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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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Poliovirus infection induces strong activation of fatty acid import.HeLa cells were infected with poliovirus at 50 PFU/cell, and at 4 h p. i. Bodipy 500/510 C4–C9 (bodipy-FA) was added for 30 min. A. low magnification view of infected vs mock-infected cells. B. FACS analysis of the fluorescence of infected (mock-infected) cells after 30 min pulse label with bodipy-FA at 4 h p. I, or control samples incubated without bodipy-FA. C. Higher magnification image showing that bodipy-FA probe is redistributed preferentially into lipid droplets in mock-infected cells and into membranes in infected cells. D. A confocal image of HeLa cells expressing pmCherry-ADRP protein (a marker for lipid droplets) and labeled with bodipy-FA for 30 min. Arrow marks a lipid droplet that did not accumulate the newly-synthesized lipids during the labeling period and also indicate lack of bodipy fluorescence leakage into the red channel. E. A confocal image of infected (mock-infected) HeLa cells labeled at 4 h p. i. with bodipy-FA and Hoechst-33342 (cell permeable DNA stain) for 30 min showing localization of bodipy-FA staining. F. A confocal image of polio-infected HeLa cells incubated for 30 min with bodipy-FA at 4 h.p.i. and processed for staining for a viral membrane-targeted protein 2B showing co-localization of the viral antigen and imported FA. Colocalization panel shows colocalized green and blue pixels identified with ImageJ software.
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ppat-1003401-g001: Poliovirus infection induces strong activation of fatty acid import.HeLa cells were infected with poliovirus at 50 PFU/cell, and at 4 h p. i. Bodipy 500/510 C4–C9 (bodipy-FA) was added for 30 min. A. low magnification view of infected vs mock-infected cells. B. FACS analysis of the fluorescence of infected (mock-infected) cells after 30 min pulse label with bodipy-FA at 4 h p. I, or control samples incubated without bodipy-FA. C. Higher magnification image showing that bodipy-FA probe is redistributed preferentially into lipid droplets in mock-infected cells and into membranes in infected cells. D. A confocal image of HeLa cells expressing pmCherry-ADRP protein (a marker for lipid droplets) and labeled with bodipy-FA for 30 min. Arrow marks a lipid droplet that did not accumulate the newly-synthesized lipids during the labeling period and also indicate lack of bodipy fluorescence leakage into the red channel. E. A confocal image of infected (mock-infected) HeLa cells labeled at 4 h p. i. with bodipy-FA and Hoechst-33342 (cell permeable DNA stain) for 30 min showing localization of bodipy-FA staining. F. A confocal image of polio-infected HeLa cells incubated for 30 min with bodipy-FA at 4 h.p.i. and processed for staining for a viral membrane-targeted protein 2B showing co-localization of the viral antigen and imported FA. Colocalization panel shows colocalized green and blue pixels identified with ImageJ software.

Mentions: The increase of phospholipid synthesis in PV-infected cells [10], [12], [13] should be sustained by sufficient supply of corresponding precursors including long chain FAs. To monitor FA import we pulse-labeled PV-infected HeLa cells with a fluorescent fatty acid Bodipy 500/510 C4, 9 (bodipy-FA) which is believed to mimic FA with 18 carbon atoms backbone. This and similar molecules are extensively used in lipid metabolism research and it was previously shown to be rapidly utilized by cellular lipid synthesis machinery and incorporated into phospholipids, triglycerides and other natural lipids [24], [25], [26], [27], [28]. The cells were infected at a multiplicity of 50 PFU/cell to ensure simultaneous development of infection, and bodipy-FA was added for 30 min at 4 hours post infection (h p.i.), in the middle of the infectious cycle. The infected cells showed strongly increased import of bodipy-FA (Fig. 1A and B). In mock-infected cells the label was distributed into multiple round structures in the cytoplasm and was also found in intracellular ER-like staining (Fig. 1C, mock). The round bright dots were identified as lipid droplets since they co-localized with a well-established lipid droplet marker ADRP [29] (Fig. 1D). Note that some ADRP-positive structures did not accumulate bodipy-FA during 30 min labeling period (Fig. 1D, arrow), consistently with the previous results that individual lipid droplets accumulate newly-synthesized lipids at different rates [30]. In infected cells, bright bodipy-FA fluorescence surrounded the nuclei and often occupied the total cytoplasmic area reflecting robust development of the poliovirus replication complexes (note pycnotic nuclei in infected cells, characteristic of polio-induced cytopathic effect)(Fig. 1E). For the experiment shown on Fig. 1 the cells were incubated in serum-free media during the labeling period, so bodipy-FA was the only fatty acid available exogenously. We also monitored FA transport when cells were incubated in normal growth media supplemented with fetal bovine serum which provides ample supply of natural FA and other lipids. As expected, the level of fluorescent signal was lower in the presence of serum, due to competition with the fatty acids from serum, but the overall picture of strong stimulation of fatty acid import upon infection was the same (not shown). Cells on Figure 1C and E are imaged directly after formaldehyde fixation without further detergent permeabilization which we found to deteriorate the fine structure of the distribution of the bodipy-FA label, especially in weakly labeled mock-infected cells. Staining of cells for a viral antigen 2B, a marker of membranous replication complexes, revealed extensive co-localization of bodipy-FA fluorescence with polio replication structures, especially in the cells where viral protein staining could still be visualized as discrete domains in the confocal plain (Fig. 1F, arrowheads, also co-localization panel). With the further development of infection staining for both viral proteins and bodipy-FA tend to occupied all available perinuclear space reflecting massive development of membranous replication complexes. Staining for other membrane-targeted poliovirus replication proteins 2C and 3A revealed similar pattern of distribution of a viral antigen and bodipy-FA label (not shown).


