This study demonstrates that VZV manipulates the JNK pathway and depends on its activity for replication. Furthermore, phospho-JNK is assembled into VZV particles and may have important functions in newly infected cells. The findings raise a number of issues about the interaction of VZV with cell signaling networks: the pattern of JNK pathway activation, the different effects of VZV in other cell types, and possible functions of activated JNK in infected cells. The major question, and the one most difficult to answer, is the mechanism of JNK activation by VZV. Related to this is the unknown antiviral mechanism of the JNK inhibitor SP600125. Despite our incomplete understanding of the molecular role of JNK in VZV infection, the results presented here show that inhibition of JNK is a plausible antiviral strategy against VZV.
The JNK pathway was activated at three levels—the MAP2K level (MKK4, MKK7), the MAPK level (JNK), and the substrate level (c-Jun, JunD)—as indicated by increased phosphorylation of these proteins and increased JNK activity. SP600125 inhibited c-Jun phosphorylation in vitro and in VZV cultures, which is evidence that no cell or viral kinases other than JNK were involved. However, VZV-infected cells had a kinase activity associated with glutathione beads that was resistant to SP600125, indicating that this kinase activity was not due to JNK. VZV encodes two viral kinases, ORF66 and ORF47, with predicted sizes of 46 and 54 kDa, respectively (34
). These are both larger than the 40-kDa phosphoprotein that appeared in the control kinase assays, so autophosphorylation is not a likely possibility. VZV or cell kinases may have bound to the beads and phosphorylated other proteins in the complex; further analysis by immunoblotting would be required to confirm this hypothesis.
The antiviral effects of SP600125 indicate how important the JNK pathway is to VZV and promote the idea that JNK is a potential therapeutic target. Targeting the JNK pathway for therapeutic benefit is a goal that both researchers and drug companies are pursuing. The JNK pathway is considered a target for the treatment of cancer and diseases caused by inflammation and neurodegeneration (12
). Several derivatives of SP600125 have been developed and are progressing in clinical trials for these applications (48
). Thus, there is potential for treating viral infections, including VZV, with JNK inhibitors. Inhibition of the JNK pathway is known to interfere with the functions of other viruses. SP600125 inhibits the activation of JNK by the hepatitis C virus protein NS3, which contributes to hepatitis C virus-related hepatocarcinogenesis (26
). For HSV-1, expression of the scaffold protein JIP-1, which effectively inhibits JNK translocation to the nucleus, decreases the HSV-1 yield by 70% (49
Observing where activated JNK was located in VZV-infected cells uncovered its predicted migration to the nucleus, its induction early in virus replication, and an unexpected appearance in cell extensions and virions. JNK is located in both the cytoplasm and nuclei of quiescent cells; stimulation by UV irradiation is known to cause accumulation of phospho-JNK-1 in the nucleus (6
). Finding phospho-JNK in discrete nuclear foci (speckles) within VZV-infected cells pointed to areas with concentrated JNK activity, perhaps where viral transcription was occurring. There is evidence that signaling kinases may form integral components of transcription complexes, thus influencing gene expression (16
). Evidence for the accumulation of JNK in the nucleus has also been shown for HSV-1 (49
). Like JNK, c-Jun migrated to the nuclei of infected cells. These translocations were specifically induced by VZV, since activated JNK and c-Jun were at basal levels in the cytoplasm of uninfected cells at the plaque borders. This is evidence that activation of the JNK pathway was not a paracrine or stress-induced effect. Activated JNK was also found in the nuclei of infected cells that expressed nuclear IE62 (early-stage infection) and that were treated with PAA to block late gene expression, supporting the contention that JNK is activated early. Moreover, phospho-JNK was present in nuclei adjacent to infected cells that did not yet show detectable levels of IE62, which could occur by way of signaling events associated with virus attachment or penetration. Alternatively, activated JNK protein could be transferred directly. Either possibility is supported by the observation that phospho-JNK was located in far-reaching cytoplasmic extensions that could fuse or deliver virions to new cells. The unforeseen association of activated JNK with VZV virions, the first report for a virus, strengthens the potential for direct transfer of this protein to new cells. Although the kinetics and mechanism of JNK activation by VZV are unclear, the events surrounding the initial activation of the JNK pathway in VZV-infected cells are of great interest and are being pursued.
Several possibilities for VZV activation of the JNK pathway are being considered and follow from the finding that MKK4 and MKK7 were heavily phosphorylated. One is that signals from membrane receptors resulted in phosphorylation of these MAP2Ks, and another is that VZV infection activated the pathway at an intermediate level. Typically, MKK7 protein kinase is activated by cytokines (tumor necrosis factor, interleukin-1), and MKK4 is activated by environmental stresses (12
). These ligands engage specific receptors that activate any number of MAP3Ks, which then activate MKK4 and MKK7. Identification of the MAP3Ks involved is severely hindered by the facts that (i) the VZV surface receptor is unknown, (ii) linking specific stimuli (such as UV irradiation) to particular MAP3Ks is very difficult, and (iii) the MAP3Ks involved in JNK activation vary by cell type. Furthermore, the physiological relevance of MAP3Ks is uncertain, and many promiscuously activate more than one pathway (12
). VZV infection could also cause MKK4, MKK7, or JNK activation by a viral or cellular kinase other than the MAP3Ks. Finally, VZV could cause the sustained activation of JNK by the modulation of phosphatases. The MAPKs, including JNK, are inactivated by a group of 10 MAPK phosphatases (31
). The balance between activating MAP2Ks, such as MKK4 and MKK7, and MAPK phosphatases determines the duration and magnitude of JNK activation. VZV disruption of this balance is manifested by the sustained and increasing JNK activation seen over 4 days of virus replication. VZV activation of the JNK pathway appears to differ from normal cell processes, since infection produced a pattern distinctly altered from that of anisomycin. Only certain isoforms of these kinases were preferentially phosphorylated in VZV-infected cells, suggesting that VZV signaling through the JNK pathway is precisely regulated. The differential phosphorylation of JNK by MKK4 and MKK7 may generate a cellular response that is unique to VZV and optimal for its replication.
