The viral DNA polymerase inhibitors (GCV, foscarnet, and cidofovir) effectively suppress virus replication, but their prolonged use leads to significant side effects and the emergence of resistant mutants (19
Compounds with specific effects on cellular pathways have been reported to inhibit HCMV replication. In general, these can be grouped into early inhibitors of HCMV replication, i.e., inhibitors of phosphoinositide 3-kinase (PI3-K) (23
), p38 mitogen-activated protein kinase (MAPK) (22
), and the tyrosine kinase inhibitor Gleevec (45
), and others active at multiple steps of virus replication, including cellular cyclin-dependent kinase inhibitor, roscovitine (41
), and leflunomide and its active metabolite FK778 (8
CG have anticancer activities, and we now provide strong evidence for their antiviral activities. We show that these compounds are potent inhibitors of HCMV replication at nanomolar concentrations, similar to the concentrations that inhibit cancer cell growth (33
). HSV-1 (alphaherpesvirus) and possibly EBV (gammaherpesvirus) were also inhibited by CG, suggesting the antiviral activities may involve cellular pathways or a viral protein shared by these herpesviruses. Because of the high sensitivity of the latently infected Akata cells to CG, the selectivity index of these compounds for EBV was less than that calculated for HCMV and HSV-1. Thus, inhibition of EBV replication requires further studies using other cell types.
Similar to the reported early inhibition of HSV-1 by CG (7
), HCMV inhibition occurred early during virus replication. The expression of IE1 and IE2 proteins was decreased in the presence of CG, suggesting either an involvement of the MIE promoter and its activation by cellular transcription factors or a process that occurs after the transcription of the IE genes. The requirement for NF-κB in the transactivation of the MIE promoter has been described (3
), and we show that CG significantly reduce the expression of NF-κB. We note that the MIE promoter has been shown to be activated by multiple transcription factors, and our data suggest that CG do not affect its transcription. Thus, the low levels of NF-κB protein measured at 12 hpi may represent an effect that occurs after the activation of the MIE.
How would CG inhibit HCMV? These compounds bind to the Na+
-ATPase that has an established role in ion transport. However, this pump has additional cellular activities, such as the regulation of cell growth and the expression of various genes (37
). Binding of CG to the Na+
-ATPase triggers a signaling cascade (signalosome), resulting in activation of the protein tyrosine kinase Src, which transactivates epidermal growth factor receptor, Ras, and MAPKs. The diverse mechanisms of control of cell proliferation have been reviewed in detail (42
). Some of the involved pathways are also used by HCMV for its efficient replication (53
), and the fact that NF-κB expression was decreased in CG-treated cells supports this interpretation. Currently, there is no evidence to support direct inhibition of a virus-specific protein by CG. Proof that these compounds indeed target a specific viral protein will require demonstration that they can select for resistant viral mutants (16
). These studies are ongoing. Alternatively, CG may target a cell protein or pathway(s) that are critical for the replication of herpesviruses.
Another mechanism that could link inhibition of cancer cells and HCMV by CG involves an interaction between the Na+
-ATPase and other ion channels. We show that HCMV significantly upregulate hERG transcription as early as 12 hpi, while CG downregulate its transcription. qRT-PCR showed that the transcription of three other potassium channels was unchanged during HCMV infection. The hERG gene has been associated with multiple cancers and appears to be important in cancer progression in vivo
. Its role in virus infections has not been studied (2
). Although MCMV infection resulted in the upregulation of mERG, treatment with CG did not inhibit virus replication, and mERG levels were unchanged during infection and treatment with CG. Interspecies differences in the effects of CG have been noted in studies using a variety of cells (12
). Transformation of human lymphocytes in vitro
by phytohemagglutinin was sensitive to ouabain, but rat cultures were relatively insensitive (38
). In addition, tumor cell sensitivity to CG was reported to be species dependent. Although nontoxic to rodent-derived tumor cell lines, CG are potent inhibitors of human tumor cells (39
). These differences in activity may be due to changes in the structure of the Na+
-ATPase among species.
At therapeutic concentrations (0.0006 to 0.0015 μM), digoxin is unlikely to inhibit HCMV replication based on the in vitro
concentrations reported here. However, work has shown that digitoxin may have anticancer effects at concentrations commonly found in cardiac patients (0.02 to 0.033 μM) (14
). Based on our in vitro
concentrations of HCMV inhibition by digitoxin (EC50
= 0.016 μM ± 0.00), digitoxin may have inhibitory effects on HCMV in vivo
. Thus, studies of additional CG with potential clinical application are needed.
In conclusion, CG are potent inhibitors of HCMV replication and IE gene expression. Their effects correlate with the transcription and translation of hERG. Future studies may characterize the signaling and chaperons involved in this interaction.