(A) HEK293 cells were transfected with indicated siRNA, and total RNA was extracted to measure by RT-qPCR the expression of indicated host genes, normalized to the levels of GAPDH. S3 Fig: Cul3 siRNA silencing restores chemokine expression via NF-κB signaling. In all figures, experiments were repeated at least three times. Wild-type HEK293 (WT) and HEK293 cells heterozygous (Het) for Cul3 were transfected with pG-LAP6-Wa-NSP1 or pG-LAP6-ST3-NSP1 and analyzed by western blot using indicated antibodies (right panel). (E) Genotyping of CRISPR-induced incomplete Cul3 knockout HEK293 cells by Sanger sequencing showing the mutated locus (frameshifts) in multiple alleles and the wild-type reference (left panel). The ratio in uninfected, control siRNA-transfected cells is set to 1. Blots were quantified and the level of β-TrCP is normalized to the loading control GAPDH. (D) MA104 cells were transfected with indicated siRNA, infected with RRV or Wa (MOI = 3, 12 hpi) and harvested for western blot analysis using indicated antibodies. (C) Same experiment as in (B) except that different siRNA was used. Western blot was performed to analyze the lysates using the indicated antibodies (FW11: FBXW11 HD1: HECTD1). (B) HEK293 cells stably expressing Wa-NSP1 were transfected with indicated siRNA, and treated with doxycycline. Western blot was performed to analyze the lysates using the indicated antibodies. (A) HEK293 cells stably expressing RRV-NSP1 were transfected with indicated siRNA, and treated with doxycycline at indicated concentrations for 24 hr. S2 Fig: Rbx1 and Cul3 are not involved in RRV-NSP1 mediated IRF3 degradation but contribute to Wa-NSP1 induced β-TrCP degradation. Our study uncovers a novel mechanism that RV employs to promote β-TrCP turnover and provides molecular insights into virus-mediated innate immunity inhibition. Mechanistically, we demonstrate that NSP1 localizes to the Golgi with the host Cul3-Rbx1 CRL complex, which targets β-TrCP and NSP1 for co-destruction at the proteasome. Importantly, inhibition of cullin-3 (Cul3) or RING-box protein 1 (Rbx1), by siRNA silencing or chemical perturbation, significantly impairs strain-specific NSP1-mediated β-TrCP degradation. Multiple Cullin-RING ubiquitin ligase (CRL) complexes were identified. Here, we utilized six human and animal RV NSP1s as baits and performed tandem-affinity purification coupled with high-resolution mass spectrometry to comprehensively characterize NSP1-host protein interaction network. RV encodes non-structural protein 1 (NSP1), a well-characterized interferon (IFN) antagonist, which facilitates virus replication by mediating the degradation of host antiviral factors including IRF3 and β-TrCP. ![]() Rotaviruses (RVs) are the leading cause of severe gastroenteritis in young children, accounting for half a million deaths annually worldwide.
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