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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

PP2A activity is not altered by DENV, but its inhibition by Okadaic acid has an antiviral effect.The PP2A activity was analyzed in Huh7 cells infected (MOI 3) with DENV 2/4 at 1, 12 and 24 hpi (A), and in Mock or DENV 2/4 infected cells treated with DMSO 0.05% (vehicle) or 10 nM Okadaic acid (O. A) for 24h (B). Activity is expressed as picomoles of phosphate (phosphates pmoles). From the same cell lysates, the levels of NS3 viral protein (lower panels) were determined by WB as infection test. * p<0.05 compared to mock vehicle-treated cells. The antiviral effect of O. A (0, 1 and 10 nM) against DENV infection was evaluated in supernatants from Huh7 cells infected (MOI 3) with DENV2 (C) and DENV4 (D) by viral yield and NS1 secretion at 24 hpi. Viral yield is expressed as Foci Forming Units (FFU) / mL. NS1 secretion was normalized respect to infected non-treated cells and expressed as fold change vs 0 mM. (E) The number of viral genome copies of DENV 2/4 infected cells treated with O. A (0, 5, 10 nM) for 24h was examined by qRT-PCR, and expressed as Log of No. Copies. * p<0.05 compared to non-treated cells. DMSO 0.05% was used as vehicle for all cases (0 nM). Data are means ± S.E of n = 3 independent experiments realized by duplicated.
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ppat.1006257.g007: PP2A activity is not altered by DENV, but its inhibition by Okadaic acid has an antiviral effect.The PP2A activity was analyzed in Huh7 cells infected (MOI 3) with DENV 2/4 at 1, 12 and 24 hpi (A), and in Mock or DENV 2/4 infected cells treated with DMSO 0.05% (vehicle) or 10 nM Okadaic acid (O. A) for 24h (B). Activity is expressed as picomoles of phosphate (phosphates pmoles). From the same cell lysates, the levels of NS3 viral protein (lower panels) were determined by WB as infection test. * p<0.05 compared to mock vehicle-treated cells. The antiviral effect of O. A (0, 1 and 10 nM) against DENV infection was evaluated in supernatants from Huh7 cells infected (MOI 3) with DENV2 (C) and DENV4 (D) by viral yield and NS1 secretion at 24 hpi. Viral yield is expressed as Foci Forming Units (FFU) / mL. NS1 secretion was normalized respect to infected non-treated cells and expressed as fold change vs 0 mM. (E) The number of viral genome copies of DENV 2/4 infected cells treated with O. A (0, 5, 10 nM) for 24h was examined by qRT-PCR, and expressed as Log of No. Copies. * p<0.05 compared to non-treated cells. DMSO 0.05% was used as vehicle for all cases (0 nM). Data are means ± S.E of n = 3 independent experiments realized by duplicated.

Mentions: PP2A dephosphorylates HMGCR and boosts its activity [34,35]. Previously in this work we showed that PP2A inhibited by OA does not alter the HMGCR activity up-regulation during DENV infection, suggesting that DENV infection does not alter the phosphatase activity of PP2A. To rule out that PP2A was also modulated in response to DENV infection, its phosphatase activity was evaluated in DENV 2/4 infected cells at 1, 12 and 24 hpi. DENV infection did not induce any effect on PP2A phosphatase activity (Fig 7A). This was corroborated in mock and DENV infected cells in the presence and absence of okadaic acid for 24h. As expected, OA-treatment diminished the PP2A phosphatase activity compared to non-treated cells but not respect to mock infected cells, which demonstrated that DENV infection does not alter PP2A phosphatase activity (Fig 7B).


DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target
PP2A activity is not altered by DENV, but its inhibition by Okadaic acid has an antiviral effect.The PP2A activity was analyzed in Huh7 cells infected (MOI 3) with DENV 2/4 at 1, 12 and 24 hpi (A), and in Mock or DENV 2/4 infected cells treated with DMSO 0.05% (vehicle) or 10 nM Okadaic acid (O. A) for 24h (B). Activity is expressed as picomoles of phosphate (phosphates pmoles). From the same cell lysates, the levels of NS3 viral protein (lower panels) were determined by WB as infection test. * p<0.05 compared to mock vehicle-treated cells. The antiviral effect of O. A (0, 1 and 10 nM) against DENV infection was evaluated in supernatants from Huh7 cells infected (MOI 3) with DENV2 (C) and DENV4 (D) by viral yield and NS1 secretion at 24 hpi. Viral yield is expressed as Foci Forming Units (FFU) / mL. NS1 secretion was normalized respect to infected non-treated cells and expressed as fold change vs 0 mM. (E) The number of viral genome copies of DENV 2/4 infected cells treated with O. A (0, 5, 10 nM) for 24h was examined by qRT-PCR, and expressed as Log of No. Copies. * p<0.05 compared to non-treated cells. DMSO 0.05% was used as vehicle for all cases (0 nM). Data are means ± S.E of n = 3 independent experiments realized by duplicated.
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ppat.1006257.g007: PP2A activity is not altered by DENV, but its inhibition by Okadaic acid has an antiviral effect.The PP2A activity was analyzed in Huh7 cells infected (MOI 3) with DENV 2/4 at 1, 12 and 24 hpi (A), and in Mock or DENV 2/4 infected cells treated with DMSO 0.05% (vehicle) or 10 nM Okadaic acid (O. A) for 24h (B). Activity is expressed as picomoles of phosphate (phosphates pmoles). From the same cell lysates, the levels of NS3 viral protein (lower panels) were determined by WB as infection test. * p<0.05 compared to mock vehicle-treated cells. The antiviral effect of O. A (0, 1 and 10 nM) against DENV infection was evaluated in supernatants from Huh7 cells infected (MOI 3) with DENV2 (C) and DENV4 (D) by viral yield and NS1 secretion at 24 hpi. Viral yield is expressed as Foci Forming Units (FFU) / mL. NS1 secretion was normalized respect to infected non-treated cells and expressed as fold change vs 0 mM. (E) The number of viral genome copies of DENV 2/4 infected cells treated with O. A (0, 5, 10 nM) for 24h was examined by qRT-PCR, and expressed as Log of No. Copies. * p<0.05 compared to non-treated cells. DMSO 0.05% was used as vehicle for all cases (0 nM). Data are means ± S.E of n = 3 independent experiments realized by duplicated.
Mentions: PP2A dephosphorylates HMGCR and boosts its activity [34,35]. Previously in this work we showed that PP2A inhibited by OA does not alter the HMGCR activity up-regulation during DENV infection, suggesting that DENV infection does not alter the phosphatase activity of PP2A. To rule out that PP2A was also modulated in response to DENV infection, its phosphatase activity was evaluated in DENV 2/4 infected cells at 1, 12 and 24 hpi. DENV infection did not induce any effect on PP2A phosphatase activity (Fig 7A). This was corroborated in mock and DENV infected cells in the presence and absence of okadaic acid for 24h. As expected, OA-treatment diminished the PP2A phosphatase activity compared to non-treated cells but not respect to mock infected cells, which demonstrated that DENV infection does not alter PP2A phosphatase activity (Fig 7B).

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