African swine fever virus B175L inhibits the type I interferon pathway by targeting STING and 2'3'-cGAMP

Abstract

African swine fever virus (ASFV), the only known DNA arbovirus, is the causative agent of African swine fever (ASF), an acutely contagious disease in pigs. ASF has recently become a crisis in the pig industry in recent years, but there are no commercially available vaccines. Studying the immune evasion mechanisms of ASFV proteins is important for the understanding the pathogenesis of ASFV and essential information for the development of an effective live-attenuated ASFV vaccines. Here, we identified ASFV B175L, previously uncharacterized proteins that inhibit type I interferon signaling by targeting STING and 2'3'-cGAMP. The conserved B175L-zf-FCS motif specifically interacted with both cGAMP and the R238 and Y240 amino acids of STING. Consequently, this interaction interferes with the interaction of cGAMP and STING, thereby inhibiting downstream signaling of IFN-mediated antiviral responses. This novel mechanism of B175L opens a new avenue as one of the ASFV virulent genes that can contribute to the advancement of ASFV live-attenuated vaccines.

Keywords: 2′3′-cGAMP; African swine fever virus (ASFV); B175L; STING and type I interferon.

Fig 1

B175L negatively regulates antiviral immune responses in stable PAM cells. PAM cells stably expressing B175L-Flag or a control plasmid were infected with ADV-GFP, HSV-GFP, or VACV-GFP (multiplicity of infection (MOI) = 1.0). The GFP images were captured at 24 hpi using fluorescence microscopy and quantified at 12 and 24 hpi using the fluorescence modulator. Virus titers of each sample were determined by standard plaque assay in A549 and Vero cells (A, C, and E). Porcine IFN-β and IL-6 concentrations in the cell culture supernatant collected at 12 hpi and 24 hpi were estimated by enzyme-linked immunosorbent assay (ELISA) (B, D, and F). (G and H) GFP images, fluorescence level, virus titer, IFN-β, and IL-6 ELISA from HSV-GFP-infected stable PIB cells. The data represent at least two independent experiments with similar results, and the values are expressed as means and SD for three biological replicates. Student’s t-test: *P  <  0.05; **P  <  0.01; ***P  <  0.001; ****P  <  0.0001; ns, not significant.

Fig 2

B175L downregulates host immune responses in PK-15 and stable MA-104 cells. PK-15 cells transiently expressing B175L-Flag or vector plasmid (A–C), as well as stable MA-104 cells (D–F), were infected with ADV-GFP, HSV-GFP, or VACV-GFP (MOI = 1.0). The virus titers of each harvested sample at the indicated time points were determined by the standard plaque assay in A549 and Vero cells. Concentrations of porcine IFN-β and IL-6 in the cell culture supernatant collected at 12 hpi and 24 hpi were estimated using ELISA. The data represent at least two independent experiments with similar results, and the values are expressed as means and SD for three biological replicates. Student’s t-test: *P  <  0.05; **P  <  0.01; ***P  <  0.001; ****P  <  0.0001; ns, not significant.

Fig 3

B175L inhibits cGAS-STING pathway signaling and the transcription of antiviral genes. (A and B) B175L-expressed PK-15 or stable PAM cells and control cells were infected with ADV-GFP (MOI = 1.0) and harvested at the indicated time points. The phosphorylated vs intact forms of TBK1, STAT1, p65, IRF3, IκBα, and the expression of B175L-Flag were detected by immunoblotting. The β-actin was used as the internal control for equal protein amounts in samples. Protein sizes are expressed in kilodaltons (kDa). All the immunoblot data are representative of at least two independent experiments, each with similar results. (C and D) PK-15 or stable PAM cells harboring B175L-Flag and control cells were infected with ADV-GFP (MOI = 1.0) and harvested at the indicated time points. The total RNA was extracted from cells, and the transcription of antiviral genes was analyzed by qRT-PCR. The mRNA induction levels in 0 h and 24 h vector- or B175L-expressing cells were compared. The mRNA expression levels were analyzed according to the delta–delta CT (2^−ΔΔCT) method, and β-actin or glyceraldehyde-3-phosphate dehydrogenase was used as an internal housekeeping gene for normalization. The data represent at least two independent experiments with similar results, and the values are expressed as means and SD for three biological replicates. Student’s t-test: *P  <  0.05; **P  <  0.01; ***P  <  0.001; ****P  <  0.0001; ns, not significant.

