Interspecies Differences in Cytomegalovirus Inhibition by Cardiac Glycosides—A Unique Role of the Alpha3 Isoform of the Na+/K+-ATPase Pump

Cardiac glycosides (CGs), historically used to treat heart failure and arrhythmias, bind to the α subunit of the Na⁺/K⁺-ATPase pump and inhibit its activity. Their anticancer and antiviral activities are of interest. The α subunit of the Na⁺/K⁺-ATPase pump has four isoforms (α1-4), each with unique tissue distribution and expression pattern; their contributions to antiviral activities have not been studied. We previously reported that CGs inhibit human CMV (HCMV) in vitro but not mouse CMV (MCMV). In addition to the low affinity of mouse α1 for CGs, we hypothesized that other isoforms contribute to the anti-CMV activities of CGs. We show here that infection with HCMV significantly induced α3 in human foreskin fibroblasts, while MCMV did not induce mouse α3. Infection with guinea pig CMV (GPCMV) in GP fibroblasts also induced α3, and CGs inhibited GPCMV replication. HCMV inhibition with digitoxin reduced α3 expression. The concentration-dependent inhibition of HCMV with digitoxin analogs also correlated with α3 expression. Intriguingly, α3 was localized to the nucleus, and changes in its expression during infection and digitoxin treatment were mostly limited to the nucleus. At 4 h post-infection, α3 colocalized with immediate early 1 (IE1) and the promyelocytic leukemia protein (PML). An interaction of α3-PML-IE1 at 24 h post-infection was disrupted by digitoxin. The mRNA levels of IE1, major immediate early promoter (MIEP)-derived IE, and antiviral cytokines were reduced in infected digitoxin-treated cells. Summarized, these findings suggest a new role for α3 in the anti-HCMV activities of CGs via nuclear antiviral signaling pathways.