Fig 1.
MGF300 genes are critical to ASFV replication in primary porcine alveolar macrophages (PAMs).
(A) Schematic diagram depicting the deletion of multigene family genes (MGFs) in the left variable region (LVR) of the ASFV HLJ/18 strain (ASFV-WT) genome. Dashed lines indicate the boundaries of the deleted MGFs relative to the ASFV-WT genome are indicated. (B and C) In vitro growth characteristics of ASFV-WT and the mutants with different deletion combinations of MGFs. PAMs were infected (MOI = 5) with each of the viruses and the virus yield was titrated at the indicated times post-infection.
Fig 2.
The MGF300-2R-deleted ASFV mutant (Del2R) induces higher production of inflammatory cytokines than does the wild-type ASFV (ASFV-WT) in PAMs.
(A) PAMs were mock infected or infected with Del2R or ASFV-WT (MOI = 5) for 12 and 20 h for RNA-seq analysis. Volcano plot of gene changes in Del2R-infected PAMs compared with the expression in ASFV-WT-infected PAMs. Red dots and blue dots denote upregulated or downregulated differentially expressed genes (DEGs), respectively. (B) Gene ontology (GO) category functional enrichment by 3 categories (Biological process, Molecular function, and Cell component) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis was performed for up-regulated genes in the group of Del2R versus ASFV-WT. (C) The heatmaps show the expression levels of DEGs in the NF-κB signaling pathway induced by Del2R versus ASFV-WT at 12 and 20 hours post-infection (hpi). (D and E) PAMs were either mock infected or infected with Del2R or ASFV-WT (MOI = 5). At 12 and 20 hpi, the mRNA levels of IL-1β and TNF-α (D) in the cell lysates were determined by RT-qPCR, and the production of IL-1β and TNF-α (E) in the cell culture supernatants were detected by commercial ELISA kits.
Fig 3.
The MGF300-2R protein interacts with IKKα and IKKβ and inhibits the NF-κB activation.
(A) HEK293T cells were transfected with increasing amounts of the expressing plasmid pHA-MGF300-2R (0.5, 1, and 1.5 μg) for 24 h and then mock-treated or treated with TNF-α (10 ng/mL) for 8 h. The protein expression of MGF300-2R was examined by western blotting and the luciferase activity was measured at 24 hours post-transfection (hpt). (B) HEK293T cells were transfected with the pFlag-MGF300-2R or empty plasmid and then treated with or without TNF-α (10 ng/mL) for 1 h. At 24 hpt, the cells were fractionated into cytoplasmic and nuclear fractions and analyzed by immunoblotting with the indicated antibodies. The p65 in the nuclear and cytoplasmic compartments was checked by western blotting. Lamin B1 and tubulin were used as nuclear and cytosolic markers, respectively. (C) PAMs were mock-infected or infected with ASFV-WT or Del2R (MOI = 5). At 20 hpi, PAMs were treated with or without TNF-α (10 ng/mL) for 1 h, the separation of cellular fractions of ASFV-infected PAMs was performed as described above. (D) Analysis of the phosphorylation levels of IκBα and p65 in PAMs mock-infected or infected with ASFV-WT, or Del2R (MOI = 5) by western blotting at 2 and 6 hpi. (E) HEK293T cells were transfected with a plasmid encoding HA-MGF300-2R along with a plasmid encoding Flag-IKKα, Flag-IKKβ, or Flag-NEMO as indicated. The cells were lysed and whole cell lysates (WCL) were immunoprecipitated with anti-HA mAb at 36 hpt. The immunoprecipitates were examined by western blotting with the indicated antibodies. (F) HEK293T cells were transfected with expressing plasmids pFlag-IKKα or pFlag-IKKβ for 36 h and lysed with NP-40 buffer. The purified GST or GST-MGF300-2R protein was used to pull down the IKKα or IKKβ in the lysates and analyzed by western blotting with the indicated antibodies. (G and H) HEK293T cells were transfected with pHA-MGF300-2R alone and cotransfected with pFlag-IKKα or pFlag-IKKβ in combination with pHA-MGF300-2R. IKKα, IKKβ, and MGF300-2R were analyzed by laser confocal microscopy. Scale bar, 10 μm. The colocalization of MGF300-2R and IKKα or IKKβ was analyzed by the Coloc2 tool of ImageJ/FIJI and shown as Pearson’s correlation coefficients.
Fig 4.
Autophagic degradation of IKKα and IKKβ can be induced by the MGF300-2R protein.
