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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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Activation of fatty acid import requires polio protein 2A.A. Schematic representation of poliovirus genome and truncated constructs used for expression of polio proteins. B. Only expression of the full P2P3 region activates import of bodipy-FA label (Arrows). HeLa cells were transfected with plasmids coding for the indicated polyprotein fragments under control of T7 promoter (empty vector for the control sample) The next day the cells were infected with vaccinia-T7 virus and labeled with bodipy-FA for 30 min at 4 h p. i. Poliovirus antigen 3A is detected as a marker of expression of viral polyprotein fragments 2A-3D (complete P2–P3), 2B-3D and 2C-3D. C. Protease activity of 2A is dispensable for activation of fatty acid import (arrows). HeLa cells were transfected with plasmids coding for the indicated polyprotein fragments under control of T7 promoter (empty vector for the control sample) The next day the cells were infected with vaccinia-T7 virus and labeled with bodipy-FA for 30 min at 4 h p. i. Poliovirus antigen 2B is detected as a marker of expression of the wt and 2A-mut containing P2–P3 polyprotein. D. Parallel samples to those shown in C were collected and analyzed for 2A protease activity and expression of viral proteins. Processing of eIF-4G (black arrow) is detected only in the sample expressing functional 2A protease (top panel). Accumulation of viral proteins detected by viral antigen 3D is comparable in all samples showing that the lack of 2A protease activity is because of the mutation, not because of the insufficient expression (lower panel).
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ppat-1003401-g006: Activation of fatty acid import requires polio protein 2A.A. Schematic representation of poliovirus genome and truncated constructs used for expression of polio proteins. B. Only expression of the full P2P3 region activates import of bodipy-FA label (Arrows). HeLa cells were transfected with plasmids coding for the indicated polyprotein fragments under control of T7 promoter (empty vector for the control sample) The next day the cells were infected with vaccinia-T7 virus and labeled with bodipy-FA for 30 min at 4 h p. i. Poliovirus antigen 3A is detected as a marker of expression of viral polyprotein fragments 2A-3D (complete P2–P3), 2B-3D and 2C-3D. C. Protease activity of 2A is dispensable for activation of fatty acid import (arrows). HeLa cells were transfected with plasmids coding for the indicated polyprotein fragments under control of T7 promoter (empty vector for the control sample) The next day the cells were infected with vaccinia-T7 virus and labeled with bodipy-FA for 30 min at 4 h p. i. Poliovirus antigen 2B is detected as a marker of expression of the wt and 2A-mut containing P2–P3 polyprotein. D. Parallel samples to those shown in C were collected and analyzed for 2A protease activity and expression of viral proteins. Processing of eIF-4G (black arrow) is detected only in the sample expressing functional 2A protease (top panel). Accumulation of viral proteins detected by viral antigen 3D is comparable in all samples showing that the lack of 2A protease activity is because of the mutation, not because of the insufficient expression (lower panel).

