Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
Viruses. 2020 Jun 26;12(6):687. doi: 10.3390/v12060687.
Live visualization of influenza A virus (IAV) structural proteins during viral infection in cells is highly sought objective to study different aspects of the viral replication cycle. To achieve this, we engineered an IAV to express a Tetra Cysteine tag (TC tag) from hemagglutinin (HA), which allows intracellular labeling of the engineered HA protein with biarsenic dyes and subsequent fluorescence detection. Using such constructs, we rescued a recombinant IAV with TC tag inserted in HA, in A/Puerto Rico/8/1934(H1N1) background (HA-TC). This recombinant HA-TC tag reporter IAV was replication-competent; however, as compared to wild type PR8 IAV, it was attenuated in multicycle replication. We confirmed expression of TC tag and biarsenical labeling of HA by immunofluorescence assay in cells infected with an HA-TC tag reporter IAV. Further, we used this reporter virus to visualize HA expression and translocation in IAV infected cells by live confocal imaging. We also tested the utility of the HA-TC IAV in testing chemical inhibitors of the HA translocation. Overall, HA-TC IAV is a versatile tool that will be useful for studying viral life cycle events, virus-host interactions, and anti-viral testing.
在细胞内感染病毒期间实时可视化甲型流感病毒 (IAV) 结构蛋白，是研究病毒复制周期不同方面的高度追求目标。为了实现这一目标，我们设计了一种甲型流感病毒，使其在血凝素 (HA) 中表达四半胱氨酸标签 (TC 标签)，这允许用双砷染料对工程化的 HA 蛋白进行细胞内标记，并随后进行荧光检测。使用这些构建体，我们在 A/Puerto Rico/8/1934(H1N1) 背景 (HA-TC) 中拯救了带有 TC 标签插入 HA 的重组 IAV。这种重组 HA-TC 标签报告 IAV 具有复制能力；然而，与野生型 PR8 IAV 相比，它在多轮复制中被削弱。我们通过感染 HA-TC 标签报告 IAV 的细胞中的免疫荧光检测证实了 TC 标签的表达和 HA 的双砷标记。此外，我们使用该报告病毒通过活细胞共焦成像实时可视化 IAV 感染细胞中的 HA 表达和易位。我们还测试了 HA-TC IAV 在测试 HA 易位的化学抑制剂中的效用。总体而言，HA-TC IAV 是一种多功能工具，将有助于研究病毒生命周期事件、病毒-宿主相互作用和抗病毒测试。
