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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

DENV infection down-regulates AMPK activity.In A, The AMPK activity, depicted as phosphorylation levels at Thr-172, was evaluated in Huh7 cells infected with DENV 2/4 (MOI 3) at 1, 12, and 24 hpi by ELISA, and NS3 viral protein levels (A, lower panel) were determined as infection test. AMPK activity was expressed as U/mL. *p<0.05 compared to mock infected cells (0 hpi). Data are means ± standard error (S.E) of n = 3 independent experiments realized by duplicate. (B) The levels of AMPK phosphorylated, AMPK total, and NS3 viral protein were analyzed by western blot in whole cell lysates obtained from Huh7 cells infected with DENV2 (MOI 0.1, 1 and 3) for 24 h. Graph represents the relative quantification of pAMPK respect to AMPK total protein. The pAMPK and total AMPK densitometry values were normalized with β-actin and pAMPK/AMPK ratios were calculated, Ratios are represented with respect to the indicated control. *p<0.05 compared to mock infected cells. Data are means ± standard error (S.E) of n = 4 independent experiments. (C) The AMPK activity and NS3 viral protein levels (C, lower panel) were determined in Mock or DENV 2/4 infected Huh7 cells treated with DMSO 0.5% (vehicle, VEH), 10 mM Metformin (MET, AMPK activator) or 10 μM Compound C (CC, AMPK inhibitor) for 24 h. *p<0.05 compared to mock VEH-treated cells, abp<0.05 compared to mock MET-treated cells. Data are means ± standard error (S.E) of n = 3 independent experiments realized by duplicate. (D) The levels of AMPK phosphorylated, AMPK total, and prM viral protein were analyzed by western blot in whole cell lysates obtained from Mock or DENV2 Huh7 infected cells (MOI 1 and 3) in the presence or absence of 10 mM metformin (MET) for 24h. Graph represents pAMPK/AMPK ratios normalized with respect to Mock infected cells with no MET treatment. pAMPK/AMPK ratios were obtained adjusting each protein with β-actin.
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ppat.1006257.g001: DENV infection down-regulates AMPK activity.In A, The AMPK activity, depicted as phosphorylation levels at Thr-172, was evaluated in Huh7 cells infected with DENV 2/4 (MOI 3) at 1, 12, and 24 hpi by ELISA, and NS3 viral protein levels (A, lower panel) were determined as infection test. AMPK activity was expressed as U/mL. *p<0.05 compared to mock infected cells (0 hpi). Data are means ± standard error (S.E) of n = 3 independent experiments realized by duplicate. (B) The levels of AMPK phosphorylated, AMPK total, and NS3 viral protein were analyzed by western blot in whole cell lysates obtained from Huh7 cells infected with DENV2 (MOI 0.1, 1 and 3) for 24 h. Graph represents the relative quantification of pAMPK respect to AMPK total protein. The pAMPK and total AMPK densitometry values were normalized with β-actin and pAMPK/AMPK ratios were calculated, Ratios are represented with respect to the indicated control. *p<0.05 compared to mock infected cells. Data are means ± standard error (S.E) of n = 4 independent experiments. (C) The AMPK activity and NS3 viral protein levels (C, lower panel) were determined in Mock or DENV 2/4 infected Huh7 cells treated with DMSO 0.5% (vehicle, VEH), 10 mM Metformin (MET, AMPK activator) or 10 μM Compound C (CC, AMPK inhibitor) for 24 h. *p<0.05 compared to mock VEH-treated cells, abp<0.05 compared to mock MET-treated cells. Data are means ± standard error (S.E) of n = 3 independent experiments realized by duplicate. (D) The levels of AMPK phosphorylated, AMPK total, and prM viral protein were analyzed by western blot in whole cell lysates obtained from Mock or DENV2 Huh7 infected cells (MOI 1 and 3) in the presence or absence of 10 mM metformin (MET) for 24h. Graph represents pAMPK/AMPK ratios normalized with respect to Mock infected cells with no MET treatment. pAMPK/AMPK ratios were obtained adjusting each protein with β-actin.

