Figure 7. Brucella infection dissociates host BORC and degradation of Bloc1s1 mRNA supports coronavirus intracellular replication.

(A) Co-immunoprecipitation (Co-IP) analysis of the interactions of BLOS1 with other proteins that form the BLOC-1 (BLOS1, BLOS2, SNAPIN, and PALLIDIN) or BORC (BLOS1, BLOS2, SNAPIN, and KXD1) complex. The nonfunctional mBloc1s1 and control cells were cultured overnight before being subjected to Co-IP assays with BLOS1 as an input. (B) Quantification of the indicated pulled-down protein levels from overnight-cultured control and mBloc1s1 cell lysates using BLOS1 as an input. (C) Co-IP assays for Brucella-infected control and mBloc1s1 cells at the indicated h.p.i. using BLOS1 (left panel) or LYSPERSIN (LYSPS, right panel) as an input. Red arrow: BLOS1. Quantification of the indicated pulled-down protein levels of SNAPIN (D) or KXD1 (E) from Brucella-infected control and mBloc1s1 cell lysates using BLOS1 or LYSPS as an input. Red asterisks: significance when compared to the control at 2 h.p.i. (F) Co-IP assays for Brucella-infected wt- or Rr-Bloc1s1 cells at the indicated h.p.i. using LYSPERSIN as an input. (G) Quantification of the indicated pulled-down protein levels from Brucella-infected cell lysates of wt- or Rr-Bloc1s1. Red asterisk: significance when compared to the wt-Bloc1s1 control at 2 h.p.i. (H) PFU (plaque-forming units) assay of coronavirus MHV infection of the mBloc1s1 and control cells treated or untreated with 4μ8C (50 μM) at the indicated h.p.i. (I) Bloc1s1 mRNA expression assay of the indicated host cells infected with MHV via qRT-PCR. Red asterisk: significance when compared to the uninfected control. (J) A proposed model describing how Brucella subverts the host RIDD-BLOS1 pathway to support intracellular parasitism by disrupting BORC-directed lysosomal trafficking. BCV: Brucella-containing vacuole. eBCV, rBCV, and aBCV: endosomal BCV, replicative BCV, and autophagic BCV, respectively. BORC: the BLOC-1-related complex. Green arrows: BCV trafficking to the ER compartment and replication. Blue arrows: BCV trafficking to peripheral lysosome and lysosomal degradation. Host cells were infected with or without Brucella or MHV, and at the indicated h.p.i., the infected or uninfected host cells were harvested for Co-IP and immunoblotting assays, or qRT-PCR assays. Blots are representative of three independent experiments. Statistical data represent the mean ± standard error of mean (SEM) from three independent experiment. *p < 0.05; **p < 0.01; ***p < 0.001. Red asterisks: Compared to the same Brucella-infected cells at 2 h.p.i.