Mentions: To implement an unbiased approach to identify acyl-CoA synthetases that support replication of poliovirus we performed screen with siRNA pools targeting all 13 long chain acyl-CoA synthetases. Only siRNA against FATP5 showed significant toxicity in HeLa cells likely due to some non-specific effect (Figure S3) since this protein is believed to be expressed only in liver [38]. Depletion of other acyl-CoA synthetases was well tolerated by the cells, the apparent slight toxic effect rather reflects somewhat slower growth of cells treated with certain siRNA pools (Figure S3). Our initial screen identified three siRNA pools that induced significant, ∼80% reduction of replication: anti-acyl-CoA synthetase Bubblegum 2 (AcsBG2), FATP3 and Acsl3 (Figure S3). Western blot analysis of the targeted proteins revealed that only effect of Acsl3 siRNA was specific. AcsBG2 was not expressed in our HeLa cells as expected, since this protein was previously shown to be specific for brain stem and testis [39], and treatment of cells with either pooled or individual siRNAs against FATP3 did not result in significant reduction of the amount of the protein (not shown). All siRNAs from the anti-Acsl3 pool resulted in reduction of the targeted protein and decreased replication of PV, siRNA #2 was the most potent. The specificity of the ACSL3 knock-down effect was confirmed by rescue of polio replication by expression of the ACSL3 with mutated siRNA #2 targeting sequence (Figure S4). The strongest reduction of polio replication with the least cellular toxicity was observed after treatment of cells with the all four anti-Acsl3 siRNAs pool (Figure 5B). We also monitored PV infection in the cells expressing recombinant protein GFP-Acsl3-HA. Accumulation of viral proteins was significantly delayed in such cells, compared to cells expressing just EGFP, or transfected with an empty pUC plasmid (Fig. 5C), suggesting that fusion protein GFP-Acsl3-HA works like a dominant-negative mutant in the context of polio infection. Note that transfection efficiency of HeLa cells is about 60–80% and protein accumulation is measured in the total cell population, therefore the actual reduction of polio replication in transfected cells only should be even stronger. Since knock-down of Acsl3 expression inhibits polio replication, it is impossible to directly examine the role of Acsl3 in activation of FA import upon infection. Thus we expressed poliovirus non-structural P2P3 polyprotein fragment (Fig. 6A) in cells treated with control or ACSL3-targeting siRNAs with the help of vaccinia virus expressing T7 RNA polymerase [40]. This system is independent of polio replication and is discussed in details in the section below. Expression of poliovirus proteins was induced by infection of cells with vaccinia-T7 virus ∼72 hours post siRNA transfection. At 4 hours post vaccinia infection bodipy-FA probe was added to the media for 30 min. The cells treated with control siRNA which were positive for a polio antigen showed strong activation of FA import (Fig. 5D, arrows), while import of bodipy-FA in the cells with ACSL3 knock-down was significantly lower (Fig. 5E, arrowheads). The statistical analysis confirmed that bodipy-FA fluorescence normalized to polio protein 2B signal strongly declined in ACSL3 knockdown cells (Fig. 5D).


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)

Activation of fatty acid import requires polio protein 2A.A. Schematic representation of poliovirus genome and truncated constructs used for expression of polio proteins. B. Only expression of the full P2P3 region activates import of bodipy-FA label (Arrows). HeLa cells were transfected with plasmids coding for the indicated polyprotein fragments under control of T7 promoter (empty vector for the control sample) The next day the cells were infected with vaccinia-T7 virus and labeled with bodipy-FA for 30 min at 4 h p. i. Poliovirus antigen 3A is detected as a marker of expression of viral polyprotein fragments 2A-3D (complete P2–P3), 2B-3D and 2C-3D. C. Protease activity of 2A is dispensable for activation of fatty acid import (arrows). HeLa cells were transfected with plasmids coding for the indicated polyprotein fragments under control of T7 promoter (empty vector for the control sample) The next day the cells were infected with vaccinia-T7 virus and labeled with bodipy-FA for 30 min at 4 h p. i. Poliovirus antigen 2B is detected as a marker of expression of the wt and 2A-mut containing P2–P3 polyprotein. D. Parallel samples to those shown in C were collected and analyzed for 2A protease activity and expression of viral proteins. Processing of eIF-4G (black arrow) is detected only in the sample expressing functional 2A protease (top panel). Accumulation of viral proteins detected by viral antigen 3D is comparable in all samples showing that the lack of 2A protease activity is because of the mutation, not because of the insufficient expression (lower panel).
