Fig. 5

YT-DRI inhibits HPIV3 replication through disrupting the RAB11A–FIP2 complex. A YT-DRI disrupts N-RAB11A interaction by competing with FIP2 for RAB11A binding. 293 T cells were individually transfected with plasmids encoding FIP2, the N protein, and RAB11A for 36 h, after which their lysates were mixed for in vitro co-IP analysis. The co-IP assays, using anti-Flag magnetic beads, showed that YT-DRI interferes with N-RAB11A binding, effectively competing with FIP2. B and C YT-DRI inhibits vRNP distribution and VLPs production. 293 T cells, transfected with relevant plasmids for 48 h, underwent cytosol and PM fractionation, with supernatants analyzed for VLPs. WB analysis, using GAPDH and Na/K ATPase as markers, revealed YT-DRI's impact on vRNP distribution (B) and VLPs production (C). D YT-DRI impedes HPIV3 infection by disrupting vRNP distribution. HeLa cells, infected with HPIV3_HA-P (MOI = 0.01), received 5 μM YT-DRI or Tat treatment for 24 h. Immunofluorescent detection of vRNP displayed altered distribution patterns, with YT-DRI hindering vRNP clusters at the PM. E The growth curve of the HPIV3 with or without treatment of YT-DRI on HeLa (WT and RAB11A-KO) cells. The HeLa cells were infected with HPIV3 (MOI = 0.05) and treated YT-DRI at a final concentration of 5 μM (∼5IC₅₀). The supernatants were assayed for viral titers at 6 h, 12 h, 18 h, 24 h, 30 h, and 36 h post-infection by TCID₅₀. F Statistical analysis of YT-DRI's antiviral impact at 24 h post-infection. The statistical significance of YT-DRI's antiviral effect in RAB11A-depleted cells was determined by two-sided unpaired t-test. All experiments were independently replicated at least twice with consistent results. Statistical significance (*p < 0.05) was determined by two-sided unpaired t-test. Data are presented as means ± SEM