Department of Microbiology, University of Iowa, Iowa City, IA, USA.
J Virol. 2013 Jan;87(2):746-55. doi: 10.1128/JVI.01634-12. Epub 2012 Oct 31.
Identification of host factors that are needed for Zaire Ebolavirus (EBOV) entry provides insights into the mechanism(s) of filovirus uptake, and these factors may serve as potential antiviral targets. In order to identify novel host genes and pathways involved in EBOV entry, gene array findings in the National Cancer Institute's NCI-60 panel of human tumor cell lines were correlated with permissivity for EBOV glycoprotein (GP)-mediated entry. We found that the gene encoding the γ2 subunit of AMP-activated protein kinase (AMPK) strongly correlated with EBOV transduction in the tumor panel. The AMPK inhibitor compound C inhibited infectious EBOV replication in Vero cells and diminished EBOV GP-dependent, but not Lassa fever virus GPC-dependent, entry into a variety of cell lines in a dose-dependent manner. Compound C also prevented EBOV GP-mediated infection of primary human macrophages, a major target of filoviral replication in vivo. Consistent with a role for AMPK in filovirus entry, time-of-addition studies demonstrated that compound C abrogated infection when it was added at early time points but became progressively less effective when added later. Compound C prevented EBOV pseudovirion internalization at 37°C as cell-bound particles remained susceptible to trypsin digestion in the presence of the inhibitor but not in its absence. Mouse embryonic fibroblasts lacking the AMPKα1 and AMPKα2 catalytic subunits were significantly less permissive to EBOV GP-mediated infection than their wild-type counterparts, likely due to decreased macropinocytic uptake. In total, these findings implicate AMPK in macropinocytic events needed for EBOV GP-dependent entry and identify a novel cellular target for new filoviral antivirals.
鉴定扎伊尔埃博拉病毒（EBOV）进入所需的宿主因素可深入了解丝状病毒摄取的机制，这些因素可能成为潜在的抗病毒靶点。为了鉴定新的宿主基因和途径，参与 EBOV 进入，国家癌症研究所 NCI-60 人类肿瘤细胞系面板中的基因阵列发现与 EBOV 糖蛋白（GP）介导的进入的易感性相关。我们发现，编码 AMP 激活蛋白激酶（AMPK）γ2 亚基的基因与肿瘤面板中的 EBOV 转导强烈相关。AMPK 抑制剂化合物 C 抑制传染性 EBOV 在 Vero 细胞中的复制，并以剂量依赖的方式减弱 EBOV GP 依赖性但不减弱拉萨热病毒 GPC 依赖性进入各种细胞系。化合物 C 还可防止 EBOV GP 介导的原代人巨噬细胞感染，这是体内丝状病毒复制的主要靶标。与 AMPK 在丝状病毒进入中的作用一致，添加时间研究表明，当化合物 C 在早期添加时，它会破坏感染，但随着时间的推移，其效果会逐渐降低。化合物 C 可防止 EBOV 假病毒内吞作用在 37°C 时发生，因为在抑制剂存在下，细胞结合的颗粒仍然易受胰蛋白酶消化，但在其不存在时则不会。缺乏 AMPKα1 和 AMPKα2 催化亚基的小鼠胚胎成纤维细胞对 EBOV GP 介导的感染的允许性明显低于其野生型对应物，这可能是由于巨胞饮作用摄取减少所致。总的来说，这些发现表明 AMPK 参与 EBOV GP 依赖性进入所需的巨胞饮事件，并鉴定出新型丝状病毒抗病毒药物的新细胞靶标。
