. Mutational analysis revealed that residue substitution at positions 356 to 360 with five alanine led to the same phenotype. The charged residues Asp356, Lys358, and Asp360 were then identified to play a role in the weak level of VP protein in A549 cells. Mutant VP bearing the D360A substitution partially rescued ZIKV growth in A549 cells. Remarkably, a single Lys-to-Arg substitution at position 358 was sufficient to abrogate VP antiviral activity against ZIKV. In conclusion, our study showed that Asp356, Lys358, and Asp360 may have an influence on biochemical properties of VP. Our major finding was that Lys358 was a key amino-acid in VP antiviral properties against ZIKV.
干扰素诱导的 viperin（VP）被认为在针对寨卡病毒（ZIKV）的先天免疫反应中发挥重要作用。人 VP 蛋白由 361 个氨基酸组成，包含一个高度保守的 C 末端区域，该区域与 VP 针对 ZIKV 的抗病毒特性有关。在本研究中，我们试图确定 VP 的最后一个 C 末端氨基酸残基是否在 VP 介导的 ZIKV 抑制中发挥作用。为了解决这个问题，通过转染在人上皮 A549 细胞中过表达重组人 viperin（rVP）。我们证实，在 ZIKV 感染之前瞬时过表达 rVP 可显著降低 A549 细胞中的病毒复制。与野生型 VP 相比，VP 的最后 17 个 C 末端氨基酸缺失导致突变蛋白的表达水平更高。突变分析表明，在位置 356 到 360 用五个丙氨酸取代残基导致相同的表型。然后确定带负电荷的残基 Asp356、Lys358 和 Asp360 在 A549 细胞中 VP 蛋白低水平表达中起作用。携带 D360A 取代的突变 VP 部分挽救了 A549 细胞中的 ZIKV 生长。值得注意的是，位置 358 处的单个 Lys 到 Arg 取代足以消除 VP 对 ZIKV 的抗病毒活性。总之，我们的研究表明，Asp356、Lys358 和 Asp360 可能对 VP 的生化特性有影响。我们的主要发现是 Lys358 是 VP 针对 ZIKV 的抗病毒特性的关键氨基酸。