There is some evidence from other reports that VZV and its close relative HSV-1 interact with the JNK pathway after entry. The HSV-1 immediate-early protein ICP27 was found to be solely necessary for the activation of JNK in the context of infection (24
). In HSV-1, ectopically overexpressed ICP0 has also been reported to activate the JNK pathway (14
). Similarly, the VZV homolog of ICP0, ORF61, activates JNK when ORF61 is expressed ectopically (61
). However, the activation of JNK by ORF61 is linked with a twofold reduction in VZV yield that could be reversed by treating the cultures with JNK inhibitor 1, a recombinant fusion of the JNK scaffold protein (islet-brain 1 and 2 proteins) and a membrane permeable peptide (HIV-Tat) (4
). That report contradicts the antiviral effects of SP600125 presented here but confirms the activation of JNK in VZV-infected cells. Differences in the cell types and inhibitors used could account for the discrepancy. Primary cells were used in this study, while melanoma cells (MeWo) were used elsewhere. Melanoma cells are a tumor cell line and thus may have oncogenic mutations that alter signaling networks and sensitivity to inhibitors. The mechanism of action of SP600125, an ATP analog, is fundamentally different from that of JNK inhibitor 1, a JNK-binding protein. Ultimately, the interaction between VZV and the JNK pathway is likely to have redundant mechanisms, as is true of HSV-1, and to be specific for each cell type studied.
Activation of the JNK pathway is common to herpesviruses, although the purpose in virus replication is not fully understood (17
). The advantages of JNK activation for herpesviruses relate to its spectrum of functions in the cell; JNK is involved in the regulation of gene expression, apoptosis, cell migration, and responses to environmental stresses. The recruitment of phospho-JNK to the nucleus points to a possible role of JNK in transcription. JNK substrates include the transcription factors c-Jun, ATF-2, and Elk-1 (41
); c-Jun and ATF-2 are needed for VZV replication (64
); therefore, VZV may activate JNK to increase viral gene expression. The JNK pathway has also been implicated in both apoptosis and cell survival, depending on the cellular context (12
). VZV induces apoptosis in certain cell types, such as HFFs (28
), but apoptosis was rare in this study (authors' observations), and no significant poly(ADP-ribose) polymerase cleavage was detected in VZV-infected MeWo cells (62
). Additionally, treatment with anisomycin, a known inducer of apoptosis (67
), showed a pattern of activation distinct from that of VZV infection. Thus, JNK activation seems unlikely to induce apoptosis in VZV-infected cells. Also, the JNK pathway is linked to cell migration (31
), and the movement of keratinocytes is impaired when they are exposed to SP600125 (29
). The pseudorabies virus US3 kinase has been shown to cause dramatic alterations in the cytoskeleton, resulting in the formation of long cell projections that are associated with enhanced spread of the virus (19
). Interestingly, VZV-induced cell extensions also contained phospho-JNK, and it would be interesting to determine whether viral kinases were also involved. Therefore, it is conceivable that JNK would be activated to facilitate viral movement within the cell. Lastly, environmental stresses activate the JNK pathway, which may actually enhance virus replication. Recently it was reported that the infectivity of HSV-1 ICP0 mutants increased after the cells were stressed by heat shock and UVC irradiation (5
). It is intriguing to speculate that these stresses activated the JNK pathway. Overall, JNK activation has various functions that may favor herpesviruses or that are even essential for replication.
Although the purpose of JNK activation for VZV replication remains to be studied, it is clear that the JNK pathway is crucial for many viruses. It is possible that upregulation of a cellular pathway determines, in part, the susceptibility of a cell to infection. For example, the Akt signaling pathway was recently found to be a key determinant for the permissiveness of human cancer cells to myxoma virus (70
). Nonpermissive tumor cells supported myxoma virus replication after expression of active Akt. Conversely, permissive cancer cells were made nonpermissive by blocking Akt activation with a dominant-negative inhibitor. Therefore, cells lacking JNK might not be susceptible to VZV infection. Following this reasoning, it would be interesting to determine whether cells lacking JNK are permissive for VZV. In conclusion, the interaction of VZV with the JNK pathway is a clear example of viral coevolution with cellular signaling networks and underscores the dependence of viruses on their hosts. The idea of using host factors, as opposed to viral proteins, as drug targets opens up new avenues for the generation of new antiviral drugs and should be considered (10