Journal: Advanced Science

Article Title: FAM134B Restricts African Swine Fever Virus Capsid Assembly via Reticulophagy and Its Antiviral Activity is Antagonized by the Viral Virulence‐Associated Factor pMGF300‐2R

doi: 10.1002/advs.77741

Figure Lengend Snippet: FAM134B targets the ASFV proteins p72 and pA137R for degradation via reticulophagy. (A–C) FAM134B interacts with p72, and pA137R. HEK293T cells were cotransfected with pFlag‐p72 (A) or pFlag‐A137R (B) together with pHA‐FAM134B. The cells were lysed and whole cell lysates (WCL) were immunoprecipitated with an anti‐HA monoclonal antibody (mAb) at 36 h post‐transfection (hpt). The immunoprecipitates were examined by immunoblotting (IB). WSL cells were infected with ASFV‐WT at a multiplicity of infection (MOI) of 1. At 24 h post‐infection (hpi), the cells were lysed for immunoprecipitation (IP) with anti‐FAM134B. Anti‐IgG antibodies were used as a negative control (C). (D–G) FAM134B degrades p72 and pA137R via the autophagy‐lysosomal pathway. WSL cells were transfected with pFlag‐FAM134B, followed by infection with ASFV‐WT at an MOI of 1 and treatment with Lac (20 µM), MG132 (20 µM), CQ (50 µM), or BafA1 (100 nM) for 8 h at 16 hpi. The cell lysates were analyzed by IB (D, E). The ATG5 ‐knockout ( ATG5‐ KO) or wild‐type (WT) HEK293T cells were transfected with pFlag‐FAM134B, followed by infection with ASFV‐P31 at an MOI of 1. At 24 hpi, the expression levels of p72, pA137R, and FAM134B were analyzed by IB (F, G). (H–L) The degradation of p72 and pA137R mediated by FAM134B depends on the LIR motif of FAM134B. WSL cells were transfected with pHA‐FAM134B or pHA‐FAM134B‐ΔLIR, followed by infection with ASFV‐WT at an MOI of 1. At 24 hpi, the expression levels of p72, pA137R, and FAM134B were analyzed by IB (H, I). Wild‐type HEK293T cells, along with FAM134B ‐KO cells reconstituted with wild‐type FAM134B (WT‐ FAM134B ‐R) or the LIR mutant (ΔLIR‐ FAM134B ‐R), were infected with ASFV‐P31 at an MOI of 1. At 24 hpi, the expression levels of p72, pA137R, and FAM134B were analyzed by IB (J, K). Additionally, viral titers were quantified (L). (M–P) Co‐localization of FAM134B with LC3B and ASFV p72 (M, N) or pA137R (O, P). HEK293T cells were cotransfected with the indicated plasmids and protein colocalization was analyzed by confocal microscopy. The data are presented as the mean ± SD from three independent experiments. Statistical significance was determined by one‐way ANOVA for the data in panels (E, G, I, K, and L), and by two‐tailed unpaired t ‐test for the data in panels (N) and (P) (ns, not significant; * p < 0.05; *** p < 0.001; **** p < 0.0001).

Article Snippet: Mouse anti‐His (AE003), rabbit anti‐ β ‐tubulin (A12289), mouse anti‐GST (AE001), anti‐Flag (AE005), anti‐HA (AE008), anti‐ glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (AC033), and anti‐GFP (AE012) and rabbit anti‐HA (AE105) antibodies were purchased from ABclonal Biotechnology Co., Ltd. Rabbit anti‐RFP (BGT‐ANT‐45233) antibodies were purchased from Biogradetech.

Techniques: Immunoprecipitation, Transfection, Western Blot, Infection, Negative Control, Knock-Out, Expressing, Mutagenesis, Confocal Microscopy, Two Tailed Test