Fig 2: The ASFV MGF-505-7R degrades JAK1 and JAK2.A, the effects of the ASFV MGF-505-7R on the expression of JAK1 in 293T cells. The 293T cells (2 × 105) were transfected with HA-JAK1 (1 µg) and Flag-MGF-505-7R (0, 0.25, 0.5, and 1.0 µg) for 24 h. Cell lysates were analyzed using western blotting with the indicated antibodies. B, the effects of the ASFV on the endogenous JAK1 in PAMs. PAMs were uninfected or infected with the ASFV for the indicated times and were then analyzed using western blotting with the indicated antibodies. C, effects of the ASFV MGF-505-7R on the expression of JAK2 in 293T cells. The experiments were similarly performed as in A. D, the effects of ASFV infection on endogenous JAK2 in PAMs. The experiments were similarly performed as in B. E, the effects of inhibitors on the MGF-505-7R-mediated destabilization of JAK1. The 293T cells (4 × 105) were transfected with the indicated plasmids. Then, 18 h after transfection, the cells were treated with the indicated inhibitors for 6 h before western blotting was performed. F, the effects of inhibitors on the MGF-505-7R-mediated destabilization of JAK2. The experiments were similarly performed as in E. Densitometry quantification of JAK2 band was determined using ImageJ (down). G, the effects of inhibitors on the ASFV-mediated destabilization of JAK1 and JAK2. The PAMs were infected with the ASFV (MOI: 0.01) for 18 h. The cells were treated with MG132 (100 µM), NH4Cl (25 mM), or 3-MA (500 ng/ml) for 6 h before western blotting was performed. Densitometry quantification of JAK1 and JAK2 bands was determined using ImageJ (down). Graphs show the mean ± SD, n = 3.

Fig 3: PB2-mediated JAK1 degradation at residues 859K and 860K.a Schematic representation of the deletion mutants of JAK1 (upper) and immunoblots of HEK293T cells transfected with JAK1 or its deletion mutants and PB2 plasmids (lower). b Ubiquitination modification online prediction of JAK1 by CPLM 1.0. c Immunoblots of HEK293T cells transfected with JAK1 or its mutants and PB2 plasmids. d Intensities analysis of the bands on the immunoblots (c) from three independent experiments. Data are presented as the mean ± SD and statistical significance was determined by unpaired two-tailed Student’s t test. ns P > 0.05. e Immunoblots of HEK293T cells transfected with JAK1 or its mutants and PB2 plasmids. f Molecular model of the JAK1 kinase domain generated by PyMOL (PDB: 4ehz). Residues K859/860 are highlighted in pink. g Ni-NTA pull-down analysis of the ubiquitination of JAK1 in HEK293T cells transfected with JAK1 or its mutants, HA-Ub and PB2 plasmids, and treated with MG132. WCL, whole-cell lysates. Data are one representative of three independent experiments.

Fig 4: Silencing of ITGB6 inhibited the JAK/STAT3 signaling pathway in CC cells. (A) GSEA analysis found that ITGB6 expression was positively correlated with HALLMARK IL6/JAK/STAT3 signaling pathway; (B) Western blotting assay was applied to detect the JAK/STAT3 signaling pathway-related proteins (JAK1, p-JAK1, JAK2, p-JAK2, p-STAT3, and STAT3) in SiHa and Hela cells. **P<0.01, compared with the si-NC group. ITGB6, Integrin ß6; CC, cervical carcinoma; GSEA, gene set enrichment analysis; IL6/JAK/STAT3, Interleukin-6/janus kinase/signal transducer and activator of transcription; si-NC, negative control siRNAs.

Fig 5: JAK1 degradation mediated by AIV PB2 significantly increases the virulence of AIV in mice.a, b The mice (n = 10 per group) were intranasally inoculated (106 EID50 per mouse) with rJY, rJYM, rCZ, or rCZM virus and monitored for 14 d for body weight loss (a) and survival (b). c Gloss lesions of lungs from the infected mice on 2 and 5 dpi. Severe pneumonia with diffuse consolidation was outlined in yellow. The images are from representative one of five mice. d Viral titers in the lungs (n = 5 per group) were determined on 2 and 5 dpi by plaque assay. e–h Hematoxylin/eosin (HE) staining (e) and scoring (f), immunohistochemistry (IHC) staining (g), and scoring of lung sections (h). Scale bar, 200 μm. The images are from representative one of five mice. The scores were calculated from five mice. i Immunoblots of lung tissue from the infected mice. j Quantification of JAK1 expression on the immunoblots (i) and normalized with actin (n = 3). k, l qPCR analysis of ISG15 (k) and IFIT1 (l) mRNA in lung from the infected mice on 2 dpi (n = 3). dpi, days post-infection. Data are presented as the mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t test in a, d, f, h, j–l or log-rank test in b.