Table 1.
Primer pairs for real-time PCR.
Fig 1.
USP26 expression is significantly increased during EV71 infection.
(A) Differential gene expression of human DUBs in RD cells infected with EV71 after 8 h were analyzed by PCR microarray. (B) RT-qPCR analysis of the USP26 mRNA levels in RD cells infected by EV71 (MOI = 1.0) at different time points. (C) Western blot analysis of the expression of USP26 and EV71/VP1 in RD cells infected by EV71 (MOI = 1.0) at different time points. The independent experiments were performed in triplicate. (D) Data were shown relative to GAPDH expression and are presented as the mean ± SD from three independent experiments. NS, not significant P>0.05, *P<0.05, ** P<0.01 and *** P<0.001.
Fig 2.
USP26 depletion inhibits EV71 infection.
(A, B) Western blot (A) and RT-qPCR (B) analysis of USP26 levels in RD cells transfected with control SiRNA (SiNC) or USP26-specific SiRNAs (SiUSP26, #1, #2 and #3) for 48 h. (C) Western blot analysis of EV71 protein levels in RD cells transfected with SiNC or SiUSP26(#2) and then infected by EV71 (MOI = 1.0) for different times. (D) RT-qPCR analysis of EV71-VP1 RNA levels in RD cells transfected with SiNC or SiUSP26 (#2) and then infected by EV71 (MOI = 1.0) for indicated times. (E) Viral titers of EV71 in RD cells with stable knockdown of USP26 were analyzed. (F) CCK8 analysis of RD cell survival after infection with EV71 (MOI = 1.0) for the indicated times. The cell survival rates at 0 h time points were normalized as 100%. (G) CCK8 analysis of RD cells survival after infection with different amount of EV71. The cell survival rates when MOI = 0 was normalized as 100%. Data were shown as the mean ± SD from three independent experiments. NS, not significant P>0.05 * P<0.05, ** P<0.01 and *** P<0.001. The independent experiments were performed in triplicate.
Fig 3.
Knocking down USP26 results in the upregulation of type I IFN production.
RD cells transfected with knockdown of USP26 or SiNC were infected with EV71(MOI = 1.0) or transfection with Poly (I:C) at 12h. (A-D) Luciferase assay was performed to analyze regulation of IFN-β, ISER, NF-κB and IRF3 promoter by knockdown of USP26. (E-H) The expression of IFN-β, ISG15, MX1 and Viperin mRNA levels were analyzed by RT-qPCR. (I) Western blot analysis of RD cells infected with EV71 for different duration of time, The cell lysates were analysed by immunoblotting using anti-pIRF3, anti-IRF3 and anti- GAPDH antibodies respectively. (J) Western blot analysis of p-IRF3 (pSer396-IRF3), total IRF3, TRAF3, TRAF6 levels in RD cells with knockdown of USP26 that were infected with EV71 (MOI = 1.0) at the indicated time. (K) RT-qPCR analysis of IFNβ mRNA levels in RD cells with knockdown of USP26 that infected with EV71 (MOI = 1.0) at the indicated time. (L) ELISA analysis of the IFN-β protein levels in RD cells with stable knockdown of USP26 that infected with EV71 (MOI = 1.0) at the indicated time. Data were shown relative to GAPDH expression and are presented as the mean ± SD from three independent experiments. NS, not significant P>0.05, ** P<0.01 and *** P<0.001. The independent experiments were performed in triplicate.
Fig 4.
USP26-deficient mice were resistant to virus infection.
WT and USP26-/- mice were infected with EV71 virus (1 × 107 PFU per mouse). (A) Survival rate of WT and USP26-/- mice after EV71 infection for indicated time. (B) Viral EV71 VP1 mRNA was determined by RT-qPCR. (C) Viral titres of EV71 virus were determined. (E) Expression of IFN-β and IL-6 was determined by qPCR. (D-G) Expression of IFN-β, ISG15, MX1 and Viperin were determined by RT-qPCR. (H) Serum levels of IFN-β was determined by ELISA at 3 days post infection. Data shown are the mean ±SD. NS, not significant P>0.05, ** P<0.01 and *** P<0.001. Data are representative of three independent experiments with similar results.
Fig 5.
USP26 can interact with TRAF3.
(A-G) Co-immunoprecipitation analysis of the interaction between USP26 and the key components of RIG-I/MAVS signaling. HEK293T cells were transfected with Flag-RIG-I or Flag-MDA5 or Flag-MAVS or Flag-TRAF6 or Flag-TRAF3 or Flag-TBK1 or Flag-IRF3, and immunoprecipitated with anti-Flag agarose beads. The eluted immunocomplex was then subjected to SDS-PAGE analysis with anti-USP26 antibody. (H) Co-immunoprecipitation analysis the interaction between endogenous USP26 and TRAF3 in HEK293T cells. The independent experiments were performed in triplicate.
Fig 6.
USP26 decreases the polyubiquitination of TRAF3.
(A) Western blot analysis of HEK293T cells transfected with Flag-TRAF3, HA-USP26 and HA-Ub for cellular ubiquitination levels; GAPDH was used as a loading control. (B) HEK293T cells transfected with Flag-TRAF3, HA-Ub, together with wild-type HA-USP26 were collected and immunoprecipitated with anti-Flag agarose beads. The eluted immunocomplex was then subjected to SDS-PAGE analysis with antiubiquitin antibody. (C) Western blot analysis of RD cells transfected with knockdown of USP26 or SiNC for 48 h, followed by treatment with EV71 (MOI = 1) for 12 h. The cell lysates were subjected to immunoprecipitation with anti-TRAF3 antibody, followed by western blot analysis of eluted immunocomplex with K63 linkage-specific ubiquitin antibodies.