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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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Modulation of long chain acyl-CoA synthetase activity in poliovirus-infected cells.A. Acyl-CoA synthetase activity is stimulated as early as 2 h p. i. and continues to increase during the time-course of infection. Acyl-CoA synthetase activity in vitro assay was performed with lysates of HeLa cells collected at indicated times post infection, the data are normalized to the activity of the lysate from mock-infected collected at 2 h p. i., p-values are shown. B. and C. Fatty import competition assay performed with the cells incubated in serum- supplemented medium during the whole experiment. HeLa cells were infected (or mock-infected) with poliovirus at 50 PFU/cell; at 4 h.p.i. bodipy-FA was added for 30 min in the presence of 125× molar excess of the indicated long chain fatty acids. No competitor fatty acid was added to control samples. The data are normalized to the signal from the mock-infected control sample; p-values are shown. D. and E. Fatty import competition assay performed with the cells incubated in serum-free medium during the whole experiment. HeLa cells were infected (or mock-infected) with poliovirus at 50 PFU/cell; at 4 h.p.i. bodipy-FA was added for 30 min in the presence of 125× molar excess of the indicated long chain fatty acids. No competitor fatty acid was added to control samples. The data are normalized to the signal from the mock-infected control sample; p-values indicating significant differences are shown.
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ppat-1003401-g003: Modulation of long chain acyl-CoA synthetase activity in poliovirus-infected cells.A. Acyl-CoA synthetase activity is stimulated as early as 2 h p. i. and continues to increase during the time-course of infection. Acyl-CoA synthetase activity in vitro assay was performed with lysates of HeLa cells collected at indicated times post infection, the data are normalized to the activity of the lysate from mock-infected collected at 2 h p. i., p-values are shown. B. and C. Fatty import competition assay performed with the cells incubated in serum- supplemented medium during the whole experiment. HeLa cells were infected (or mock-infected) with poliovirus at 50 PFU/cell; at 4 h.p.i. bodipy-FA was added for 30 min in the presence of 125× molar excess of the indicated long chain fatty acids. No competitor fatty acid was added to control samples. The data are normalized to the signal from the mock-infected control sample; p-values are shown. D. and E. Fatty import competition assay performed with the cells incubated in serum-free medium during the whole experiment. HeLa cells were infected (or mock-infected) with poliovirus at 50 PFU/cell; at 4 h.p.i. bodipy-FA was added for 30 min in the presence of 125× molar excess of the indicated long chain fatty acids. No competitor fatty acid was added to control samples. The data are normalized to the signal from the mock-infected control sample; p-values indicating significant differences are shown.

Mentions: Import of FAs is inextricably connected to activity of acyl-CoA synthetases [23]. Transport of saturated or unsaturated long-chain fatty acids containing 18 or fewer carbons across biological membranes is rapid and not thought to be rate-limiting [31], [32]. Thus the increased uptake of FFA probe suggests that long chain acyl-CoA synthetase activity must be up-regulated upon infection. To measure this activity we prepared lysates from HeLa cells infected at the multiplicity of 50 PFU/cell and incubated without serum for different times post-infection. The infected cells demonstrated elevated level of acyl-CoA synthetase activity as early as 2 h p.i which steadily increased at later times (Fig. 3A).


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)

Modulation of long chain acyl-CoA synthetase activity in poliovirus-infected cells.A. Acyl-CoA synthetase activity is stimulated as early as 2 h p. i. and continues to increase during the time-course of infection. Acyl-CoA synthetase activity in vitro assay was performed with lysates of HeLa cells collected at indicated times post infection, the data are normalized to the activity of the lysate from mock-infected collected at 2 h p. i., p-values are shown. B. and C. Fatty import competition assay performed with the cells incubated in serum- supplemented medium during the whole experiment. HeLa cells were infected (or mock-infected) with poliovirus at 50 PFU/cell; at 4 h.p.i. bodipy-FA was added for 30 min in the presence of 125× molar excess of the indicated long chain fatty acids. No competitor fatty acid was added to control samples. The data are normalized to the signal from the mock-infected control sample; p-values are shown. D. and E. Fatty import competition assay performed with the cells incubated in serum-free medium during the whole experiment. HeLa cells were infected (or mock-infected) with poliovirus at 50 PFU/cell; at 4 h.p.i. bodipy-FA was added for 30 min in the presence of 125× molar excess of the indicated long chain fatty acids. No competitor fatty acid was added to control samples. The data are normalized to the signal from the mock-infected control sample; p-values indicating significant differences are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675155&req=5

ppat-1003401-g003: Modulation of long chain acyl-CoA synthetase activity in poliovirus-infected cells.A. Acyl-CoA synthetase activity is stimulated as early as 2 h p. i. and continues to increase during the time-course of infection. Acyl-CoA synthetase activity in vitro assay was performed with lysates of HeLa cells collected at indicated times post infection, the data are normalized to the activity of the lysate from mock-infected collected at 2 h p. i., p-values are shown. B. and C. Fatty import competition assay performed with the cells incubated in serum- supplemented medium during the whole experiment. HeLa cells were infected (or mock-infected) with poliovirus at 50 PFU/cell; at 4 h.p.i. bodipy-FA was added for 30 min in the presence of 125× molar excess of the indicated long chain fatty acids. No competitor fatty acid was added to control samples. The data are normalized to the signal from the mock-infected control sample; p-values are shown. D. and E. Fatty import competition assay performed with the cells incubated in serum-free medium during the whole experiment. HeLa cells were infected (or mock-infected) with poliovirus at 50 PFU/cell; at 4 h.p.i. bodipy-FA was added for 30 min in the presence of 125× molar excess of the indicated long chain fatty acids. No competitor fatty acid was added to control samples. The data are normalized to the signal from the mock-infected control sample; p-values indicating significant differences are shown.
Mentions: Import of FAs is inextricably connected to activity of acyl-CoA synthetases [23]. Transport of saturated or unsaturated long-chain fatty acids containing 18 or fewer carbons across biological membranes is rapid and not thought to be rate-limiting [31], [32]. Thus the increased uptake of FFA probe suggests that long chain acyl-CoA synthetase activity must be up-regulated upon infection. To measure this activity we prepared lysates from HeLa cells infected at the multiplicity of 50 PFU/cell and incubated without serum for different times post-infection. The infected cells demonstrated elevated level of acyl-CoA synthetase activity as early as 2 h p.i which steadily increased at later times (Fig. 3A).

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