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Increased long chain acyl-Coa synthetase activity and fatty acid import is linked to membrane synthesis for development of picornavirus replication organelles.

Nchoutmboube JA, Viktorova EG, Scott AJ, Ford LA, Pei Z, Watkins PA, Ernst RK, Belov GA - PLoS Pathog. (2013)

Bottom Line: Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity.Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes.Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process.

View Article: PubMed Central - PubMed

Affiliation: Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT
All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be relevant for other (+)RNA viruses as well.

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Cleavage and redistribution of long chain acyl-CoA synthetases in infected cells and requirement of functional Acsl3 for polio replication and FA import.A. HeLa cells infected at 50 PFU/cells were incubated for 2, 4, and 6 hours post infection and collected for Western blot after permeabilization with digitonin for 5 min at room temperature (lanes 5–8); control cells (lanes 1–4) underwent the same treatment but without the detergent. Proteins were detected by multiple western blots of the same membrane after stripping of previous antibodies. Actin is shown as loading control. Results from a representative experiment are shown. Arrows indicate cleavage products detected with anti-FATP3 and Acsl3 antibodies. Arrowhead points to the loss of FATP3 after digitonin treatment from infected cells. B. Acsl3 knock-down severely impairs polio replicon replication (top panel) while showing minimal cytotoxicity (lower panel). siRNA knock-down efficiency of Acsl3 protein is shown. C. Expression of a fusion protein GFP-Acsl3-HA reduces poliovirus replication. HeLa cells were transfected overnight with either empty pUC plasmid, pEGFP-N1 plasmid or pGFP-Acsl3-HA plasmid. Cells were infected (V) with poliovirus at 50 PFU/cell or mock-infected (M) and collected for analysis at 4 h p.i. Polio 2C band intensity is normalized to the EGFP expressing sample. Expression of GFP-Acsl3 protein is detected with either anti-Acsl3 antibodies (second panel) or anti-GFP antibodies (third panel) which also show expression of EGFP (forth panel). Actin is shown as loading control. D. Knock-down of ACSL3 expression reduces activation of FA import upon expression of poliovirus proteins. HeLa cells were transfected with control or ACSL-3-targeting siRNA and 48 h later they were transfected with the plasmid pTM-2A-3D coding for the entire poliovirus non-structural polyprotein fragment P2P3. The next day expression of polio proteins was induced by infection of cells with vaccinia-T7 virus. Bodipy-FA label was added for 30 min at 4 h post vaccinia-T7 infection. Statistical analysis of ∼150 cells from each sample shows bodipy-FA signal normalized to poliovirus antigen 2B fluorescence, p value is shown. Western blot shows ACSL3 knock-down, actin is shown as a loading control.
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ppat-1003401-g005: Cleavage and redistribution of long chain acyl-CoA synthetases in infected cells and requirement of functional Acsl3 for polio replication and FA import.A. HeLa cells infected at 50 PFU/cells were incubated for 2, 4, and 6 hours post infection and collected for Western blot after permeabilization with digitonin for 5 min at room temperature (lanes 5–8); control cells (lanes 1–4) underwent the same treatment but without the detergent. Proteins were detected by multiple western blots of the same membrane after stripping of previous antibodies. Actin is shown as loading control. Results from a representative experiment are shown. Arrows indicate cleavage products detected with anti-FATP3 and Acsl3 antibodies. Arrowhead points to the loss of FATP3 after digitonin treatment from infected cells. B. Acsl3 knock-down severely impairs polio replicon replication (top panel) while showing minimal cytotoxicity (lower panel). siRNA knock-down efficiency of Acsl3 protein is shown. C. Expression of a fusion protein GFP-Acsl3-HA reduces poliovirus replication. HeLa cells were transfected overnight with either empty pUC plasmid, pEGFP-N1 plasmid or pGFP-Acsl3-HA plasmid. Cells were infected (V) with poliovirus at 50 PFU/cell or mock-infected (M) and collected for analysis at 4 h p.i. Polio 2C band intensity is normalized to the EGFP expressing sample. Expression of GFP-Acsl3 protein is detected with either anti-Acsl3 antibodies (second panel) or anti-GFP antibodies (third panel) which also show expression of EGFP (forth panel). Actin is shown as loading control. D. Knock-down of ACSL3 expression reduces activation of FA import upon expression of poliovirus proteins. HeLa cells were transfected with control or ACSL-3-targeting siRNA and 48 h later they were transfected with the plasmid pTM-2A-3D coding for the entire poliovirus non-structural polyprotein fragment P2P3. The next day expression of polio proteins was induced by infection of cells with vaccinia-T7 virus. Bodipy-FA label was added for 30 min at 4 h post vaccinia-T7 infection. Statistical analysis of ∼150 cells from each sample shows bodipy-FA signal normalized to poliovirus antigen 2B fluorescence, p value is shown. Western blot shows ACSL3 knock-down, actin is shown as a loading control.