Mentions: The reduction of the HMGCR phosphorylation is related with an increased activity [38]. Previously, our group reported that DENV infection decreases the phosphorylated levels of HMGCR at early time post-infection in Huh7 cells [33], which suggest that during DENV infection this enzyme is activated. The main upstream kinase involved in HMGCR phosphorylation is the 5' adenosine AMP-activated protein (AMPK). AMPK activity is modulated through the phosphorylation in the threonine 172. To evaluate if DENV infection modulates the AMPK activity, the phosphorylation levels of AMPK at Thr-172 were evaluated by ELISA in mock and DENV infected cells (MOI 3) at different hour post-infection (hpi). The time course of AMPK activity in DENV infected cells (serotype 2 and 4) showed a reduction of AMPK phosphorylation (AMPKαT172) at 12 and 24 hpi compared to mock cells (Fig 1A). NS3 viral protein was evaluated as evidence of infection (Fig 1A, lower panel). The reduction of AMPKα phosphorylation induced by DENV at 24 hpi was also observed by western blot using antibodies directed against AMPKα phosphorylated form and total AMPKα. A MOI-dependent reduction of AMPK phosphorylation was observed in DENV2 infected cells at MOI of 1 and 3. (Fig 1B). Next, we confirmed these results using a pharmacological approach. Mock and DENV 2/4 infected cells were treated with DMSO 0.5% (vehicle, VEH), compound C (CC, AMPK inhibitor), and metformin (MET, AMPK activator) for 24h, and AMPK phosphorylation was analyzed by ELISA (Fig 1C). As expected, under control condition (mock cells) the CC-treatment inhibited the AMPK activity (39.57 ± 2.21 U/mL), while MET treatment enhanced the AMPK activation (263.16 ± 21.9 U/mL) compared to the mock VEH-treated cells (56.76±1.61 U/mL). Similarly, AMPK activity decayed in DENV 2/4 infected cells treated with VEH at 24 hpi (DENV2: 40.35 ± 2.3 U/mL, and DENV4: 34.9 ± 2.14 U/mL) respect to mock VEH-treated cells. This activity was similar to the observed with CC-treatment in mock cells (39.57 ± 2.21 U/mL). However, CC-treatment of infected cells did not cause a further reduction in the AMPK activity (DENV2: 38.97 ± 3.45, and DENV4: 32.11 ± 2.70). In contrast, MET-treatment enhanced the AMPK activity in DENV 2/4 infected cells (DENV2: 87.28 ± 9.85, and DENV4: 89.48 ± 10.21) compared to mock VEH-treated cells, but this increase was significantly lower (up to 2-fold) than the one observed in mock MET-treated cells (Fig 1C). Levels of NS3 viral protein were determined as infection evidence. Interestingly, MET-treated infected cells reduced the NS3 levels compared to VEH-treated infected cells (Fig 1C, lower panel). The AMPK phosphorylation was confirmed by western blot in mock or DENV2 infected cells treated with metformin for 24 h (Fig 1D). Likewise, the prM viral protein levels were reduced by metformin treatment (Fig 1D). These results demonstrate that DENV infection decreased the AMPK activity and suggest that the activation of AMPK induces a strong anti-DENV effect.


DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target
DENV infection down-regulates AMPK activity.In A, The AMPK activity, depicted as phosphorylation levels at Thr-172, was evaluated in Huh7 cells infected with DENV 2/4 (MOI 3) at 1, 12, and 24 hpi by ELISA, and NS3 viral protein levels (A, lower panel) were determined as infection test. AMPK activity was expressed as U/mL. *p<0.05 compared to mock infected cells (0 hpi). Data are means ± standard error (S.E) of n = 3 independent experiments realized by duplicate. (B) The levels of AMPK phosphorylated, AMPK total, and NS3 viral protein were analyzed by western blot in whole cell lysates obtained from Huh7 cells infected with DENV2 (MOI 0.1, 1 and 3) for 24 h. Graph represents the relative quantification of pAMPK respect to AMPK total protein. The pAMPK and total AMPK densitometry values were normalized with β-actin and pAMPK/AMPK ratios were calculated, Ratios are represented with respect to the indicated control. *p<0.05 compared to mock infected cells. Data are means ± standard error (S.E) of n = 4 independent experiments. (C) The AMPK activity and NS3 viral protein levels (C, lower panel) were determined in Mock or DENV 2/4 infected Huh7 cells treated with DMSO 0.5% (vehicle, VEH), 10 mM Metformin (MET, AMPK activator) or 10 μM Compound C (CC, AMPK inhibitor) for 24 h. *p<0.05 compared to mock VEH-treated cells, abp<0.05 compared to mock MET-treated cells. Data are means ± standard error (S.E) of n = 3 independent experiments realized by duplicate. (D) The levels of AMPK phosphorylated, AMPK total, and prM viral protein were analyzed by western blot in whole cell lysates obtained from Mock or DENV2 Huh7 infected cells (MOI 1 and 3) in the presence or absence of 10 mM metformin (MET) for 24h. Graph represents pAMPK/AMPK ratios normalized with respect to Mock infected cells with no MET treatment. pAMPK/AMPK ratios were obtained adjusting each protein with β-actin.
