We have previously reported that HCMV infection induces numerous changes to the metabolic network; among these changes is the activation of glycolysis (22
). In this study, we began to elucidate the mechanisms responsible for HCMV-mediated glycolytic activation. We find that viral envelope fusion and tegument protein delivery are not sufficient for glycolytic activation; instead, de novo
protein synthesis is necessary for HCMV-mediated glycolytic activation (Fig. ). While it is currently unclear which viral factors are responsible for the activation of glycolysis, our results indicating that inhibition of viral DNA replication does not affect glycolytic activation suggest that late proteins, whose expression is dependent on DNA replication, are not involved.
We find that inhibition of CaMKK but not PKA or CaMKII ablates HCMV-induced glycolytic activation (Fig. ). HCMV has been shown to mobilize cellular calcium stores (14
), which suggests that HCMV infection should activate calmodulin-dependent kinase cascades. Viral proteins that have been implicated in calcium signaling, e.g., UL
), could play a role in CaMKK activation and therefore viral induction of glycolysis.
Interestingly, inhibitors of CaMKII did not block HCMV-mediated glycolytic activation compared to the level for DMSO-treated infected cells. This would suggest that CaMKII is not important for HCMV-mediated glycolytic activation. However, strict conclusions from this result are difficult since treatment with the CaMKII inhibitor KN-62 resulted in the elevation of glycolysis in mock-infected cells (Fig. ). This was surprising given the reports suggesting that CaMKII can activate glycolysis (3
). One possibility is that HCMV infection attenuates CaMKII activity, resulting in increased glycolysis. In this scenario, the magnitude of virally mediated glycolytic activation might be insensitive to pharmaceutical inhibition of CaMKII since it is already inhibited by HCMV infection. Another scenario is that CaMKII inhibition elevates the basal levels of glycolysis for both mock- and HCMV-infected cells, potentially to a high enough level that HCMV cannot induce it further. Such scenarios as well as their importance for HCMV infection could be tested through future analysis of CaMKII activity during the HCMV viral life cycle.
An alternative to CaMKII-dependent signaling that is consistent with our results would be CaMKK-mediated activation of CaMKIV. CaMKIV can activate CREB, resulting in transcriptional activation of cAMP response element (CRE)-containing genes (3
). CREB has been reported to activate several glycolytic enzymes (16
), and we have previously reported that viral infection activates the expression of several glycolytic enzymes as well (22
). Taken together, these results suggest a potential scenario in which upstream CaMKK-mediated transcriptional activation of glycolytic genes could be responsible for HCMV-mediated activation of glycolysis. Other mechanisms of CaMKK-mediated activation of glycolysis include CaMKK-mediated activation of CaMKI. CaMKI has been reported to phosphorylate phosphfrucktokinase-2 (PFK-2) (26
), a glycolytic control enzyme whose phosphorylation results in glycolytic activation through activation of PFK-1 activity (reviewed in reference 28
). Additionally, the AMP-activated kinase is another enzyme that can be activated by CaMKK and is capable of phosphorylating/activating PFK-2 (reviewed in reference 36
). These possibilities are not mutually exclusive, and future work will determine their respective roles in HCMV-mediated glycolytic activation.
Our results indicate that pharmaceutical inhibition of CaMKK attenuates viral infection (Fig. ). Additionally, expression of a kinase-dead variant of CaMKK also attenuated HCMV infection, albeit to a lesser extent (Fig. ). The discrepancy between these two methods of CaMKK inhibition poses a challenge with respect to data interpretation. On one hand, small-molecule pharmaceuticals can have off-target effects which can be controlled for through analysis of secondary methods of inhibition. On the other hand, results involving a kinase-dead variant, i.e., an ATP-binding mutant, are totally dependent on the extent to which the mutant allele actually inhibits wild-type CaMKK activity. Such inhibition requires both substantial overexpression of the dominant-negative mutant and the subsequent dilution of crucial concentration-limited secondary interacting factors that are necessary for wild-type CaMKK activity. With respect to this point, to our knowledge, there have been no reports indicating that expression of a kinase-dead CaMKK variant can attenuate CaMKK's glycolytic phenotypes. Our results indicating that CaMKK-KD expression did not affect HCMV-mediated glycolysis support this possibility. Regardless, both our pharmaceutical approaches and our genetic-based approaches indicate that targeted inhibition of CaMKK attenuates HCMV replication. Further analysis of players downstream of CaMKK, using additional pharmaceutical and genetic tools, will further illuminate the role of this pathway during HCMV infection.
We find that pharmaceutical inhibition of glycolysis results in a magnitude of viral growth inhibition similar to that observed for pharmaceutical CaMKK inhibition. These results are consistent with a scenario in which HCMV infection is dependent on CaMKK activity for glycolytic activation. It is currently unclear what aspects of glycolysis contribute to viral replication. Non-mutually exclusive possibilities include an increased rate of ATP generation, production of glycosylation moieties for proteins, and production of acetyl-CoA for fatty acid biosynthesis, which we have previously reported to be increased during HCMV infection (23
). Another possibility is that the viral induction of glycolysis is necessary to maintain a basal level or floor of pentose-phosphate pathway flux, an activity we find is actually decreased during HCMV infection (23
). This could be required so that the virus can maintain pentose production for nucleotide biosynthesis. Further dissection of these metabolic pathways with subsequent analysis of their impact on the metabolic network and on viral replication will begin to answer these questions.
Our data indicate a correlation between CaMKK inhibition, glycolytic inhibition, and viral growth. Despite this correlation between the viral attenuation resulting from the inhibition of CaMKK and glycolysis, the impact on viral replication in the face of CaMKK inhibition may be independent of its impact on virally induced glycolysis. Additionally, at this point we also cannot rule out the possibility that CaMKK inhibition may block HCMV-mediated glycolytic induction by preventing the accumulation of a viral factor whose expression is necessary for glycolytic activation. It does appear, however, that continued CaMKK activity is necessary for maintenance of glycolytic activation, as we find that delayed treatment with STO-609 allows virally mediated glycolysis activation at 48 h postinfection but subsequently inhibits glycolytic activation at 72 h postinfection. Our data also indicate that HCMV infection induces the accumulation of CaMKK mRNA and protein (Fig. ). While the exact mechanism through which CaMKK contributes to HCMV replication needs to be elucidated, the attenuation of HCMV replication in the face of CaMKK inhibition and the induction of CaMKK expression observed during HCMV infection suggest that CaMKK is targeted by HCMV infection.
Our results indicate that, like HCMV infection, HSV-1 infection induces glycolysis (Fig. ). However, HSV-1 mediated induction of glycolysis is independent of CaMKK inhibition (Fig. ). Pharmaceutical inhibition of CaMKK also resulted in a reduction in HSV-1 viral replication (Fig. ), but to a much smaller extent than that of the observed impact on HCMV replication (Fig. ). These results suggest that HSV-1 and HCMV employ different mechanisms to activate cellular glycolytic flux.
In conclusion, CaMKK is an important cellular factor for both HCMV-mediated activation of glycolysis and HCMV replication, and the expression of CaMKK is induced by viral infection. Future work will elucidate the mechanisms through which CaMKK contributes to both virally mediated glycolytic activation and viral replication. Given the dependence of viral replication on these mechanisms, their specific identification could have therapeutic potential.