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

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Activated HMGCR is required for the formation of DENV-replication complexes and the maintenance of its architecture.The distribution of HMGCR and components of viral replication complexes (NS4A and E viral proteins) was evaluated by confocal microscopy in Huh7 cells infected with DENV2 (MOI 3) and treated with DMSO 0.5% (vehicle), 10 mM Metformin or 50 μM lovastatin (HMGCR inhibitor) for 24h. The integrity of replication complexes is depicted as the co-localization between NS4A and E proteins. In A is indicated the distribution of HMGCR (red), NS4A (light blue), and E protein (green) as well as the colocalization per infected cell of NS4A/HMGCR (B) and NS4A/E (C) represented by mean ± S.E of the colocalization of 60 infected cells per condition. D and E represent the mean fluorescence intensity of NS4A protein (D) and HMGCR (E) analyzed by flow cytometry. Graphs represent the mean fluorescence intensity ± S.E of three independent experiments, the histograms indicate the fluorescence intensity of a representative experiment.
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ppat.1006257.g004: Activated HMGCR is required for the formation of DENV-replication complexes and the maintenance of its architecture.The distribution of HMGCR and components of viral replication complexes (NS4A and E viral proteins) was evaluated by confocal microscopy in Huh7 cells infected with DENV2 (MOI 3) and treated with DMSO 0.5% (vehicle), 10 mM Metformin or 50 μM lovastatin (HMGCR inhibitor) for 24h. The integrity of replication complexes is depicted as the co-localization between NS4A and E proteins. In A is indicated the distribution of HMGCR (red), NS4A (light blue), and E protein (green) as well as the colocalization per infected cell of NS4A/HMGCR (B) and NS4A/E (C) represented by mean ± S.E of the colocalization of 60 infected cells per condition. D and E represent the mean fluorescence intensity of NS4A protein (D) and HMGCR (E) analyzed by flow cytometry. Graphs represent the mean fluorescence intensity ± S.E of three independent experiments, the histograms indicate the fluorescence intensity of a representative experiment.

Mentions: Next, to determine if the increased HMGCR activity mediated by AMPK inhibition during DENV infection plays a role in the replicative complex structure, DENV infected cells were treated with DMSO 0.5% (vehicle), metformin (AMPK activator) or lovastatin (HMGCR inhibitor) for 24h, and the HMGCR co-localization with the viral proteins NS4A and E, as well as replicative complex integrity were analyzed by confocal microscopy. In infected vehicle-treated cells, HMGCR showed co-localization with NS4A (co-localization = 0.28 ± 0.026, Fig 4B) as well as with NS3 (co-localization = 0.36 ± 0.04, S2A and S2B Fig). The replicative complexes integrity depicted as the co-localization between NS4A and E viral proteins or NS3 and E, was maintained in infected vehicle-treated cells showing a co-localization of 0.36 ± 0.05 between NS4A and E (Fig 4C) and a colocalization of 0.41 ± 0.06 between NS3 and E (S2A and S2C Fig)


DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target
Activated HMGCR is required for the formation of DENV-replication complexes and the maintenance of its architecture.The distribution of HMGCR and components of viral replication complexes (NS4A and E viral proteins) was evaluated by confocal microscopy in Huh7 cells infected with DENV2 (MOI 3) and treated with DMSO 0.5% (vehicle), 10 mM Metformin or 50 μM lovastatin (HMGCR inhibitor) for 24h. The integrity of replication complexes is depicted as the co-localization between NS4A and E proteins. In A is indicated the distribution of HMGCR (red), NS4A (light blue), and E protein (green) as well as the colocalization per infected cell of NS4A/HMGCR (B) and NS4A/E (C) represented by mean ± S.E of the colocalization of 60 infected cells per condition. D and E represent the mean fluorescence intensity of NS4A protein (D) and HMGCR (E) analyzed by flow cytometry. Graphs represent the mean fluorescence intensity ± S.E of three independent experiments, the histograms indicate the fluorescence intensity of a representative experiment.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5383345&req=5

ppat.1006257.g004: Activated HMGCR is required for the formation of DENV-replication complexes and the maintenance of its architecture.The distribution of HMGCR and components of viral replication complexes (NS4A and E viral proteins) was evaluated by confocal microscopy in Huh7 cells infected with DENV2 (MOI 3) and treated with DMSO 0.5% (vehicle), 10 mM Metformin or 50 μM lovastatin (HMGCR inhibitor) for 24h. The integrity of replication complexes is depicted as the co-localization between NS4A and E proteins. In A is indicated the distribution of HMGCR (red), NS4A (light blue), and E protein (green) as well as the colocalization per infected cell of NS4A/HMGCR (B) and NS4A/E (C) represented by mean ± S.E of the colocalization of 60 infected cells per condition. D and E represent the mean fluorescence intensity of NS4A protein (D) and HMGCR (E) analyzed by flow cytometry. Graphs represent the mean fluorescence intensity ± S.E of three independent experiments, the histograms indicate the fluorescence intensity of a representative experiment.
Mentions: Next, to determine if the increased HMGCR activity mediated by AMPK inhibition during DENV infection plays a role in the replicative complex structure, DENV infected cells were treated with DMSO 0.5% (vehicle), metformin (AMPK activator) or lovastatin (HMGCR inhibitor) for 24h, and the HMGCR co-localization with the viral proteins NS4A and E, as well as replicative complex integrity were analyzed by confocal microscopy. In infected vehicle-treated cells, HMGCR showed co-localization with NS4A (co-localization = 0.28 ± 0.026, Fig 4B) as well as with NS3 (co-localization = 0.36 ± 0.04, S2A and S2B Fig). The replicative complexes integrity depicted as the co-localization between NS4A and E viral proteins or NS3 and E, was maintained in infected vehicle-treated cells showing a co-localization of 0.36 ± 0.05 between NS4A and E (Fig 4C) and a colocalization of 0.41 ± 0.06 between NS3 and E (S2A and S2C Fig)

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus