Identification of anti-viral activity of the cardenolides, Na + /K + -ATPase inhibitors, against porcine transmissible gastroenteritis virus

Abstract

A series of naturally occurring cardenolides that exhibit potent anti-transmissible gastroenteritis virus (TGEV) activity in swine testicular (ST) cells has been identified. In an immunofluorescence assay, these cardenolides were found to diminish the expressions of TGEV nucleocapsid and spike protein, which was used as an indication for viral replication; block TGEV infection induced apoptosis and cytopathic effects; and impart the same trend of inhibitory activity against Na/K-ATPase as for anti-TGEV activity. The viral titer inhibition was found to take place in a dose-dependent manner. Knocking down expression of Na/K-ATPase, the cellular receptor of cardenolides, in ST cells was found to significantly impair the susceptibility of ST cells to TGEV infectivity. Thus, we have identified Na/K-ATPase as an anti-viral drug target and its antagonists, cardenolides, a novel class of anti- TGEV agents.

The cardenolide ouabain suppresses coronaviral replication via augmenting a Na+/K+-ATPase-dependent PI3K_PDK1 axis signaling

“…The Taiwan field isolated virulent strain of TGEV was propagated in ST cells cultured with MEM and 2% FBS. IFA and studies on the mechanistic signaling effectors regarding ouabain and the other pharmacological inhibitors in TGEV infected ST cells were performed as described (Yang et al, 2017a;Yang et al, 2017b). Briefly, the ST cells in 96-well plates, with or without pretreatment of test compounds, were infected with TGEV at an MOI of 7.…”

“…ST cells harbored ATP1A1 shRNA (clone ID: TRCN0000444902), PIK3CA shRNA#1 (clone ID:TRCN0000196582), PIK3CA shRNA#2: (clone ID: TRCN0000196795), PDPK1 shRNA#1 (clone ID: TRCN0000221540), PDPK1 shRNA #2: (clone ID:TRCN0000196933) or negative control-shRNAs (shLacZ, clone ID: TRCN0000231722 for ATP1A1; and shLacZ, clone ID: TRCN0000072224 for PIK3CA and PDPK1) (Academia Sinica, Taiwan) respectively for specific gene silencing were prepared as described previously (Yang et al, 2017a). After validation of Na + /K + -ATPase α1, PI3Kα or PDK1 knockdown expression, the selected cells were used for the followed experiments.…”

“…(Diederich et al, 2017). However, only a few reports have investigated the antiviral mechanisms of cardenolides (Burkard et al, 2015;Yang et al, 2017a).…”

“…Swine testicular (ST) cells have been used as a host cell line for TGEV in many studies since the 1990s because ST cells express aminopeptidase N and are highly susceptible to TGEV (Delmas et al, 1992;Oh et al, 2003;Weingartl and Derbyshire, 1995;Weiss and Navas-Martin, 2005). Previously, we reported that a variety of cardenolides (including ouabain) exert potent anti-TGEV activities (Yang et al, 2017a). While knocking down the expression of Na + /K + -ATPase decreases TGEV infectivity in ST cells (Yang et al, 2017a), the exact associated effective pathways remain to be investigated.…”

“…Previously, we reported that a variety of cardenolides (including ouabain) exert potent anti-TGEV activities (Yang et al, 2017a). While knocking down the expression of Na + /K + -ATPase decreases TGEV infectivity in ST cells (Yang et al, 2017a), the exact associated effective pathways remain to be investigated.…”