The data presented show for the first time that HCMV infection of HFFs is associated with complex changes in the expression of multiple proteins of the Wnt pathway and that modulators of this pathway, monensin, nigericin, and salinomycin, are potent inhibitors of HCMV replication without causing toxicity to HFFs. These three compounds may share a similar mechanism of HCMV inhibition, reflected by their slope, SI, and antiviral assays. The add-on and removal assays suggest that inhibition of HCMV replication occurs at several stages. Since the slopes of the dose-response curves of the three compounds were close to 1 (), their effects may involve a viral protein that associates with a cellular target which is critical throughout the full replication cycle. Studies are ongoing to determine whether a viral target is indeed involved in the activity of these compounds. While the tested compounds did not affect IE1 expression at 24, 48, or 72 hpi, the early late gene UL44 and IE2 were significantly decreased. The inhibitory effect was noted to augment itself as replication proceeded: DNA replication was inhibited at 48 hpi, pp65 gene expression was undetectable at 72 hpi, and a yield reduction assay revealed a complete absence of plaques in infected HFFs treated with the three compounds. In contrast to the activation of Wnt/β-catenin by the oncogenic EBV and KSHV, HCMV infection resulted in inhibition of several components of the Wnt pathway, while axin 1 expression was enhanced at all tested time points. Moreover, HCMV inhibition with Wnt modulators was associated with significant and further downregulation of several components of both the canonical and noncanonical Wnt pathways. The expression of Wnt5a, which activates the β-catenin-independent pathway but negatively regulates β-catenin-dependent activity (18
), was significantly decreased throughout HCMV infection of HFFs and even more so after treatment with nigericin.
Several anticancer agents (imatinib [Gleevec], roscovitine, rapamycin) were reported to inhibit HCMV replication, likely by interfering with one or more cell signaling pathways (19
). The drawback of these agents is their toxicity to cancer cells as well as to noncancerous primary HFFs. We have shown that the HCMV inhibitors, artemisinin dimers, have strong anticancer activities but are nontoxic to HFFs at concentrations that inhibit HCMV replication (22
). A similar differential effect was reported with the Wnt modulators: while strongly inhibiting cancer cells, no toxic effects were found in the surrounding healthy cells (12
). In agreement with these reports, we show here that cancer cells are exceedingly more sensitive to monensin, nigericin, and salinomycin than are primary HFFs.
Wnt signaling plays a crucial role in embryonic development and cancer, processes that are affected by HCMV. Originally, Wnt signals were classified into canonical (β-catenin dependent) and noncanonical (β-catenin independent). However, this pathway is now known to be more complex, Wnt action is context dependent, and multiple intracellular cascades can be triggered, some a blend of canonical and noncanonical components (25
). The key characteristics of canonical signaling are the requirement for the LRP5/6 coreceptor to enable β-catenin accumulation and the involvement of LEF/TCF transcription factors. LRP6 phosphorylation by glycogen synthase kinase 3 (GSK-3) and casein kinase 1γ (CK1) is crucial for activation of the canonical Wnt/β-catenin signaling (27
). When a Wnt ligand binds to the Fz (Frizzled) receptor and its coreceptor LRP6, this complex, together with the scaffolding protein Dishevelled (Dvl), results in LRP6 phosphorylation, activation, and recruitment of the axin complex to the receptors. These events lead to inhibition of axin-mediated β-catenin phosphorylation and stabilization of β-catenin, which accumulates and travels to the nucleus to activate Wnt target gene expression. The noncanonical Wnts avoid LRP coreceptors and β-catenin stabilization to activate intracellular kinases and regulate distinct β-catenin-independent pathways. These include the planar cell polarity (PCP) pathway and the Wnt/calcium pathway. The PCP pathway, mediated by Fz and Dvl, activates c-Jun N-terminal kinase (JNK) and Rho-associated kinase. Noncanonical Wnts binding to Fz can also stimulate an increase in intracellular Ca2+
levels, thereby activating calcium-sensitive proteins, such as Ca2+
/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) (28
Monensin, an ionophorous antibiotic isolated from Streptomyces cinnamonensis
and used as an antibiotic in dairy cattle, was recently reported as a novel antineoplastic compound in prostate cancer cells (24
). Salinomycin, structurally similar to monensin, was identified as a breast cancer stem cell inhibitor in vitro
and in vivo
). It also reduced cancer and cancer stem cell growth in leukemias and uterine sarcoma cells (23
). Nigericin and salinomycin inhibited Wnt signaling by blocking the phosphorylation of the Wnt coreceptor LRP6 and inducing its degradation (12
). Monensin was reported in the past to block protein transport from the Golgi apparatus to the cell membrane and to inhibit HSV-1, HSV-2, and HCMV (35
). Monensin treatment inhibited transport of progeny virus to the surface of infected cells, while viral protein synthesis and DNA replication were not inhibited. The reduction of extracellular virus release was more significant for HSV-2 than HSV-1 (35
). In the case of HCMV, monensin inhibited DNA replication and generation of virus progeny in HFFs and its activity was MOI dependent (37
). These results correlate with our findings and suggest differences in activities of Wnt modulators between HSV- and HCMV-infected cells. Although the reports on monensin predated the discovery of Wnt and stem cell cancer, a correlation between the Wnt pathway and transport of proteins from the Golgi apparatus in HCMV-infected cells remains to be studied.
HCMV infection of HFFs resulted in decreased expression of multiple members (both canonical and noncanonical) of the Wnt pathway. Intriguingly, even further inhibition of Wnt signaling by monensin, nigericin, or salinomycin resulted in HCMV inhibition, suggesting a very fine balance between virus and Wnt components that either maintains or abrogates lytic replication. A recent study reported on dysregulation of the canonical Wnt/β-catenin signaling pathway by HCMV. Infection induced degradation of β-catenin, which resulted in a decrease in its transcriptional activity in response to Wnt ligand stimulation (39
). HCMV also inhibited Wnt/β-catenin signaling in human extravillous cytotrophoblasts. Similarly, we found that HCMV infection resulted in decreased expression of total β-catenin. Another study showed that overexpression of HCMV-encoded US28 in intestinal epithelial cells inhibited GSK-3β function, promoted accumulation of β-catenin, and increased expression of Wnt target genes involved in the control of the cell proliferation (40
). These studies and ours suggest that the Wnt pathway is tightly regulated by HCMV. Virus replication mostly targets this pathway for inhibition of its components (with the exception of axin 1), and further inhibition of specific components results in virus inhibition.
HCMV infection of the developing brain results in long-term neurological sequelae. How brain damage is induced by HCMV is not well understood, but neural stem cells in the fetal brain appear to be an important cell type affected by the virus (41
). The Wnt pathway may play an important role in neural stem cell development, differentiation, and migration. In a murine CMV (MCMV) model, MCMV inhibited neuronal differentiation and decreased expression of Wnt-1 and neurogenin 1 (42
We conclude that HCMV targets components of the Wnt pathway. While the gammaherpesviruses KSHV and EBV activate the canonical Wnt/β-catenin pathway, HCMV exerts different and mostly inhibitory effects on this pathway. While the described compounds may not be useful for HCMV therapy, studies with additional small molecules that modulate the Wnt pathway may assist in dissecting the complex interaction between HCMV and Wnt.