AMP-Activated Protein Kinase Is Required for the Macropinocytic Internalization of Ebolavirus

Abstract

c 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 ␥2 subunit of AMPactivated 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 AMPK␣1 and AMPK␣2 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.

Suppression of Kaposi's Sarcoma-Associated Herpesvirus Infection and Replication by 5′-AMP-Activated Protein Kinase

“…KSHV entry into cells is primarily through clathrin-mediated endocytosis in HUVEC and fibroblasts (34,46) and macropinocytosis in dermal microvascular endothelial cells (DMVEC) (47). Because other reports showed that AMPK activation enhanced macropi- nocytosis (14,15), we examined the role of AMPK activation on KSHV entry and infection. We found that inhibition of AMPK activation by either AMPK␣1 knockdown or the use of inhibitor compound C did not affect KSHV entry and trafficking.…”

“…Several other RNA viruses, including Sindbis virus (SINV), vesicular stomatitis virus (VSV), and Kunjin virus (KUNV), which depend on cellular membrane modifications and fatty acid synthesis, are also restricted by AMPK (13). In contrast to RNA viruses, DNA viruses Zaire Ebolavirus and vaccinia virus rely on the AMPK activity for actin polymerization and the induction of macropinocytosis during entry (14,15).…”

Ebola Virus Potential Drug Targets and Prospects for Small Molecule Drug Discovery

“…Molecular modeling studies using the crystal structure of EBOV GP as well as mutational analysis suggest a hydrophobic pocket at or near the interface of GP1 and GP2 as a hypothetical binding site for compound 11 (Basu et al, 2011). The AMP-activated protein kinase (AMPK) inhibitor, dorsomorphin (12), has been demonstrated to inhibit Ebola virus entry and infection in vitro, indicating role of host AMPK in EBOV infection and its potential as a target for antiviral drug development (Kondratowicz et al, 2013). p38 MAP kinase is also thought to play an important role in EBOV entry since inhibitors of the enzyme have been demonstrated to impair viral entry in human dendritic cells .…”

EPS8 facilitates uncoating of influenza A virus

“…The National Cancer Institute-60 (NCI-60) panel consists of 59 distinct cell lines with well-characterized transcriptomic profiles (Shankavaram et al, 2007;Weinstein and Pommier, 2003). The diversity of cell types and the depth of transcriptomic data permit high confidence genome-wide correlations between cellular gene expression and infection susceptibility (Kondratowicz et al, 2013;Lenaerts et al, 2012;Schowalter et al, 2012). We therefore inoculated the NCI-60 panel of cell lines with a singlecycle variant of A/WSN/1933 (H1N1, WSN) encoding GFP (WSN-GFP) ( Figure 1A).…”