Fig 1.
Proteomics analysis of rotavirus NSP1-host interactions.
(A) Workflow diagram of affinity purification-mass spectrometry (AP-MS) pipeline used to identify NSP1-interacting host proteins. (B) Representative silver-stained SDS-PAGE of elutes (post TEV cleavage) from HEK293 cell lines stably expressing NSP1s derived from RV strains: RRV (simian), ETD (murine), or Wa (human). (C) Network representation of the bait protein Wa-NSP1 (yellow node) and some high-confidence host interaction partners (cyan nodes). Solid blue lines represent interactions identified in this study. Dotted grey lines indicate curated PPIs in public proteomics databases. (D) Heat map summary of a few host proteins (IRF3, β-TrCP, and CRL complex) interacting with different NSP1s (RRV and SA11-5S: simian RVs; WA and ST3: human RVs; UK: bovine RV; ETD: murine RV). The color corresponds to the number of peptides identified in the AP-MS experiments. (E) (Western blot (WB) of lysates using WT HEK293 cells and HEK293 stable cell lines, with or without doxycycline (dox) treatment, probed for indicated antibodies (FL: full-length MAVS; mini: mini-MAVS).
Fig 2.
Cul3 and Rbx1 interact with NSP1 and contribute to β-TrCP degradation.
(A) Lysates of HEK293 cells co-transfected with GFP-NSP1, Myc-Cul3, and HA-Rbx1 were subject to immunoprecipitation using α-Myc or α-HA antibody and probed for α-GFP antibody. (B) HEK293-Wa-NSP1 stable cell lines were treated with 1 μg/ml of doxycycline for 24 hr and harvested for immunoprecipitation using α-GFP antibody and probed for indicated antibodies. (C) Lysates of mock or Wa infected HEK293 cells (MOI = 3, 24 hpi) were subject to immunoprecipitation using control rabbit polyclonal or α-Cul3 antibody, and probed for indicated antibodies. (D) HEK293 cells stably expressing RRV or ETD-NSP1 were transfected with indicated siRNA, treated with doxycycline, and harvested for western blot using indicated antibodies. (E) Same experiment as in (D) except that HEK293 cells stably expressing Wa-NSP1 were used instead (HD1: HECTD1). Blots were quantified and the level of β-TrCP is normalized to loading control GAPDH. The ratio in mock-treated cells is set to 1. (F) Same experiment as in (D) except that ST3, UK, or SA11-5S NSP1s were used (HD1: HECTD1). (G) HEK293 cells stably expressing Wa-NSP1 were treated with MLN4924 and doxycycline, harvested for western blot using indicated antibodies. In all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*p≤0.05; **p≤0.01; ***p≤0.001).
Fig 3.
Cul3 attenuation restores IκB degradation and chemokine expression.
(A) HEK293 cells stably expressing Wa-NSP1 were transfected with indicated siRNA, treated with doxycycline, and stimulated with TNF-α (10 ng/ml) for 15 min. Lysates were harvested for western blot using indicated antibodies. (B) Same experiment as in (A) except that cells were also stimulated with IL-1β (10 ng/ml) for 15 min and examined for the localization of NF-κB p65 subunit (red), Wa-NSP1 (green) and nucleus (DAPI, blue) by confocal microscopy. Panels are single z slices with a scale bar of 5 μm. (C) HEK293 cells stably expressing Wa-NSP1 were transfected with indicated siRNA, treated with doxycycline, and stimulated with TNF-α (10 ng/ml) or IFN-β (100 U/ml) for 6 hr. RNA was extracted to measure by RT-qPCR the expression of CXCL10 and RSAD2, normalized to the housekeeping gene GAPDH. (D) Same experiment as in (C) except that HEK293 cells stably expressing UK-NSP1 were used. (E) HEK293 cells were transfected with indicated siRNA, infected with RRV or Wa (MOI = 1, 3 dpi), and harvested for RT-qPCR analysis examining the expression of viral gene NSP5, normalized to GAPDH. (F) Supernatants from (E) were collected at indicated time points and titrated by standard plaque forming unit assay. In all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*p≤0.05; **p≤0.01; ***p≤0.001).
Fig 4.
NSP1 co-localizes with CRL components at the Golgi apparatus.
(A) HEK293 cells were transfected with pG-LAP6-Wa-NSP1 (green), and analyzed by confocal microscopy for the localization of the Golgi apparatus (GM130, red) and nucleus (DAPI, blue). Single cell zoom-in is shown in the inset. Co-localization (yellow) is highlighted by white arrowheads. Panels are single z slices with a scale bar of 15 μm. (B) Same experiment as in (A) except that cells were super-infected with Wa (MOI = 3, 12 hpi) prior to fixation. Single cell zoom-in is shown in the inset. Co-localization (yellow) is highlighted by white arrowheads. Panels are single z slices with a scale bar of 15 μm. (C) Lysates of Wa-infected HEK293 cells (MOI = 3, 24 hpi) were fractionated over a sucrose gradient using ultracentrifugation. Fractions were analyzed by western blot for the indicated antibodies. In all figures, experiments were repeated at least three times.
