Figure 1.
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.
Figure 2.
In infected cells synthesis of neutral lipids is shut down and imported fatty acids are directed to strongly stimulated synthesis of phosphatidylcholine.
HeLa cells were infected (V) or mock-infected (M) with poliovirus at 50 PFU/cell and incubated in medium with or without serum as indicated. Bodipy-FA was added for 30 min at 4 h p. i.; after that total lipids were extracted and resolved by thin layer chromatography. A. Neutral lipids resolved in hexane∶ether∶acetic acid (80∶20∶1) system. Bodipy-FA-lipids represent fluorescent fatty acid-containing lipids synthesized during 30 min labeling period. Total lipids show all neutral lipids stained with bromothymol. B. Polar lipids resolved in chloroform∶ethanol∶water∶triethylamine (30∶35∶7∶35) system. Bodipy-FA-lipids represent fluorescent fatty acid-containing lipids synthesized during 30 min labeling period. Total lipids show all phospholipids stained with Phostain. PC: 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (phosphatidylcholine marker).
Figure 3.
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.
Figure 4.
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.
Figure 5.
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.
Figure 6.
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).
Figure 7.
Activation of long chain fatty acid import is a general phenomenon of picornavirus infection.
A–C. 293HEK (human embryonic kidney), SH-S5Y5 (human neuroblastoma) or Vero (green African kidney) cells were infected with poliovirus at 50 PFU/cell and incubated for 4 hours before 30 min label with bodipy-FA. Fluorescent and phase contrast images of infected and mock-infected cells are shown. D. Higher magnification of Vero cells infected with poliovirus and labeled with bodipy-FA like in C, showing that bodipy-FA accumulation is activated only in cells actively expressing viral proteins (arrow). E, F. HeLa cells were infected with either Coxsackie B3 virus or encephalomyocarditis virus at 50 PFU/cell and incubated for 4 hours before 30 min label with bodipy-FA. Fluorescent and phase contrast images of infected and mock-infected cells are shown.
Figure 8.
A model of the structural development of picornavirus replication complexes based on activation of cellular acyl-CoA synthetase activity by viral proteins (2A is required, but is not sufficient in case of polio).
Elevated acyl-CoA synthetase activity leads to increased import of fatty acids (FA) form the media and also to utilization of FA form intracellular sources (FAS- fatty acid synthase)resulting in upregulated synthesis of altered species of phosphatidylcholines (PC). PCs with short fatty acid moieties preferentially synthesized in polio-infected cells spontaneously assemble into characteristic tightly packed convoluted tubular membranes structures constituting structural scaffold of poliovirus replication organelles.