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ppat.1006257.g001: DENV infection down-regulates AMPK activity.In A, The AMPK activity, depicted as phosphorylation levels at Thr-172, was evaluated in Huh7 cells infected with DENV 2/4 (MOI 3) at 1, 12, and 24 hpi by ELISA, and NS3 viral protein levels (A, lower panel) were determined as infection test. AMPK activity was expressed as U/mL. *p<0.05 compared to mock infected cells (0 hpi). Data are means ± standard error (S.E) of n = 3 independent experiments realized by duplicate. (B) The levels of AMPK phosphorylated, AMPK total, and NS3 viral protein were analyzed by western blot in whole cell lysates obtained from Huh7 cells infected with DENV2 (MOI 0.1, 1 and 3) for 24 h. Graph represents the relative quantification of pAMPK respect to AMPK total protein. The pAMPK and total AMPK densitometry values were normalized with β-actin and pAMPK/AMPK ratios were calculated, Ratios are represented with respect to the indicated control. *p<0.05 compared to mock infected cells. Data are means ± standard error (S.E) of n = 4 independent experiments. (C) The AMPK activity and NS3 viral protein levels (C, lower panel) were determined in Mock or DENV 2/4 infected Huh7 cells treated with DMSO 0.5% (vehicle, VEH), 10 mM Metformin (MET, AMPK activator) or 10 μM Compound C (CC, AMPK inhibitor) for 24 h. *p<0.05 compared to mock VEH-treated cells, abp<0.05 compared to mock MET-treated cells. Data are means ± standard error (S.E) of n = 3 independent experiments realized by duplicate. (D) The levels of AMPK phosphorylated, AMPK total, and prM viral protein were analyzed by western blot in whole cell lysates obtained from Mock or DENV2 Huh7 infected cells (MOI 1 and 3) in the presence or absence of 10 mM metformin (MET) for 24h. Graph represents pAMPK/AMPK ratios normalized with respect to Mock infected cells with no MET treatment. pAMPK/AMPK ratios were obtained adjusting each protein with β-actin.
Mentions: The reduction of the HMGCR phosphorylation is related with an increased activity [38]. Previously, our group reported that DENV infection decreases the phosphorylated levels of HMGCR at early time post-infection in Huh7 cells [33], which suggest that during DENV infection this enzyme is activated. The main upstream kinase involved in HMGCR phosphorylation is the 5' adenosine AMP-activated protein (AMPK). AMPK activity is modulated through the phosphorylation in the threonine 172. To evaluate if DENV infection modulates the AMPK activity, the phosphorylation levels of AMPK at Thr-172 were evaluated by ELISA in mock and DENV infected cells (MOI 3) at different hour post-infection (hpi). The time course of AMPK activity in DENV infected cells (serotype 2 and 4) showed a reduction of AMPK phosphorylation (AMPKαT172) at 12 and 24 hpi compared to mock cells (Fig 1A). NS3 viral protein was evaluated as evidence of infection (Fig 1A, lower panel). The reduction of AMPKα phosphorylation induced by DENV at 24 hpi was also observed by western blot using antibodies directed against AMPKα phosphorylated form and total AMPKα. A MOI-dependent reduction of AMPK phosphorylation was observed in DENV2 infected cells at MOI of 1 and 3. (Fig 1B). Next, we confirmed these results using a pharmacological approach. Mock and DENV 2/4 infected cells were treated with DMSO 0.5% (vehicle, VEH), compound C (CC, AMPK inhibitor), and metformin (MET, AMPK activator) for 24h, and AMPK phosphorylation was analyzed by ELISA (Fig 1C). As expected, under control condition (mock cells) the CC-treatment inhibited the AMPK activity (39.57 ± 2.21 U/mL), while MET treatment enhanced the AMPK activation (263.16 ± 21.9 U/mL) compared to the mock VEH-treated cells (56.76±1.61 U/mL). Similarly, AMPK activity decayed in DENV 2/4 infected cells treated with VEH at 24 hpi (DENV2: 40.35 ± 2.3 U/mL, and DENV4: 34.9 ± 2.14 U/mL) respect to mock VEH-treated cells. This activity was similar to the observed with CC-treatment in mock cells (39.57 ± 2.21 U/mL). However, CC-treatment of infected cells did not cause a further reduction in the AMPK activity (DENV2: 38.97 ± 3.45, and DENV4: 32.11 ± 2.70). In contrast, MET-treatment enhanced the AMPK activity in DENV 2/4 infected cells (DENV2: 87.28 ± 9.85, and DENV4: 89.48 ± 10.21) compared to mock VEH-treated cells, but this increase was significantly lower (up to 2-fold) than the one observed in mock MET-treated cells (Fig 1C). Levels of NS3 viral protein were determined as infection evidence. Interestingly, MET-treated infected cells reduced the NS3 levels compared to VEH-treated infected cells (Fig 1C, lower panel). The AMPK phosphorylation was confirmed by western blot in mock or DENV2 infected cells treated with metformin for 24 h (Fig 1D). Likewise, the prM viral protein levels were reduced by metformin treatment (Fig 1D). These results demonstrate that DENV infection decreased the AMPK activity and suggest that the activation of AMPK induces a strong anti-DENV effect.

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