African swine fever virus enhances viral replication by increasing intracellular reduced glutathione levels, which suppresses stress granule formation

Figures

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  Figure 1. ASFV inhibits SGs formation during infection. (A) ASFV inhibits SGs formation at 3, 12, and 24 hpi (MOI = 1). (B) ASFV inhibits Ars-induced SGs formation at 3, 12, and 24 hpi (MOI = 1). Red arrows indicate SG-negative and ASFV-positive PAMs.

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  Figure 2. Intracellular GSH levels were elevated following ASFV infection. (A) Intracellular GSH levels were measured using specific probes, with signal intensity (white light) shown at 3, 12, and 24 hp with an MOI = 5. (B) Thioltracker signal intensity in individual PAMs was quantified and visualised using GraphPad Prism. Each dot represents the values for individual PAM. The longer horizontal lines indicate the mean values, while the error bars represent the standard deviation (SD). (C) PAMs were either mock-infected or ASFV-infected and treated with or without Ars at 12 hpi, with an MOI = 1. The SGs were detected using an anti-G3BP1 antibody, while intracellular GSH levels were labelled with the Thioltracker probe. Red arrows highlight that ASFV-infected PAMs exhibit higher GSH levels than mock-infected or Ars-treated PAMs. (D) GSH intensity was quantified and visualised.

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  Figure 3. Effect of depleting or supplying GSH on ASFV-mediated (MOI = 1) PAMs resistance to Ars-induced SGs formation at 12 hpi. (A) PAMs were mock- or ASFV-infected and treated with PBS, Ars, BSO, or Ars and BSO. The impact of BSO-mediated GSH depletion on SGs formation. Red arrows indicate SGs formation in ASFV-infected PAMs under BSO treatment. (B) The number of SG-positive, mock-infected, or ASFV-infected PAMs was recorded and quantified from at least five fields for each of the three biological repeats. (C) PAMs were mock-infected or ASFV-infected. PAMs were also treated with NAC (10 mM) with or without Ars (0.5 mM) and then fixed and processed for IFA.

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  Figure 4. Indicated GSH-related factors determination of mock- and ASFV-infected PAMs (MOI = 1). Lysates harvested from mock-infected or ASFV-infected PAMs were analysed at various times post-infection to determine levels of GSH-related factors. (A) NRF2, the GSH synthesis and regeneration pathways related factors, and (B) GSH-associated ferroptosis related factors via immunoblotting.

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  Figure 5. AIFM1 is an important target for regulating the intracellular antioxidant capacity of ASFV-infected PAMs (MOI = 1). (A) Lysates were harvested from PAMs infected with PBS or ASFV and treated with PBS or Ars, followed by WB at 24 hpi. (B) PAMs were transfected with si-AIFM1 for AIFM1-knockdown and followed by mock- or ASFV-infection. Staining with anti-AIFM1 (green) and anti-p30 (red) was used to visualise the results. (C) SGs formation in AIFM1-knockdown PAMs, with staining using anti-AIFM1 (red) and anti-G3BP1 (green). (D) PAMs were transfected with si-NC or si-AIFM1 for 24 h followed by mock- or ASFV-infection. PAMs were treated with Ars or PBS at 24 hpi followed by a confocal assay using anti-G3BP1 (green) and anti-p30 (red). Red arrows indicate obvious SGs formation in ASFV-infected PAMs under AIFM1-knockdown.

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  Figure 6. Impact of GSH depletion or supplementation on ASFV replication in PAMs (MOI = 1). (A and B) cp204l levels, (C) p30 levels, and (D) viral titers at 12 and 24 hpi under various concentrations of BSO treatments. (E and F) cp204l levels, (G) p30 levels, and (H) viral titers at 12 and 24 hpi under various concentrations of NAC treatments.

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  Figure 7. The schematic diagram of the mechanism by which ASFV elevates intracellular GSH levels through multiple pathways, thereby antagonising the intracellular SGs formation and creating an optimised viral replication environment (created with Biorender).