Infection-specific long-chain fatty acid metabolism as a broad anti-enterovirus target

Figure 1.. Anti-viral effect of polyunsaturated fatty acids correlates with their conformation. A. A scheme of the experiment for assessing the effect of exogenously added LCFAs in viral infection and bond-line structures of C18 LCFAs used in these experiments. B. Western blot analysis of the accumulation of poliovirus protein 2C in HeLa cells infected at an MOI of 50 and incubated in serum-free medium supplemented with the indicated LCFAs from 2 to 4.5 h p.i. Actin is shown as a loading control. C. Left panels: percentage of the Sytox green-positive nuclei indicating membrane damage in cells treated with 200 μM of the indicated LCFAs as in B. Quantifications for each sample were performed from four randomly chosen fields of view with an average of ~1000 nuclei in each. Right panels: representative images of control cells and those incubated with ODPA showing Sytox green and DAPI signals used for quantifications. D. Top: a scheme of the experiment to measure the development of lipid peroxides using the C11 probe in cells treated with 200 μM of the indicated LCFAs after infection as in B. Bottom: quantitation of the C11 signal ratio at 581 nm (oxidized) to that of the original compound at…

Figure 2.. Effect of serum- and LD-derived LCFAs on poliovirus replication. A. Cytotoxicity assay (ATP level) of HeLa cells incubated in normal growth medium supplemented with the indicated LCFAs (concentration in μM is shown on X axis) for 6 h. B. Poliovirus yield in HeLa cells infected with an MOI of 5 or 0.5 and incubated after virus adsorption in normal growth medium supplemented with 200 μM of the indicated LCFAs for 6 h. Experiments were performed with three biological replicates. C. Top left: toxicity assay (ATP level) of HeLa cells incubated overnight in normal growth medium supplemented with 200 μM of the indicated LCFAs. Right: quantitation of LDs in these cells, each dot represents an individual randomly chosen field of view with ~50 nuclei each. Bottom: representative images of Oil Red O staining of LDs used for quantitation. D. Poliovirus yield in HeLa cells incubated overnight in normal growth medium supplemented with 200 μM of the indicated LCFAs, infected with poliovirus at an MOI of 5 or 0.5, and incubated after the virus adsorption in a serum-free medium for 6 h p.i. Experiments were performed with three biological replicates.

Figure 3.. Lipid synthesis pathways in infected cells are affected by exogenously added LCFAs. A. A scheme of experiments to trace the active LCFA targeting pathways with the FFA probe. B. Representative images showing live HeLa cells infected with poliovirus with an MOI of 50 (or mock-infected controls)and incubated in the presence of 200 μM of the indicated LCFAs for 4 h plus 30 min with the same fatty acids and 0.4 μM of the FFA probe. The fluorescent signal shows cellular structures where FFA is incorporated during 30 min of incubation. C. Quantitation of LDs in cells treated as in A. At least ~40 cells are quantified for each sample. D. Western blot with lysates of cells infected and incubated with LCFAs as in B. Actin is shown as a loading control.

Figure 4.. Inhibition of Acyl-CoA cholesterol acyltransferase (ACAT) activity blocks neutral lipid synthesis in PUFA-treated infected cells. A. A scheme of LD and the major activities synthesizing triglycerides and cholesterol esters. Green LCFA indicates FFA probe. B. A scheme of the experiments used to evaluate the effect of inhibitors on neutral lipid synthesis. C-D representative images of live HeLa cells infectedwith poliovirus with an MOI of 50 (or mock-infected controls) and incubated in the presence of 200 μM of the indicated LCFAs for 4 h plus 30 min with the same fatty acids and 0.4 μM of the FFA probe (top) and quantitation of LDs (bottom). Dots represent data for individual randomly chosen fields of view, with at least ~25 cells per field.

Figure 5.. PUFAs can affect multiple steps of the poliovirus life cycle. A. Western blots from HeLa cells infected with poliovirus with an MOI of 50 and incubated after virus adsorption with 200 μM of the LCFAs and 50 μM of ACTA inhibitor as indicated for 4 h p.i. B. Toxicity assay (ATP level) of cells infected and incubated with LCFAs and ACAT inhibitor as in A. C. Top: a scheme of the experiment to evaluate the effect of PUFAs in the presence of ACAT inhibitor on virion penetration/uncoating and initial translation of the viral RNA. Bottom: Western blots of HeLa cells infected with the indicated MOIs (or mock-infected controls) and incubated with (lanes 1–7) or without Gua, an inhibitor of enterovirus replication (lane 9). The indicated PUFA (200 μM) and ACAT inhibitor (50 μM) were added to samples 5 and 6 after virus adsorption or at 1 h p.i., respectively; all cells were lysed at 2 h p.i. D. Top: a scheme of the experiment to evaluate the effect of PUFAs in the presence of ACAT inhibitor on the translation/replication step of the viral life cycle. Bottom: Western blots of HeLa cells infected (or mock-infected controls) with an MOI of 50 of poliovirus and incubated for 1 h p.i. in the…

Figure 6.. Broad anti-enterovirus activity of select PUFAs is refractory to the emergence of resistance. A. HeLa (top) or A549 cells (bottom) were infected with an MOI of 10 of CVB3, poliovirus, or EVD68 and incubated after the virus adsorption in normal growth medium supplemented with 200 μM of stearic acid (control), linoleic acid, or ODPA in the presence of 50 μM of ACAT inhibitor for 6 h (poliovirus) or 8 h (other enteroviruses). Graphs show total virus yield; experiments were performed with three biological replicates. B. Differentiated human airway epithelial cultures were infected at the apical surface with RVA16 and incubated for ~24h in the presence of 50 μM of ACAT inhibitor and 200 μM of either linoleic acid or ODPA (or with a corresponding amount of DMSO vehicle in control). Virus replication was determined by the quantification of intracellular RNA. The basal medium after incubation was used for the toxicity assay (LDH release), in which a lower value indicates less toxicity. Data from cultures differentiated from each donor are indicated by individual symbols. C. Poliovirus was serially propagated in HeLa cells in the presence of 200 μM of either linoleic acid or ODPA and 50…