Fig 5.
Wa-NSP1 interacts with host proteins Cul3 and β-TrCP.
(A) Schematic of structural and functional domains within WT Wa-NSP1 and illustration of Wa-NSP1 point mutations and deletion mutants. (B) Lysates of HEK293 cells transfected with WT and mutant Wa-NSP1s were subject to immunoprecipitation using α-GFP antibody and probed with α-Cul3 antibody. (C) Lysates of HEK293 cells transfected with WT and mutant Wa-NSP1s were analyzed by western blot using indicated antibodies. (D) HEK293 cells were co-transfected with PRDII-luc, pRL-TK and Wa-NSP1 mutants, stimulated with TNF-α (10 ng/ml) for 6 hr, and harvested for Dual-Glo luciferase assay. Arbitrary units were determined by the ratio of firefly luciferase (FFL) to renilla luciferase (RL). (E) Lysates of HEK293 cells transfected with indicated pG-LAP6 plasmids and treated with 10 μM of lactacystin for 6 hr were subject to immunoprecipitation using α-GFP antibody and probed with α-β-TrCP antibody (Rmut: RING mutant, H61RC63AC66A; Cmut: CTERM mutant, D479AS480AS483A; DM: Double-Mutant, Rmut+Cmut). (F) Lysates of HEK293 cells transfected with indicated pG-LAP6 plasmids were analyzed by western blot using indicated antibodies. In all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*p≤0.05; **p≤0.01; ***p≤0.001).
Fig 6.
NSP1 and β-TrCP are targeted for proteasomal co-degradation.
(A) HEK293 cells were transfected with control or Cul3 siRNA, and co-transfected with Flag-β-TrCP, HA-tagged Ubiquitin (Ub) and GFP-Wa-NSP1. Lysates were subject to immunoprecipitation using α-Flag antibody and probed with α-HA antibody. Top panel blots were quantified and the ubiquitination intensities were expressed as a percentage of the Ub level in the Ub+NSP1+ctrl siRNA lane, which is set to 100. (B) HEK293 cells were transfected with Flag-β-TrCP and infected with human RV Wa (MOI = 3, 24 hpi). Lysates were subject to immunoprecipitation using α-Flag antibody and probed with α-poly-Ub antibody (UI: uninfected). (C) HEK293 cells were transfected with pG-LAP6-Wa-NSP1, treated with lactacystin (10 μM) or MLN4924 (0.1 or 1 μM) for 24 hr, and harvested for western blot using indicated antibodies. (D) HEK293 cells were mock or Wa infected (MOI = 3, 12 hpi), treated with MLN4924 (1 μM) for 11 hr, and harvested for western blot using indicated antibodies. (E) HEK293 cells were co-transfected with HA-tagged APOBEC3G, pG-LAP6-Wa-NSP1 or pCMV6-Entry vector encoding Flag-tagged indicated viral proteins, treated with lactacystin (10 μM) or MLN4924 (1 μM) for 24 hr, and harvested for western blot using indicated antibodies. In all figures, experiments were repeated at least three times.
Fig 7.
CRL inhibition reduces β-TrCP degradation by RV Wa infection in human intestinal enteroids.
(A) Brightfield images of human enteroids cultured in 3D Matrigel matrix. Scale bar, 100 μm. (B) Enteroids were infected with human RV Wa strain (MOI = 3) for 16 hr and analyzed by confocal microscopy for viral antigen VP6 (green), cytoskeleton (phalloidin staining, red), and nucleus (DAPI, blue). Scale bar, 100 μm. (C) Enteroids were infected with human RV Wa strain (MOI = 1, 24 hpi) with or without MLN4924 treatment (1 μM, 24 hr). Trypsin-digested single cells were stained with viral antigen VP6, epithelium marker EpCAM and β-TrCP, and analyzed by flow cytometry. Fluorescent intensity of β-TrCP in EpCAM, VP6 double positive cells was quantified in FlowJo v8.8. In all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*p≤0.05; **p≤0.01; ***p≤0.001).
Fig 8.
Model for NSP1 hijacking of Cul3-CRL to target β-TrCP for degradation.
During RV infection, NSP1 co-localizes with Cul3 and Rbx1 at the Golgi apparatus, interacts with Cul3 and β-TrCP using its N- and C-terminal domains respectively, and promotes the co-degradation of β-TrCP and NSP1 at the proteasome. Blocking Cul3 (MLN4924) or proteasome (lactacystin) prevented β-TrCP turnover by RV NSP1. Cul3 inhibition leads to the restoration of IκB levels and NF-κB-driven gene expression.