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ppat-1003401-g006: Activation of fatty acid import requires polio protein 2A.A. Schematic representation of poliovirus genome and truncated constructs used for expression of polio proteins. B. Only expression of the full P2P3 region activates import of bodipy-FA label (Arrows). HeLa cells were transfected with plasmids coding for the indicated polyprotein fragments under control of T7 promoter (empty vector for the control sample) The next day the cells were infected with vaccinia-T7 virus and labeled with bodipy-FA for 30 min at 4 h p. i. Poliovirus antigen 3A is detected as a marker of expression of viral polyprotein fragments 2A-3D (complete P2–P3), 2B-3D and 2C-3D. C. Protease activity of 2A is dispensable for activation of fatty acid import (arrows). HeLa cells were transfected with plasmids coding for the indicated polyprotein fragments under control of T7 promoter (empty vector for the control sample) The next day the cells were infected with vaccinia-T7 virus and labeled with bodipy-FA for 30 min at 4 h p. i. Poliovirus antigen 2B is detected as a marker of expression of the wt and 2A-mut containing P2–P3 polyprotein. D. Parallel samples to those shown in C were collected and analyzed for 2A protease activity and expression of viral proteins. Processing of eIF-4G (black arrow) is detected only in the sample expressing functional 2A protease (top panel). Accumulation of viral proteins detected by viral antigen 3D is comparable in all samples showing that the lack of 2A protease activity is because of the mutation, not because of the insufficient expression (lower panel).
Mentions: To implement an unbiased approach to identify acyl-CoA synthetases that support replication of poliovirus we performed screen with siRNA pools targeting all 13 long chain acyl-CoA synthetases. Only siRNA against FATP5 showed significant toxicity in HeLa cells likely due to some non-specific effect (Figure S3) since this protein is believed to be expressed only in liver [38]. Depletion of other acyl-CoA synthetases was well tolerated by the cells, the apparent slight toxic effect rather reflects somewhat slower growth of cells treated with certain siRNA pools (Figure S3). Our initial screen identified three siRNA pools that induced significant, ∼80% reduction of replication: anti-acyl-CoA synthetase Bubblegum 2 (AcsBG2), FATP3 and Acsl3 (Figure S3). Western blot analysis of the targeted proteins revealed that only effect of Acsl3 siRNA was specific. AcsBG2 was not expressed in our HeLa cells as expected, since this protein was previously shown to be specific for brain stem and testis [39], and treatment of cells with either pooled or individual siRNAs against FATP3 did not result in significant reduction of the amount of the protein (not shown). All siRNAs from the anti-Acsl3 pool resulted in reduction of the targeted protein and decreased replication of PV, siRNA #2 was the most potent. The specificity of the ACSL3 knock-down effect was confirmed by rescue of polio replication by expression of the ACSL3 with mutated siRNA #2 targeting sequence (Figure S4). The strongest reduction of polio replication with the least cellular toxicity was observed after treatment of cells with the all four anti-Acsl3 siRNAs pool (Figure 5B). We also monitored PV infection in the cells expressing recombinant protein GFP-Acsl3-HA. Accumulation of viral proteins was significantly delayed in such cells, compared to cells expressing just EGFP, or transfected with an empty pUC plasmid (Fig. 5C), suggesting that fusion protein GFP-Acsl3-HA works like a dominant-negative mutant in the context of polio infection. Note that transfection efficiency of HeLa cells is about 60–80% and protein accumulation is measured in the total cell population, therefore the actual reduction of polio replication in transfected cells only should be even stronger. Since knock-down of Acsl3 expression inhibits polio replication, it is impossible to directly examine the role of Acsl3 in activation of FA import upon infection. Thus we expressed poliovirus non-structural P2P3 polyprotein fragment (Fig. 6A) in cells treated with control or ACSL3-targeting siRNAs with the help of vaccinia virus expressing T7 RNA polymerase [40]. This system is independent of polio replication and is discussed in details in the section below. Expression of poliovirus proteins was induced by infection of cells with vaccinia-T7 virus ∼72 hours post siRNA transfection. At 4 hours post vaccinia infection bodipy-FA probe was added to the media for 30 min. The cells treated with control siRNA which were positive for a polio antigen showed strong activation of FA import (Fig. 5D, arrows), while import of bodipy-FA in the cells with ACSL3 knock-down was significantly lower (Fig. 5E, arrowheads). The statistical analysis confirmed that bodipy-FA fluorescence normalized to polio protein 2B signal strongly declined in ACSL3 knockdown cells (Fig. 5D).

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