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)

Poliovirus infection induces strong activation of fatty acid import.HeLa cells were infected with poliovirus at 50 PFU/cell, and at 4 h p. i. Bodipy 500/510 C4–C9 (bodipy-FA) was added for 30 min. A. low magnification view of infected vs mock-infected cells. B. FACS analysis of the fluorescence of infected (mock-infected) cells after 30 min pulse label with bodipy-FA at 4 h p. I, or control samples incubated without bodipy-FA. C. Higher magnification image showing that bodipy-FA probe is redistributed preferentially into lipid droplets in mock-infected cells and into membranes in infected cells. D. A confocal image of HeLa cells expressing pmCherry-ADRP protein (a marker for lipid droplets) and labeled with bodipy-FA for 30 min. Arrow marks a lipid droplet that did not accumulate the newly-synthesized lipids during the labeling period and also indicate lack of bodipy fluorescence leakage into the red channel. E. A confocal image of infected (mock-infected) HeLa cells labeled at 4 h p. i. with bodipy-FA and Hoechst-33342 (cell permeable DNA stain) for 30 min showing localization of bodipy-FA staining. F. A confocal image of polio-infected HeLa cells incubated for 30 min with bodipy-FA at 4 h.p.i. and processed for staining for a viral membrane-targeted protein 2B showing co-localization of the viral antigen and imported FA. Colocalization panel shows colocalized green and blue pixels identified with ImageJ software.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675155&req=5

ppat-1003401-g001: Poliovirus infection induces strong activation of fatty acid import.HeLa cells were infected with poliovirus at 50 PFU/cell, and at 4 h p. i. Bodipy 500/510 C4–C9 (bodipy-FA) was added for 30 min. A. low magnification view of infected vs mock-infected cells. B. FACS analysis of the fluorescence of infected (mock-infected) cells after 30 min pulse label with bodipy-FA at 4 h p. I, or control samples incubated without bodipy-FA. C. Higher magnification image showing that bodipy-FA probe is redistributed preferentially into lipid droplets in mock-infected cells and into membranes in infected cells. D. A confocal image of HeLa cells expressing pmCherry-ADRP protein (a marker for lipid droplets) and labeled with bodipy-FA for 30 min. Arrow marks a lipid droplet that did not accumulate the newly-synthesized lipids during the labeling period and also indicate lack of bodipy fluorescence leakage into the red channel. E. A confocal image of infected (mock-infected) HeLa cells labeled at 4 h p. i. with bodipy-FA and Hoechst-33342 (cell permeable DNA stain) for 30 min showing localization of bodipy-FA staining. F. A confocal image of polio-infected HeLa cells incubated for 30 min with bodipy-FA at 4 h.p.i. and processed for staining for a viral membrane-targeted protein 2B showing co-localization of the viral antigen and imported FA. Colocalization panel shows colocalized green and blue pixels identified with ImageJ software.
Mentions: The increase of phospholipid synthesis in PV-infected cells [10], [12], [13] should be sustained by sufficient supply of corresponding precursors including long chain FAs. To monitor FA import we pulse-labeled PV-infected HeLa cells with a fluorescent fatty acid Bodipy 500/510 C4, 9 (bodipy-FA) which is believed to mimic FA with 18 carbon atoms backbone. This and similar molecules are extensively used in lipid metabolism research and it was previously shown to be rapidly utilized by cellular lipid synthesis machinery and incorporated into phospholipids, triglycerides and other natural lipids [24], [25], [26], [27], [28]. The cells were infected at a multiplicity of 50 PFU/cell to ensure simultaneous development of infection, and bodipy-FA was added for 30 min at 4 hours post infection (h p.i.), in the middle of the infectious cycle. The infected cells showed strongly increased import of bodipy-FA (Fig. 1A and B). In mock-infected cells the label was distributed into multiple round structures in the cytoplasm and was also found in intracellular ER-like staining (Fig. 1C, mock). The round bright dots were identified as lipid droplets since they co-localized with a well-established lipid droplet marker ADRP [29] (Fig. 1D). Note that some ADRP-positive structures did not accumulate bodipy-FA during 30 min labeling period (Fig. 1D, arrow), consistently with the previous results that individual lipid droplets accumulate newly-synthesized lipids at different rates [30]. In infected cells, bright bodipy-FA fluorescence surrounded the nuclei and often occupied the total cytoplasmic area reflecting robust development of the poliovirus replication complexes (note pycnotic nuclei in infected cells, characteristic of polio-induced cytopathic effect)(Fig. 1E). For the experiment shown on Fig. 1 the cells were incubated in serum-free media during the labeling period, so bodipy-FA was the only fatty acid available exogenously. We also monitored FA transport when cells were incubated in normal growth media supplemented with fetal bovine serum which provides ample supply of natural FA and other lipids. As expected, the level of fluorescent signal was lower in the presence of serum, due to competition with the fatty acids from serum, but the overall picture of strong stimulation of fatty acid import upon infection was the same (not shown). Cells on Figure 1C and E are imaged directly after formaldehyde fixation without further detergent permeabilization which we found to deteriorate the fine structure of the distribution of the bodipy-FA label, especially in weakly labeled mock-infected cells. Staining of cells for a viral antigen 2B, a marker of membranous replication complexes, revealed extensive co-localization of bodipy-FA fluorescence with polio replication structures, especially in the cells where viral protein staining could still be visualized as discrete domains in the confocal plain (Fig. 1F, arrowheads, also co-localization panel). With the further development of infection staining for both viral proteins and bodipy-FA tend to occupied all available perinuclear space reflecting massive development of membranous replication complexes. Staining for other membrane-targeted poliovirus replication proteins 2C and 3A revealed similar pattern of distribution of a viral antigen and bodipy-FA label (not shown).

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