Mentions: The human genome contains genes for 13 long and very long chain acyl-CoA synthetases that may facilitate FA uptake by the cells [22]. The data on expression profiles of these proteins as well as on their contribution to cellular metabolism are still very fragmentary and controversial [37].First, we monitored by western blot several long-chain acyl-CoA synthetases for which the reliable antibodies were available. We observed specific proteolytic cleavage of FATP3 and to a lesser extent Acsl3 proteins in infected cells, suggesting that their activity is being actively regulated (Figure 5A, arrows). Western blots of Acsl5 and FATP4 did not reveal obvious modifications of these enzymes in infected cells (Figure 5A). To see if association of acyl-CoA synthetases with cellular components is changed upon infection we treated the cells with digitonin. The membrane-targeted viral proteins 2C and 2BC were virtually totally recovered from the permeabilized cells. At the same soluble proteins 3D and 3CD were mostly lost upon cell permeabilization confirming optimal permeabilization conditions (Fig. 5A). Some amount of 3D and 3CD is expected to remain associated with the membrane-bound viral replication complexes. We observed significant loss of FATP3 protein from permeabilized cells at 4 and 6 h p.i. indicating that its association with cellular components is changing (Figure 5A, arrowhead). FATP3 was previously shown to be an-ER-localized protein with its N-terminus inserted into the ER lumen [21], and would be expected to remain in cells after digitonin treatment, as we see in the mock-infected cells. Thus its loss from the infected samples demonstrates that association of this protein with cellular structures is changing upon infection. Interestingly, FATP3 is one of the two long chain acyl-CoA synthetases undergoing proteolytic processing upon infection.


Increased long chain acyl-Coa synthetase activity and fatty acid import is linked to membrane synthesis for development of picornavirus replication organelles.

Nchoutmboube JA, Viktorova EG, Scott AJ, Ford LA, Pei Z, Watkins PA, Ernst RK, Belov GA - PLoS Pathog. (2013)

Cleavage and redistribution of long chain acyl-CoA synthetases in infected cells and requirement of functional Acsl3 for polio replication and FA import.A. HeLa cells infected at 50 PFU/cells were incubated for 2, 4, and 6 hours post infection and collected for Western blot after permeabilization with digitonin for 5 min at room temperature (lanes 5–8); control cells (lanes 1–4) underwent the same treatment but without the detergent. Proteins were detected by multiple western blots of the same membrane after stripping of previous antibodies. Actin is shown as loading control. Results from a representative experiment are shown. Arrows indicate cleavage products detected with anti-FATP3 and Acsl3 antibodies. Arrowhead points to the loss of FATP3 after digitonin treatment from infected cells. B. Acsl3 knock-down severely impairs polio replicon replication (top panel) while showing minimal cytotoxicity (lower panel). siRNA knock-down efficiency of Acsl3 protein is shown. C. Expression of a fusion protein GFP-Acsl3-HA reduces poliovirus replication. HeLa cells were transfected overnight with either empty pUC plasmid, pEGFP-N1 plasmid or pGFP-Acsl3-HA plasmid. Cells were infected (V) with poliovirus at 50 PFU/cell or mock-infected (M) and collected for analysis at 4 h p.i. Polio 2C band intensity is normalized to the EGFP expressing sample. Expression of GFP-Acsl3 protein is detected with either anti-Acsl3 antibodies (second panel) or anti-GFP antibodies (third panel) which also show expression of EGFP (forth panel). Actin is shown as loading control. D. Knock-down of ACSL3 expression reduces activation of FA import upon expression of poliovirus proteins. HeLa cells were transfected with control or ACSL-3-targeting siRNA and 48 h later they were transfected with the plasmid pTM-2A-3D coding for the entire poliovirus non-structural polyprotein fragment P2P3. The next day expression of polio proteins was induced by infection of cells with vaccinia-T7 virus. Bodipy-FA label was added for 30 min at 4 h post vaccinia-T7 infection. Statistical analysis of ∼150 cells from each sample shows bodipy-FA signal normalized to poliovirus antigen 2B fluorescence, p value is shown. Western blot shows ACSL3 knock-down, actin is shown as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675155&req=5