Fig 4

B175L interacts with STING. (A) Silver staining for the B175L interactome assay followed by mass spectrometry. The arrow indicates the protein expression of B175L-Strep and the expected protein expression of STING (UniProt: Q86WV6). (B) HEK293T cells were co-expressed with IFN-β promoter (firefly), TK-Renilla (internal control), stimulators [Poly (dA:dT), or STING, or TBK1, or IKKε], and increasing doses (50, 100, 200, 400 ng) of B175L-Flag for 24 h. The luciferase activity of each sample was quantified with a dual-luciferase reporter assay system. To measure cGAS and cGAMP-induced luciferase activities, 93-Dual hSTING-A162 cells were stimulated with 3× Flag cGAS or 4 µg/mL of cGAMP for 12 h. The IFN-β-dependent expression of Lucia luciferase was estimated by QUANTI-Luc. The data represent at least three independent experiments with similar results, and the values are expressed as means and SD for three biological replicates. Student’s t-test: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. (C) HEK293T cells transfected with B175L-Flag and STING-Strep plasmids were subjected to immunoprecipitation with an anti-Strep antibody followed by immunoblotting with the indicated antibodies. (D) PK-15 cells transfected with B175L-Flag and control plasmids or stable PAM cells harboring B175L-Flag or control plasmids were stimulated with HSV-GFP (MOI = 1.0). After 24 hpi, the cells were harvested and subjected to immunoprecipitation and immunoblotting. Protein sizes are expressed in kilodaltons (kDa). All the immunoblot data are representative of at least two independent experiments, each with similar results. (E) Confocal microscopy assays were used to examine the co-localization of B175L and STING in PK-15 cells upon VACV wild-type (MOI = 1.0) stimulation and in HeLa cells. The nuclei were stained with DAPI (blue). The arrow indicates the co-localized B175L and STING proteins at the endogenous level.

Fig 5

B175L inhibits STING and cGAMP interaction. (A) GST-tagged B175L domains (aa 1–60 and aa 1–110) with or without the zf-FCS motif (left). Immunoprecipitation of GST-tagged B175L and its domain constructs with STING-Strep (right). Protein expressions were determined by anti-GST and anti-Strep antibodies. (B and C) Domain analysis to find the interface of STING that interacts with the zf-FCS motif of B175L-Flag. Control plasmid, GST-tagged STING (wild type), and its constructs (aa 1–145, aa 1–185, aa 1–340, aa 185–235, aa 185–270, aa 185–330, and aa 185–340) were transfected into HEK293T cells and subjected to GST immunoprecipitation followed by immunoblotting with anti-Flag and anti-GST antibodies. (D) In vitro competition assay with cGAMP, STING, and B175L. 2′3′-cGAMP-Cy5 conjugate or 2′3′-cGAMP-Biotin conjugate was incubated with affinity-purified STING and B175L (1, 3, 6, and 10 µg). After the incubation, the reaction mixture was pulled down with Strep beads or Dynabeads M-280 Streptavidin and subjected to immunoblotting analysis. Protein sizes are expressed in kilodaltons (kDa). All the immunoblot data are representative of at least two independent experiments, each with similar results.

Fig 6

B175L interaction requires R238A and Y240A of STING. (A) PAM cells that stably express B175L inhibit cGAMP-induced phosphorylation of TBK1 and IRF3. Stable PAM cells were stimulated with 1, 3, and 5 µM of 2′3′-cGAMP. After 12 h post-transfection, the phosphorylated vs nonphosphorylated TBK1 and IRF3 and the expression of B175L-Flag were determined by immunoblotting. (B) The sequence alignment shows well-studied cGAMP-specific binding amino acids of STING R238, Y240, and N242 that share a conserved interface among human (hSTING), porcine (poSTING), and mouse (mSTING). (C and D) STING R238A and Y240 abolished B175L and STING binding. Expression plasmids encoding B175L, full-length STING, and its mutants were transfected into HEK293T cells, followed by immunoprecipitation and immunoblotting. (E) B175L inhibited STING polymerization in a dose-dependent manner. HEK293 cells transfected with STING-Flag expression plasmids and increased doses of B175L-Strep plasmids were stimulated with 4 µM of cGAMP. After 4 h, cell lysates were resolved by Semi-denaturating detergent agarose gel electrophoresis (SDD-AGE) (top) or SDS-PAGE (bottom) with the indicated antibodies. (F) cGAMP interacts with B175L. 2′3′-cGAMP-Cy5 conjugate was incubated at 37°C with affinity-purified proteins of B175L-GST or its constructs. After 2 h, the reaction mixture was pulled down with an anti-Cy5 antibody and subjected to SDS-PAGE. Protein sizes are expressed in kilodaltons (kDa). All the immunoblot data are representative of at least two independent experiments, each with similar results. (G) Graphical summary of B175L immune evasion. ASFV B175L, a previously uncategorized protein, evades antiviral immune responses targeting the central immune molecule STING and its ligand cGAMP, which cause inhibition of downstream immune signaling via TBK1 and IRF3.