(A to C) HEK293T cells were cotransfected with each of pFlag-IKKα, pFlag-IKKβ, or pFlag-NEMO, and an increasing amount of pHA-MGF300-2R (0, 1.0, 2.0, and 3.0 μg) for 24 h. The cell lysates were analyzed by immunoblotting. (D to F) Analysis by western blotting of IKKα (D), IKKβ (E), NEMO (F), and ASFV-A137R levels at 12 and 20 hpi in PAMs mock-infected or infected with ASFV-WT, or Del2R (MOI = 5). (G) HEK293T cells were cotransfected with pFlag-IKKα and pHA-MGF300-2R for 18 h, then the cells were treated with Lac (10 μM), MG132 (10 mM), BafA1 (0.4 μM), 3-MA (50 μM), or LY294002 (20 mM) for 6 h. The cell lysates were analyzed by immunoblotting. (H) HEK293T cells were cotransfected with pFlag-IKKβ and pHA-MGF300-2R for 18 h, then the cells were treated with Lac (10 μM), MG132 (10 mM), BafA1 (0.4 μM), 3-MA (50 μM), or LY294002 (20 mM) for 6 h. The cell lysates were analyzed by immunoblotting. (I) Flag-IKKα and HA-MGF300-2R were cotransfected into the autophagy-related protein 5 (ATG5)-knockout (ATG5-/-) or wild-type (WT) HeLa cells for 36 h, and the expression of IKKα or MGF300-2R were analyzed by immunoblotting with the indicated antibodies. (J) Flag-IKKβ and HA-MGF300-2R were cotransfected into the ATG5-/- or WT HeLa cells for 36 h, and the expression of IKKβ or MGF300-2R were analyzed by immunoblotting with the indicated antibodies.
Fig 5.
MGF300-2R promotes the autophagic degradation of IKKα and IKKβ by the cargo receptor TOLLIP.
(A to C) HEK293T cells were cotransfected with each of pFlag-MGF300-2R, pFlag-IKKα, pFlag-IKKβ, and HA-tagged cargo receptors as indicated for 24 h, followed by immunoprecipitation with protein A/G beads. The WCL and IP precipitates were analyzed by immunoblotting with the indicated antibodies. (D and E) The p62-knockout (p62-/-) or wild-type (WT) HEK293T cells were cotransfected with Flag-IKKα, Flag-IKKβ, and Flag-MGF300-2R for 36 h, and the expression of IKKα, IKKβ or MGF300-2R was analyzed by immunoblotting. (F and G) The TOLLIP-knockout (TOLLIP-/-) or wild-type (WT) HEK293T cells were cotransfected with pFlag-IKKα, pFlag-IKKβ, and pFlag-MGF300-2R for 36 h, and the expression of IKKα, IKKβ, or MGF300-2R was analyzed by immunoblotting.
Fig 6.
MGF300-2R facilitates the K27-linked ubiquitination and degradation of IKKα and IKKβ.
(A and B) MGF300-2R induces the ubiquitination of IKKα and IKKβ. HEK293T cells were transfected with pFlag-IKKα or pFlag-IKKβ and pHis-Ub together with pHA-MGF300-2R. At 24 hpt, cells were processed for IP with anti-Flag magnetic beads. WCLs and precipitated proteins were analyzed by western blotting with the indicated antibodies. (C and D) HEK293T cells were transfected with pFlag-IKKα or pFlag-IKKβ, pHA-MGF300-2R, and pHA-Ub or its mutants (HA-K6, HA-K11, HA-K27, HA-K29, HA-K33, HA-K48, and HA-K63). At 24 hpt, cells were processed for IP with anti-Flag magnetic beads. WCLs and precipitated proteins were analyzed by western blotting with the indicated antibodies. (E and F) HEK293T cells were transfected with pFlag-IKKα or pFlag-IKKβ, pHA-MGF300-2R, pHA-Ub or its mutants (HA-K6R, HA-K11R, HA-K27R, HA-K29R, HA-K33R, HA-K48R, and HA-K63R). At 24 hpt, cells were processed for IP with anti-Flag magnetic beads. WCLs and precipitated proteins were analyzed by western blotting with the indicated antibodies.
Fig 7.
MGF300-2R is a virulence factor involved in the pathogenicity of ASFV.
(A) The pigs were inoculated intramuscularly with Del2R (n = 4, 102.0 or 103.0 TCID50) or ASFV-WT (n = 3, 103.0 TCID50) or mock inoculated intramuscularly with RPMI 1640 (n = 3, 1 mL), and the sera and blood were collected at 0, 1, 3, 5, 7, 14, and 21 days post-inoculation. The rectal temperature (B) and survival rates (C) of the different groups of pigs. (D) Viremia for the different groups of pigs infected with ASFV-WT or Del2R was detected by qPCR. (E) Viral loads in different tissues of the pigs inoculated with Del2R or ASFV-WT were detected by qPCR. LN1: submandibular lymph nodes, LN2: mesenteric lymph nodes.
Fig 8.
The MGF300-2R-deleted ASFV mutant (Del2R) induces higher pro-inflammatory cytokines and host antibody responses in pigs than does the wild-type ASFV (ASFV-WT).
The protein levels of IL-1β (A), TNF-α (B), and IFN-α (C) in the serum samples were measured by commercial ELISA kits. Serum antibodies against p72 (D) or p30 (E) in the inoculated pigs were detected by ELISA. The results were shown as a blocking percentage. For the anti-p72 antibodies, the blocking rate above 50% was considered positive, while below 40% was considered negative; for the anti-p30 antibodies, the blocking rate below 40% was considered positive, while above 50% was considered negative. Samples with blocking between 40 and 50% were considered doubtful.
Fig 9.
A working model for the regulation of the NF-κB signaling pathway by the ASFV MGF300-2R protein.
Black arrows indicate the NF-κB signaling pathway. Upon infection, ASFV expresses the MGF300-2R protein, which interacts with both IKKα and IKKβ and promotes the K27-linked ubiquitination of IKKα and IKKβ for the TOLLIP-dependent autophagic degradation, serving as a suppressor to prevent the activation of the NF-κB signaling pathway.