Digitoxin Suppresses Human Cytomegalovirus Replication via Na ⁺ , K ⁺ /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

Abstract

Host-directed therapeutics for human cytomegalovirus (HCMV) requires elucidation of cellular mechanisms that inhibit HCMV. We report a novel pathway used by cardiac glycosides to inhibit HCMV replication: induction of AMP-activated protein kinase (AMPK) activity and autophagy flux through the Na,K/ATPase α1 subunit. Our data illustrate an intricate balance between the autophagy regulators AMPK, mammalian target of rapamycin (mTOR), and ULK1 during infection and treatment with the cardiac glycoside digitoxin. Both infection and digitoxin induced AMPK phosphorylation, but ULK1 was differentially phosphorylated at unique sites leading to opposing effects on autophagy. Suppression of autophagy during infection occurred via ULK1 phosphorylation at Ser757 by enhanced mTOR activity. Digitoxin continuously phosphorylated AMPK, leading to ULK1 phosphorylation at Ser317, and suppressed mTOR, resulting in increased autophagy flux and HCMV inhibition. In ATG5-deficient human fibroblasts, digitoxin did not inhibit HCMV, supporting autophagy induction as a mechanism for virus inhibition. Drug combination studies with digitoxin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) further confirmed the role of autophagy activation in HCMV inhibition. Individually, each compound phosphorylated AMPK, but their combination reduced autophagy rather than inducing it and was antagonistic against HCMV, resulting in virus replication. The initial ULK1 activation by digitoxin was counteracted by AICAR, which prevented the downstream interaction of Beclin1 and phosphatidylinositol 3-kinase class III (PI3K-CIII), further supporting digitoxin-mediated HCMV inhibition through autophagy. Finally, the α1 subunit was required for autophagy induction, since in α1-deficient cells neither AMPK nor autophagy was activated and HCMV was not inhibited by digitoxin. In summary, induction of a novel pathway (α1-AMPK-ULK1) induces autophagy as a host-directed strategy for HCMV inhibition. Infection with human cytomegalovirus (HCMV) creates therapeutic challenges in congenitally infected children and transplant recipients. Side effects and selection of resistant mutants with the limited drugs available prompted evaluation of host-directed therapeutics. We report a novel mechanism of HCMV inhibition by the cardiac glycoside digitoxin. At low concentrations that inhibit HCMV, digitoxin induced signaling through the α1 subunit of the Na,K/ATPase pump and the cellular kinase AMPK, resulting in binding and phosphorylation of ULK1 (Ser317) and autophagy activation. HCMV suppressed autophagy through ULK1 phosphorylation (Ser757) by activating the mTOR kinase. The pump-autophagy pathway was required for HCMV inhibition, since in α1- or ATG5-deficient cells the virus was not inhibited. Furthermore, the AMPK activator AICAR antagonized digitoxin activity against HCMV, a phenomenon resulting from opposing effects downstream in the autophagy pathway, at the Beclin1 stage. In summary, autophagy may provide a strategy for harnessing HCMV replication.

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function

“…These pathways include mitogen-activated kinase (MAPK) signalling both via extracellular signal-regulated kinase (ERK) 1 and 2 including RAF1 (MAPKKK upstream of ERK) as well as via p38 MAPK [168][169][170][171][172]. Other kinases thought to be involved in the IE phase of HCMV infection include adenosine monophosphate-activated protein kinase (AMPK) [173], hematopoietic cell kinase (a src family kinase) [174], cyclin-dependent kinases (CDKs) [175], protein kinase A [176] and mitogen and stress activated kinase (MSK) [128]. The activation of kinase signalling pathways in the initial infection phase comes with multiple, mostly beneficial consequences for the virus including major IE gene activation.…”

“…Alternate mechanisms of action are based on the ability of these compounds to modulate cell signalling pathways [370]. For example, cardiac glycoside digitoxin has been reported to inhibit HCMV through induction of cellular autophagy following activation of the regulatory kinase AMPK via a novel NA + ,K + -ATPase subunit α1-AMPK-ULK1 pathway [173]. In addition to inhibiting HCMV, cardiac glycosides act as antivirals against a range of clinically important DNA and RNA viruses.…”

AMP-Activated Protein Kinase and Host Defense against Infection

“…HCMV-mediated AMPK activation is dependent on CaMKK, and inhibition of AMPK activity abrogated HCMV replication and DNA synthesis [ 55 ]. Furthermore, the cardiac glycoside digitoxin induces phosphorylation of AMPK/ULK1, whereas it suppresses mTOR activity to increase autophagic flux and inhibit HCMV replication [ 56 ]. Moreover, HCMV induces production of the host protein viperin [ 57 ], which is required for AMPK activation, transcriptional activation of GLUT4 and lipogenic enzymes, and lipid synthesis [ 58 ].…”

Elucidation of the mechanism of anti-herpes action of two novel semisynthetic cardenolide derivatives

“…The mechanism by which cardenolides affect several steps of HSV replication could be related to the inhibition of Na + / K + -ATPase in host cells. Many studies have demonstrated the modulation of Na + /K + -ATPase functions in host cells by DNA viruses (adenoviruses [18], cytomegalovirus [23,[70][71][72], and HSV [27,28]), and RNA viruses (chikungunya virus [19,73], coronaviruses [20,74,75], respiratory syncytial virus [76,77], Ebola virus [78,79], influenza virus [33,80,81], and HIV [30,31,82]). By activating signaling cascades or by altering the concentration of intracellular ions, the binding of cardenolides to Na + /K + -ATPase seems to create an unfavorable environment for viral replication.…”