DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target

Dengue is the most common mosquito-borne viral disease in humans. Changes of lipid-related metabolites in endoplasmic reticulum of dengue virus (DENV) infected cells have been associated with replicative complexes formation. Previously, we reported that DENV infection inhibits HMGCR phosphorylation generating a cholesterol-enriched cellular environment in order to favor viral replication. In this work, using enzymatic assays, ELISA, and WB we found a significant higher activity of HMGCR in DENV infected cells, associated with the inactivation of AMPK. AMPK activation by metformin declined the HMGCR activity suggesting that AMPK inactivation mediates the enhanced activity of HMGCR. A reduction on AMPK phosphorylation activity was observed in DENV infected cells at 12 and 24 hpi. HMGCR and cholesterol co-localized with viral proteins NS3, NS4A and E, suggesting a role for HMGCR and AMPK activity in the formation of DENV replicative complexes. Furthermore, metformin and lovastatin (HMGCR inhibitor) altered this co-localization as well as replicative complexes formation supporting that active HMGCR is required for replicative complexes formation. In agreement, metformin prompted a significant dose-dependent antiviral effect in DENV infected cells, while compound C (AMPK inhibitor) augmented the viral genome copies and the percentage of infected cells. The PP2A activity, the main modulating phosphatase of HMGCR, was not affected by DENV infection. These data demonstrate that the elevated activity of HMGCR observed in DENV infected cells is mediated through AMPK inhibition and not by increase in PP2A activity. Interestingly, the inhibition of this phosphatase showed an antiviral effect in an HMGCR-independent manner. These results suggest that DENV infection increases HMGCR activity through AMPK inactivation leading to higher cholesterol levels in endoplasmic reticulum necessary for replicative complexes formation. This work provides new information about the mechanisms involved in host lipid metabolism during DENV replicative cycle and identifies new potential antiviral targets for DENV replication.
Author summary DENV replicative complexes formation is associated with changes of lipid-related metabolites in endoplasmic reticulum, such as an increase in cholesterol synthesis. This increase correlates with a significant augment in the activity of HMGCoA reductase (the limiting enzyme in cholesterol synthesis), favoring a cholesterol-enriched cellular environment. The augment in the activity of the HMGCR observed in infected cells is caused by a decrease in the phosphorylation level of the HMGCR, associated with the inactivation of AMPK. In agreement, AMPK activation by metformin reduces HMGCR activity and affects viral replication. The role HMGCR and AMPK activity in DENV replicative complexes formation was confirmed by the co-localization of HMGCR and cholesterol with the viral proteins NS3, NS4A and E. Furthermore, metformin and lovastatin (HMGCR inhibitor) treatments altered this co-localization as well as replicative complexes formation supporting that active HMGCR is required for replicative complexes formation. The results show that during DENV infection, an increase in the HMGCR activity occurs through AMPK inactivation, leading to higher cholesterol levels in endoplasmic reticulum necessary for replicative complexes formation. This work provides new information about the mechanisms involved in host lipid metabolism during DENV replicative cycle and identifies potential new antiviral targets for DENV replication.