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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

Metformin induces an antiviral effect in DENV infected cells.In A and B, The antiviral effect of metformin-treatment (0, 1 and 10 mM) against DENV infection was evaluated in supernatants from Huh7 cells infected (MOI 3) with DENV2 (A) and DENV4 (B) at 24 hpi through determination of viral yield by foci assay, and NS1 secretion by ELISA. 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. (C) The percentage of infected cells after 10 mM metformin-treatment was determined by flow cytometry using a mouse anti-E monoclonal antibody-4G2 to detect the E viral protein in mock or DENV 2/4 infected cells. Upper histograms show the fluorescence of infected cells at 24h (gray filled histograms) or 48h (dark filled histograms) respect to mock infected cells (non-filled histograms). Lower histograms display the fluorescence of DENV infected cells treated with metformin (gray filled histograms) respect to vehicle-treated infected cells (dark filled histograms). (D) The Mean Fluorescence intensity (MFI) is presented on Graphs. (E) DENV 2/4 infected cells treated with 10mM metformin (MET) were visualized at 24 hpi by confocal microscopy using a mouse anti-E monoclonal antibody-4G2 (green). Nuclei were stained with Hoechst (blue). Scale bar 50 μm. Images correspond to one experiment representative of n = 3. (F) The number of viral genome copies of DENV 2/4 infected cells treated with metformin (0, 1, 10 mM) for 24h was examined by qRT-PCR, and expressed as Log of No. Copies. DMSO 0.5% was used as vehicle for all cases (0 mM). Data are means ± S.E of n = 3 independent experiments realized by duplicated. * p<0.05 compared to non-treated cells.
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ppat.1006257.g006: Metformin induces an antiviral effect in DENV infected cells.In A and B, The antiviral effect of metformin-treatment (0, 1 and 10 mM) against DENV infection was evaluated in supernatants from Huh7 cells infected (MOI 3) with DENV2 (A) and DENV4 (B) at 24 hpi through determination of viral yield by foci assay, and NS1 secretion by ELISA. 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. (C) The percentage of infected cells after 10 mM metformin-treatment was determined by flow cytometry using a mouse anti-E monoclonal antibody-4G2 to detect the E viral protein in mock or DENV 2/4 infected cells. Upper histograms show the fluorescence of infected cells at 24h (gray filled histograms) or 48h (dark filled histograms) respect to mock infected cells (non-filled histograms). Lower histograms display the fluorescence of DENV infected cells treated with metformin (gray filled histograms) respect to vehicle-treated infected cells (dark filled histograms). (D) The Mean Fluorescence intensity (MFI) is presented on Graphs. (E) DENV 2/4 infected cells treated with 10mM metformin (MET) were visualized at 24 hpi by confocal microscopy using a mouse anti-E monoclonal antibody-4G2 (green). Nuclei were stained with Hoechst (blue). Scale bar 50 μm. Images correspond to one experiment representative of n = 3. (F) The number of viral genome copies of DENV 2/4 infected cells treated with metformin (0, 1, 10 mM) for 24h was examined by qRT-PCR, and expressed as Log of No. Copies. DMSO 0.5% was used as vehicle for all cases (0 mM). Data are means ± S.E of n = 3 independent experiments realized by duplicated. * p<0.05 compared to non-treated cells.

Mentions: The replicative complexes disruption promoted by the activation of AMPK supports our hypothesis that this compound could inflict important inhibition of DENV infection. To further analysis of the antiviral effect induced by the activation of AMPK, DENV2 infected Huh7 cells were treated for 24 h with increasing concentrations of direct and indirect AMPK activators (MET, TMPA and A-769662) and lovastatin and infection was evaluated by percentage of infected cells (FACS) and viral yield (foci assay). The reduction in amount of infected cells promoted by all the drugs was concentration-dependent, The discrepancies between drugs could be explained by the concentrations used of each drug (S5A and S5B Fig). Cell viability assay in the presence of the different drugs is presented in supplemental material (S7 Fig). Additionally, viral yield was reduced by AMPK activation and lovastatin. Particularly, A769662 reduced viral yield for 2 log meanwhile other drugs reduced viral progeny for 1 log, however this drug just reduced the amount of infected cells just in 15% suggesting that the analog of AMP has an important effect in viral particle assembly or secretion (S5C and S5D Fig). From the AMPK activators tested in this work, the only drug approved by FDA is MET, then, we considered to evaluate the antiviral effect of this drug in a deeper way. It was confirmed that MET-treatment declined in a dose-dependent manner the viral yield up to one logarithm and the NS1 secretion up to 90% in infected cells with both serotype DENV2 (Fig 6A) and DENV4 (Fig 6B) at 24 hpi. In addition, the inhibition of DENV infection was demonstrated by flow cytometry, confocal microscopy and viral genome amplification (qRT-PCR). The 10 mM MET-treatment decreased the amount of infected cells with both serotype DENV2 and DENV4 at 24 and 48 hpi compared to non-treated cells (Fig 6C lower panels darker histograms and 6D). Images by confocal microscopy showed a significant reduction in DENV2 and DENV4 infected cells (Fig 6E, green color cells) after 10 mM metformin-treatment at 24 hpi compared to non-treated cells. Finally, a dose-dependent reduction in viral genome copies (up to 0.7 logarithm for DENV2, and 1.5 logarithm for DENV4) was observed in infected cells after metformin-treatment at 24 hpi respect to non-treated cells (Fig 6F). These data demonstrate that MET has a significant anti-DENV effect without affects the cell viability (S7 Fig).


DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target
Metformin induces an antiviral effect in DENV infected cells.In A and B, The antiviral effect of metformin-treatment (0, 1 and 10 mM) against DENV infection was evaluated in supernatants from Huh7 cells infected (MOI 3) with DENV2 (A) and DENV4 (B) at 24 hpi through determination of viral yield by foci assay, and NS1 secretion by ELISA. 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. (C) The percentage of infected cells after 10 mM metformin-treatment was determined by flow cytometry using a mouse anti-E monoclonal antibody-4G2 to detect the E viral protein in mock or DENV 2/4 infected cells. Upper histograms show the fluorescence of infected cells at 24h (gray filled histograms) or 48h (dark filled histograms) respect to mock infected cells (non-filled histograms). Lower histograms display the fluorescence of DENV infected cells treated with metformin (gray filled histograms) respect to vehicle-treated infected cells (dark filled histograms). (D) The Mean Fluorescence intensity (MFI) is presented on Graphs. (E) DENV 2/4 infected cells treated with 10mM metformin (MET) were visualized at 24 hpi by confocal microscopy using a mouse anti-E monoclonal antibody-4G2 (green). Nuclei were stained with Hoechst (blue). Scale bar 50 μm. Images correspond to one experiment representative of n = 3. (F) The number of viral genome copies of DENV 2/4 infected cells treated with metformin (0, 1, 10 mM) for 24h was examined by qRT-PCR, and expressed as Log of No. Copies. DMSO 0.5% was used as vehicle for all cases (0 mM). Data are means ± S.E of n = 3 independent experiments realized by duplicated. * p<0.05 compared to non-treated cells.
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ppat.1006257.g006: Metformin induces an antiviral effect in DENV infected cells.In A and B, The antiviral effect of metformin-treatment (0, 1 and 10 mM) against DENV infection was evaluated in supernatants from Huh7 cells infected (MOI 3) with DENV2 (A) and DENV4 (B) at 24 hpi through determination of viral yield by foci assay, and NS1 secretion by ELISA. 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. (C) The percentage of infected cells after 10 mM metformin-treatment was determined by flow cytometry using a mouse anti-E monoclonal antibody-4G2 to detect the E viral protein in mock or DENV 2/4 infected cells. Upper histograms show the fluorescence of infected cells at 24h (gray filled histograms) or 48h (dark filled histograms) respect to mock infected cells (non-filled histograms). Lower histograms display the fluorescence of DENV infected cells treated with metformin (gray filled histograms) respect to vehicle-treated infected cells (dark filled histograms). (D) The Mean Fluorescence intensity (MFI) is presented on Graphs. (E) DENV 2/4 infected cells treated with 10mM metformin (MET) were visualized at 24 hpi by confocal microscopy using a mouse anti-E monoclonal antibody-4G2 (green). Nuclei were stained with Hoechst (blue). Scale bar 50 μm. Images correspond to one experiment representative of n = 3. (F) The number of viral genome copies of DENV 2/4 infected cells treated with metformin (0, 1, 10 mM) for 24h was examined by qRT-PCR, and expressed as Log of No. Copies. DMSO 0.5% was used as vehicle for all cases (0 mM). Data are means ± S.E of n = 3 independent experiments realized by duplicated. * p<0.05 compared to non-treated cells.
Mentions: The replicative complexes disruption promoted by the activation of AMPK supports our hypothesis that this compound could inflict important inhibition of DENV infection. To further analysis of the antiviral effect induced by the activation of AMPK, DENV2 infected Huh7 cells were treated for 24 h with increasing concentrations of direct and indirect AMPK activators (MET, TMPA and A-769662) and lovastatin and infection was evaluated by percentage of infected cells (FACS) and viral yield (foci assay). The reduction in amount of infected cells promoted by all the drugs was concentration-dependent, The discrepancies between drugs could be explained by the concentrations used of each drug (S5A and S5B Fig). Cell viability assay in the presence of the different drugs is presented in supplemental material (S7 Fig). Additionally, viral yield was reduced by AMPK activation and lovastatin. Particularly, A769662 reduced viral yield for 2 log meanwhile other drugs reduced viral progeny for 1 log, however this drug just reduced the amount of infected cells just in 15% suggesting that the analog of AMP has an important effect in viral particle assembly or secretion (S5C and S5D Fig). From the AMPK activators tested in this work, the only drug approved by FDA is MET, then, we considered to evaluate the antiviral effect of this drug in a deeper way. It was confirmed that MET-treatment declined in a dose-dependent manner the viral yield up to one logarithm and the NS1 secretion up to 90% in infected cells with both serotype DENV2 (Fig 6A) and DENV4 (Fig 6B) at 24 hpi. In addition, the inhibition of DENV infection was demonstrated by flow cytometry, confocal microscopy and viral genome amplification (qRT-PCR). The 10 mM MET-treatment decreased the amount of infected cells with both serotype DENV2 and DENV4 at 24 and 48 hpi compared to non-treated cells (Fig 6C lower panels darker histograms and 6D). Images by confocal microscopy showed a significant reduction in DENV2 and DENV4 infected cells (Fig 6E, green color cells) after 10 mM metformin-treatment at 24 hpi compared to non-treated cells. Finally, a dose-dependent reduction in viral genome copies (up to 0.7 logarithm for DENV2, and 1.5 logarithm for DENV4) was observed in infected cells after metformin-treatment at 24 hpi respect to non-treated cells (Fig 6F). These data demonstrate that MET has a significant anti-DENV effect without affects the cell viability (S7 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