Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
J Virol. 2014 Jun;88(12):6714-28. doi: 10.1128/JVI.00530-14. Epub 2014 Apr 2.
Viruses modulate cellular signaling pathways at almost every step of the infection cycle. Cellular signaling pathways activated at later times of influenza infection have previously been investigated; however, early influenza virus-host cell interactions remain understudied. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that regulates phosphatidylinositol 3-kinase (PI3K) activation and actin reorganization, two critical processes during influenza A virus (IAV) infection in most cell types. Using 6 influenza A virus strains (A/Puerto Rico/8/1934, A/Aichi/2/1968 × A/Puerto Rico/8/1934 reassortant [X-31], A/California/04/2009, mouse-adapted A/California/04/2009, A/WSN/1933, and A/New Caledonia/20/1999), we examined the role of FAK during IAV entry. We found that influenza virus attachment induced PI3K-dependent FAK-Y397 phosphorylation. Pharmacological FAK inhibition or expression of a kinase-dead mutant of FAK led to disruption of the actin meshwork that resulted in sequestration of IAV at the cell periphery and reduced virion localization to early endosomes. Additionally, FAK inhibition impeded viral RNA replication at later times of infection and ultimately resulted in significantly reduced viral titers in both A549 and differentiated normal human bronchial epithelial (NHBE) cells. Although not all tested strains activated FAK, all of them exhibited a reduction in viral replication in response to inhibition of FAK signaling. These findings highlight novel biphasic roles of FAK activation during IAV infection and indicate that FAK serves as a central link between receptor-mediated PI3K activation and actin reorganization during IAV infection.
We found that FAK links early activation of PI3K and actin reorganization, thereby regulating influenza virus entry. Surprisingly, we also found that FAK can regulate viral RNA replication independently of its role in entry. Our study addresses a knowledge gap in the understanding of signaling events triggered by influenza virus that mediate its internalization and initiation of the infection cycle. Understanding of these fundamental molecular events will be necessary to identify novel host targets, such as FAK, and development of future anti-influenza virus therapeutics.
病毒在感染周期的几乎每个步骤中都调节细胞信号通路。先前已经研究了在流感感染后期被激活的细胞信号通路；然而，流感病毒与宿主细胞的早期相互作用仍未得到充分研究。粘着斑激酶（FAK）是一种细胞质酪氨酸激酶，可调节磷酸肌醇 3-激酶（PI3K）的激活和肌动蛋白重组，这是大多数细胞类型中甲型流感病毒（IAV）感染的两个关键过程。我们使用了 6 株流感病毒（A/Puerto Rico/8/1934、A/Aichi/2/1968×A/Puerto Rico/8/1934 重组体[X-31]、A/California/04/2009、鼠适应的 A/California/04/2009、A/WSN/1933 和 A/New Caledonia/20/1999），研究了 FAK 在 IAV 进入过程中的作用。我们发现，流感病毒附着诱导了 PI3K 依赖性的 FAK-Y397 磷酸化。使用药理学 FAK 抑制或表达 FAK 的激酶失活突变体，导致肌动蛋白网格的破坏，从而导致 IAV 被隔离在细胞外周，并减少病毒粒子定位到早期内体。此外，FAK 抑制在感染后期阻碍了病毒 RNA 的复制，最终导致 A549 和分化的正常人类支气管上皮（NHBE）细胞中的病毒滴度显著降低。虽然并非所有测试的菌株都能激活 FAK，但它们都表现出对 FAK 信号转导抑制的病毒复制减少。这些发现突出了 FAK 在 IAV 感染过程中的新型双相激活作用，并表明 FAK 是受体介导的 PI3K 激活和肌动蛋白重组在 IAV 感染过程中的中心联系。
我们发现 FAK 连接了 PI3K 的早期激活和肌动蛋白的重组，从而调节了流感病毒的进入。令人惊讶的是，我们还发现 FAK 可以独立于其在进入中的作用来调节病毒 RNA 的复制。我们的研究解决了流感病毒引发的信号事件理解中的一个知识空白，这些信号事件介导了其内化和感染周期的启动。对这些基本分子事件的理解对于确定新型宿主靶点（如 FAK）以及开发未来的抗流感病毒治疗方法是必要的。
