We have previously established that HCMV infection induces numerous changes to the host-cell metabolic network 
. Induction of glycolysis has also been found to be critical for high-titer HCMV replication 
. Here we report that HCMV activates AMPK, a metabolic stress kinase, and that HCMV depends on its activity for high-titer replication. HCMV requires AMPK activation to increase glucose import and drive increased glycolytic flux (). Inhibition of AMPK attenuated both early and late gene expression and markedly reduced viral DNA replication. These results suggest that AMPK is an important cellular factor for HCMV replication.
Model of HCMV-mediated manipulation of the AMPK pathway.
Unstressed, uninfected cells do not normally utilize AMPK to activate glycolysis 
. Our results support this view, as the inhibition of AMPK did not impact the import of glucose or the FBP labeling rate in uninfected cells (). In contrast to uninfected cells, HCMV infection induces the activation of AMPK, which is critical for HCMV-mediated glycolytic activation. Interestingly, activation of AMPK would be predicted to have several consequences that are detrimental to infection including inhibition of protein translation and fatty acid biosynthesis 
. AMPK-mediated inhibition of translation occurs through induction of the TSC1/2 complex which in turn negatively regulates translation through inhibition of mTOR 
. It has recently been shown that the HCMV UL
38 protein can bind to the TSC1/2 complex and prevent its inhibitory activity on mTOR and translation initiation 
. Taken together, it appears that HCMV infection induces AMPK activation which in turn drives glycolytic activation, yet blocks the anti-viral effects of AMPK activation through the action of specific gene products such as UL
While the UL
38 protein appears sufficient to block the inhibitory effects of AMPK activation on mTOR activity, it is less clear how HCMV infection blocks AMPK's inhibitory effects on fatty acid biosynthesis. We have previously found that HCMV induces fatty acid biosynthesis, and specifically induces the activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid biosynthesis 
. ACC, and consequently fatty acid biosynthesis, is negatively regulated by activated AMPK 
. Given that HCMV requires activated fatty acid biosynthesis and ACC activity for viral replication, it is likely that HCMV infection blocks the negative impact of activated AMPK on ACC activity, potentially through the activity of an HCMV viral protein.
Our results suggest that inhibition of CaMKK blocks the down-stream effects associated with activated AMPK (). Previous reports suggest that pharmaceutical inhibition of CaMKK using STO-609 blocks CaMKK-mediated activation of AMPK but does not impact LKB1- mediated AMPK activation or activation of AMPK upon energetic stress, for example, upon treatment with glycolysis inhibitors 
. Taken together, these results suggest that HCMV infection requires CaMKK activity to activate AMPK, though the exact mechanism responsible is unclear. Our results suggest that inhibition of CaMKK reduces the amount of Thr172-phosphorylated AMPK, a known CaMKK phosphorylation site, as well as the total amount of AMPK. Both of these effects would be predicted to contribute to a decrease in AMPK activity during HCMV infection. Other AMPK phosphorylation sites have been implicated in the regulation of AMPK activity 
, thus CaMKK could potentially be modulating AMPK activity through phosphorylation of sites other than Thr172 as well.
We have previously reported that inhibition of CaMKK blocks high-titer virus production 
. Our current findings that AMPK inhibition blocks HCMV replication to a similar extent as CaMKK inhibition is consistent with a model in which HCMV-mediates AMPK activation through CaMKK (). How HCMV infection induces CaMKK activity still remains to be determined, although it has previously been reported that HCMV infection induces Ca2+
release from ER stores and we have found that HCMV infection induces CaMKK expression 
, both of which would be predicted to increase CaMKK activity.
Glycolysis has been shown to be important for HCMV replication, and glycolytic inhibition has a similar impact on HCMV as inhibition of AMPK and CaMKK 
. The similarity is both quantitative, in terms of the magnitude of reduction in viral titers, as well as qualitative, in blocking viral DNA replication and late gene expression, attenuating early gene expression and having no detectable impact on immediate early gene expression 
. Given these similarities, and combined with the observed necessity of AMPK and CaMKK for HCMV-induced glycolysis, the simplest model would be that CaMKK and AMPK activation are important for HCMV replication due to their activation of glycolysis. Despite these correlations, it remains to be determined how much of HCMV's reliance on CaMKK and AMPK activity is due to their activation of glycolysis. The possibility that these kinases contribute to viral infection through phosphorylation of other cellular or viral targets cannot be ruled out.
In summary, we find that HCMV infection activates AMPK, which is required for HCMV-mediated glycolytic activation and high-titer HCMV replication. As AMPK activation signals metabolic stress to normal cells, HCMV has evolved mechanisms to block the anti-viral consequences of metabolic stress pathway activation. While some of these mechanisms are known, such as UL
38 maintaining mTOR activation through interaction and inhibition of the TSC complex, others remain to be elucidated, such as maintenance of fatty acid biosynthesis. This stress response balancing act is representative of a recurrent theme in virus-host evolution. Replicating viruses must create a cellular environment conducive to viral replication, the efforts of which host cells have evolved to resist. In the case of AMPK, the evolutionary struggle is for the keys to the host-cell metabolic machinery. As the AMPK pathway is not normally activated in uninfected cells and inhibition of AMPK activity is tolerated in animal models 
, targeting this pathway clinically could be therapeutically beneficial for preventing HCMV-associated disease.