Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects
Elena Bekerman, … , Glenn Randall, Shirit Einav
Elena Bekerman, … , Glenn Randall, Shirit Einav
Published February 27, 2017
Citation Information: J Clin Invest. 2017;127(4):1338-1352. https://doi.org/10.1172/JCI89857.
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Research Article Therapeutics Virology

Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects

Abstract

Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses.

Authors

Elena Bekerman, Gregory Neveu, Ana Shulla, Jennifer Brannan, Szu-Yuan Pu, Stanley Wang, Fei Xiao, Rina Barouch-Bentov, Russell R. Bakken, Roberto Mateo, Jennifer Govero, Claude M. Nagamine, Michael S. Diamond, Steven De Jonghe, Piet Herdewijn, John M. Dye, Glenn Randall, Shirit Einav

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Figure 1

AP1 and AP2 cotraffic with HCV and orchestrate infection.

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AP1 and AP2 cotraffic with HCV and orchestrate infection. (A) Confirmati...
(A) Confirmation of gene expression knockdown by Western blot in Huh7.5 cells stably expressing AP shRNA or nontargeting control (NT). (B) Entry of HCV pseudoparticles (HCVpp) was measured by luciferase assays at 48 hours after infection. (C) HCV RNA replication measured via luciferase assays 72 hours after HCV RNA electroporation. (D) HCV infectivity measured via luciferase assays by inoculation of naive cells with lysates (intracellular) and supernatants (extracellular) from electroporated cells. (E) AP1 ectopic expression following transfection of Huh7.5 cells with GLuc-tagged WT, T144A AP1, or an empty control; blotted with anti-GLuc antibody. (F) HCV intra- and extracellular infectivity in AP1-overexpressing cells versus control. Shown are means ± SD (n = 3–10). (G) Representative live cell fluorescence microscopy montages of TC-core HCV (green) cotrafficking with AP1- and AP2-mCherry (red). Distance traveled (μm) and time elapsed (min:s) during video acquisition are indicated. (H) Quantification of motile TC-core puncta cotrafficking with AP1, AP2, and LC3. (I) Quantification of distance traveled per acquisition of WT or Y136A mutant TC-core HCV associated with AP2. (J) Quantification of distance traveled per acquisition of TC-core HCV associated with AP1 or AP2 upon treatment with sunitinib (4 μM) and erlotinib (10 μM). Results in B–D and F represent data pooled from at least 2 independent experiments each with 6–10 biological replicates. H–J are representative experiments out of at least 3 conducted. Shown are means ± SD; ***P < 0.001 relative to corresponding NT (B–D), empty vector control (F), WT TC-core (I), or vehicle control (J) by 1-way ANOVA, followed by Dunnett’s (B, D, and J) or Tukey’s (F) multiple comparisons test or 2-tailed unpaired t test (I).