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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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Shift in phosphatidylcholine spectrum following infection demonstrated by TLC-MALDI.A. Schematic representation of TLC-MALDI, blue spots represent phospholipid migration on TLC plate, hash marks represent stepwise MALDI data capture. B. HeLa cells were infected with poliovirus at 50 PFU/cell and processed for the total lipid extraction at the indicated time points post infection. TLC-MALDI data shown at phosphatidylcholine (PC) migration (Rf) range, fatty acid chain lengths noted, intensity of signal at respective mass to charge ratios (m/z) (blue scale). C. PC diversity (unique m/z signatures) shifts in abundance from higher Rf to lower Rf during time course of infection. D. Percent change of PC subset/PC total ratio compared to mock. Results from a representative experiment are shown.
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ppat-1003401-g004: Shift in phosphatidylcholine spectrum following infection demonstrated by TLC-MALDI.A. Schematic representation of TLC-MALDI, blue spots represent phospholipid migration on TLC plate, hash marks represent stepwise MALDI data capture. B. HeLa cells were infected with poliovirus at 50 PFU/cell and processed for the total lipid extraction at the indicated time points post infection. TLC-MALDI data shown at phosphatidylcholine (PC) migration (Rf) range, fatty acid chain lengths noted, intensity of signal at respective mass to charge ratios (m/z) (blue scale). C. PC diversity (unique m/z signatures) shifts in abundance from higher Rf to lower Rf during time course of infection. D. Percent change of PC subset/PC total ratio compared to mock. Results from a representative experiment are shown.

Mentions: The competition experiments suggest that the pool of acyl-CoAs available for new phospholipid synthesis should be different in infected and mock-infected cells. To investigate the changes in the spectrum of PC molecules, TLC-MALDI was used to couple the power of solvent resolution of phospholipids by TLC to the mass identification capacity of matrix assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Fig. 4A). PC was first identified by characteristic TLC migration, and reflectron positive mode MALDI-TOF-MS was used to scan the TLC lane. The mass to charge ratio (m/z) was used to secondarily identify the major PC molecules and acyl variants (Fig. 4B). We observed a substantial drop in the diversity of the PC molecules containing long C18 chains, running in the high Rf chromatography zone, and correspondingly a rapid increase in the PCs with shorter acyl chains from the low Rf zone upon infection (Fig. 4C). The analysis of the individual PC classes demonstrates a fast shift in the composition of PCs during infection. At 2 h p. i. there is already a significant increase in PCs with C18/C18 acyl chains, as well as C16/C18 ones, accompanied by a noticeable drop in the abundance of the C14/C16 and C16/C16 PCs, compared to mock-infected cells. This general trend continues later in infection with an especially strong increase in the C16/C18 PC species at 4 h p. i. (Fig. 4D). Changes in lipid abundance at 6 h.p.i. do not follow the general trends observed at 2 and 4 h.p.i. likely due to the significant degree of cell lysis observed at this late stage of infection at high MOI. It should be noted that while the competition assay showed a strong preference for import of C14 myristic acid to infected cells, it only reflects the changes in the prevalent cellular acyl-CoA synthetase activity induced by polio infection, and cannot be directly interpreted as that myristic acid is the predominant imported FA in natural conditions. The actual composition of intracellular acyl-CoA pool will be shaped by the availability of the corresponding FA substrates. The resolution of TLC-MALDI is not sufficient to separate PC molecules with saturated and unsaturated FA chains with the same number of carbon atoms. Thus, PV infection does not only up-regulates the overall synthesis of PC but specifically changes the molecular composition of this structural phospholipid indicating that membranes of PV replication complexes are significantly different from the pre-existing cellular membranes.


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)

Shift in phosphatidylcholine spectrum following infection demonstrated by TLC-MALDI.A. Schematic representation of TLC-MALDI, blue spots represent phospholipid migration on TLC plate, hash marks represent stepwise MALDI data capture. B. HeLa cells were infected with poliovirus at 50 PFU/cell and processed for the total lipid extraction at the indicated time points post infection. TLC-MALDI data shown at phosphatidylcholine (PC) migration (Rf) range, fatty acid chain lengths noted, intensity of signal at respective mass to charge ratios (m/z) (blue scale). C. PC diversity (unique m/z signatures) shifts in abundance from higher Rf to lower Rf during time course of infection. D. Percent change of PC subset/PC total ratio compared to mock. Results from a representative experiment are shown.
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getmorefigures.php?uid=PMC3675155&req=5

ppat-1003401-g004: Shift in phosphatidylcholine spectrum following infection demonstrated by TLC-MALDI.A. Schematic representation of TLC-MALDI, blue spots represent phospholipid migration on TLC plate, hash marks represent stepwise MALDI data capture. B. HeLa cells were infected with poliovirus at 50 PFU/cell and processed for the total lipid extraction at the indicated time points post infection. TLC-MALDI data shown at phosphatidylcholine (PC) migration (Rf) range, fatty acid chain lengths noted, intensity of signal at respective mass to charge ratios (m/z) (blue scale). C. PC diversity (unique m/z signatures) shifts in abundance from higher Rf to lower Rf during time course of infection. D. Percent change of PC subset/PC total ratio compared to mock. Results from a representative experiment are shown.
Mentions: The competition experiments suggest that the pool of acyl-CoAs available for new phospholipid synthesis should be different in infected and mock-infected cells. To investigate the changes in the spectrum of PC molecules, TLC-MALDI was used to couple the power of solvent resolution of phospholipids by TLC to the mass identification capacity of matrix assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Fig. 4A). PC was first identified by characteristic TLC migration, and reflectron positive mode MALDI-TOF-MS was used to scan the TLC lane. The mass to charge ratio (m/z) was used to secondarily identify the major PC molecules and acyl variants (Fig. 4B). We observed a substantial drop in the diversity of the PC molecules containing long C18 chains, running in the high Rf chromatography zone, and correspondingly a rapid increase in the PCs with shorter acyl chains from the low Rf zone upon infection (Fig. 4C). The analysis of the individual PC classes demonstrates a fast shift in the composition of PCs during infection. At 2 h p. i. there is already a significant increase in PCs with C18/C18 acyl chains, as well as C16/C18 ones, accompanied by a noticeable drop in the abundance of the C14/C16 and C16/C16 PCs, compared to mock-infected cells. This general trend continues later in infection with an especially strong increase in the C16/C18 PC species at 4 h p. i. (Fig. 4D). Changes in lipid abundance at 6 h.p.i. do not follow the general trends observed at 2 and 4 h.p.i. likely due to the significant degree of cell lysis observed at this late stage of infection at high MOI. It should be noted that while the competition assay showed a strong preference for import of C14 myristic acid to infected cells, it only reflects the changes in the prevalent cellular acyl-CoA synthetase activity induced by polio infection, and cannot be directly interpreted as that myristic acid is the predominant imported FA in natural conditions. The actual composition of intracellular acyl-CoA pool will be shaped by the availability of the corresponding FA substrates. The resolution of TLC-MALDI is not sufficient to separate PC molecules with saturated and unsaturated FA chains with the same number of carbon atoms. Thus, PV infection does not only up-regulates the overall synthesis of PC but specifically changes the molecular composition of this structural phospholipid indicating that membranes of PV replication complexes are significantly different from the pre-existing cellular membranes.

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