ppat-1003401-g005: Cleavage and redistribution of long chain acyl-CoA synthetases in infected cells and requirement of functional Acsl3 for polio replication and FA import.A. HeLa cells infected at 50 PFU/cells were incubated for 2, 4, and 6 hours post infection and collected for Western blot after permeabilization with digitonin for 5 min at room temperature (lanes 5–8); control cells (lanes 1–4) underwent the same treatment but without the detergent. Proteins were detected by multiple western blots of the same membrane after stripping of previous antibodies. Actin is shown as loading control. Results from a representative experiment are shown. Arrows indicate cleavage products detected with anti-FATP3 and Acsl3 antibodies. Arrowhead points to the loss of FATP3 after digitonin treatment from infected cells. B. Acsl3 knock-down severely impairs polio replicon replication (top panel) while showing minimal cytotoxicity (lower panel). siRNA knock-down efficiency of Acsl3 protein is shown. C. Expression of a fusion protein GFP-Acsl3-HA reduces poliovirus replication. HeLa cells were transfected overnight with either empty pUC plasmid, pEGFP-N1 plasmid or pGFP-Acsl3-HA plasmid. Cells were infected (V) with poliovirus at 50 PFU/cell or mock-infected (M) and collected for analysis at 4 h p.i. Polio 2C band intensity is normalized to the EGFP expressing sample. Expression of GFP-Acsl3 protein is detected with either anti-Acsl3 antibodies (second panel) or anti-GFP antibodies (third panel) which also show expression of EGFP (forth panel). Actin is shown as loading control. D. Knock-down of ACSL3 expression reduces activation of FA import upon expression of poliovirus proteins. HeLa cells were transfected with control or ACSL-3-targeting siRNA and 48 h later they were transfected with the plasmid pTM-2A-3D coding for the entire poliovirus non-structural polyprotein fragment P2P3. The next day expression of polio proteins was induced by infection of cells with vaccinia-T7 virus. Bodipy-FA label was added for 30 min at 4 h post vaccinia-T7 infection. Statistical analysis of ∼150 cells from each sample shows bodipy-FA signal normalized to poliovirus antigen 2B fluorescence, p value is shown. Western blot shows ACSL3 knock-down, actin is shown as a loading control.
Mentions: The human genome contains genes for 13 long and very long chain acyl-CoA synthetases that may facilitate FA uptake by the cells [22]. The data on expression profiles of these proteins as well as on their contribution to cellular metabolism are still very fragmentary and controversial [37].First, we monitored by western blot several long-chain acyl-CoA synthetases for which the reliable antibodies were available. We observed specific proteolytic cleavage of FATP3 and to a lesser extent Acsl3 proteins in infected cells, suggesting that their activity is being actively regulated (Figure 5A, arrows). Western blots of Acsl5 and FATP4 did not reveal obvious modifications of these enzymes in infected cells (Figure 5A). To see if association of acyl-CoA synthetases with cellular components is changed upon infection we treated the cells with digitonin. The membrane-targeted viral proteins 2C and 2BC were virtually totally recovered from the permeabilized cells. At the same soluble proteins 3D and 3CD were mostly lost upon cell permeabilization confirming optimal permeabilization conditions (Fig. 5A). Some amount of 3D and 3CD is expected to remain associated with the membrane-bound viral replication complexes. We observed significant loss of FATP3 protein from permeabilized cells at 4 and 6 h p.i. indicating that its association with cellular components is changing (Figure 5A, arrowhead). FATP3 was previously shown to be an-ER-localized protein with its N-terminus inserted into the ER lumen [21], and would be expected to remain in cells after digitonin treatment, as we see in the mock-infected cells. Thus its loss from the infected samples demonstrates that association of this protein with cellular structures is changing upon infection. Interestingly, FATP3 is one of the two long chain acyl-CoA synthetases undergoing proteolytic processing upon infection.

Bottom Line: Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity.Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes.Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process.

View Article: PubMed Central - PubMed

Affiliation: Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT
All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be relevant for other (+)RNA viruses as well.

Show MeSH
Related in: MedlinePlus