Peptides targeting RAB11A–FIP2 complex inhibit HPIV3, RSV, and IAV replication as broad-spectrum antivirals

Fig. 1. Actin plays a critical role in the late stages of HPIV3 infection. A–D Impact of cytoskeleton inhibitors on HPIV3 replication. HeLa cells infected with HPIV3 for 18 h were treated with Nocodazole (NCZ) or Cytochalasin D (Cyto D). At 24 h post infection (hpi), the virions released in the cell supernatant (A) and the quantity of intracellular progeny virus particles (B) were determined by TCID50 assays. Cells were collected for RT-qPCR analysis of intracellular viral RNA (C), and Western Blot (WB) analysis of intracellular viral protein (D). E Influence of cytoskeleton inhibitors on HPIV3 VLPs production. 293T cells transfected with indicated plasmids for 30 h were treated with NCZ or Cyto D for 6 h. Cell lysates and VLPs were prepared and analyzed by WB. F Impact of cytoskeleton inhibitors on vRNP distribution. HeLa cells infected with HPIV3HA-P (MOI = 0.01) for 18 h were treated with NCZ or Cyto D for 6 h. Fluorescent microscopy was employed to examine vRNP distribution using an anti-HA antibody (green), plasma membrane stained by Dil (red), and nuclei were stained with DAPI. Arrowheads indicate plasma membrane. Scale bar, 50 μm. G Progeny virions released via actin filaments.…

Fig. 2. A RAB11A-dependent mechanism governs the progresses of HPIV3 infection: from vRNP transport to budding. A Impact of RAB11A depletion on HPIV3 replication in HeLa cells. WT and RAB11A-depleted HeLa cells infected with HPIV3 (MOI = 0.01) for 24 h were analyzed for viral protein by WB, and viral titers in the cell supernatant were determined by TCID50. B RAB11A is not required for HPIV3 binding and internalization. Analysis of the virions binding and internalized into HeLa of HPIV3 to WT and RAB11A-depleted HeLa cells using RT-qPCR. Cells were incubated with the virus at an MOI of 5 for 1 h at 4 °C, and then washed 3 times with ice-cold PBS to remove unbound viral particles, and then RNA could be extracted for binding analysis. For internalization assay, fresh complete DMEM was added following binding processes, and the cells were incubated at 37 °C for another 1 h to allow virus internalization. HPIV3 RNA copy numbers were normalized to the mean values for WT cells in each assay. C Disruption of vRNP trafficking due to RAB11A depletion. WT and RAB11A-depleted HeLa cells infected with HPIV3HA-P (MOI = 0.01) for 24 h were immunofluorescently labeled for vRNP (green), plasma membranes…

Fig. 3. Formation of RAB11A–FIP2 complex is necessary to HPIV3 infection. A N protein orchestrates an exclusive interaction with active RAB11A. 293T cells, individually or jointly transfected with plasmids encoding N-Flag, Myc-RAB11A, Myc-RAB11ADN, or Myc-RAB11ACA, were subjected to co-IP assays at 36 h post-transfection. Immunoprecipitation with anti-Flag magnetic beads followed by WB revealed the specific interaction. B Diverse RAB11A forms and their co-localization with vRNP. HeLa cells expressing RAB11A, RAB11ADN, or RAB11ACA were infected with HPIV3HA-P (MOI = 0.01) for 24 h. Immunofluorescent detection of vRNP (green) and RAB11A (red) showcased distinct co-localization patterns. Scale bar, 50 μm. C Impact of RAB11A variants on vRNP distribution and VLPs production. 293T cells transfected with RAB11A variants were fractionated into cytosol and plasma membrane (PM) fractions. WB analysis of vRNP and VLPs in different cellular compartments demonstrated the influence of RAB11A forms. Tubulin and Na/K ATPase served as cytosol or PM markers. D RAB11ADN and RAB11ACA act as suppressors of HPIV3 replication. Hela cells overexpressing RAB11A, RAB11ADN, or RAB11ACA were infected with…

Fig. 4. Antiviral activity of peptides targeting RAB11A–FIP2 complex against respiratory RNA viruses infection in cells. A Overexpression RBD outcompetes FIP2 for RAB11A binding. 293T cells, transfected with specific plasmids for 36 h, underwent co-IP assays using anti-Myc magnetic beads. Subsequent WB analysis revealed the competitive binding of RBD with FIP2 to RAB11A. B Overexpression RBD hinders N protein interaction with RAB11A. 293T cells, transfected with relevant plasmids for 36 h, were subjected to co-IP assays. Utilizing anti-Myc magnetic beads, the assays demonstrated RBD-mediated suppression of N protein binding to RAB11A. C Overexpression RBD inhibits HPIV3 infection. Hela cells, transfected with Flag-mCherry or Flag-mCherry-RBD plasmids, were infected with HPIV3 (MOI = 0.01) for 24 h. WB analysis showcased reduced viral protein levels, and TCID50 quantification confirmed lower viral titers in the presence of RBD. D and E Dose-dependent inhibition of HPIV3 infection by RBD and Tat peptides. HeLa cells, infected with HPIV3 (MOI = 0.01), received escalating concentrations of RBD (D) or Tat (E). At 24 hpi, WB analysis depicted the dose-dependent impact on viral protein levels,…

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 HPIV3HA-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…

Fig. 6. In Vivo evaluation of YT-DRI against respiratory viral infections. A Experimental design for HPIV3 infection. 6–8-week nude BALB/c mice were intranasally infected with 2 × 106 PFU of HPIV3 per mouse and treated with PBS or YT-DRI (2 mg/kg) via intraperitoneal injection, administered once per day from Day 0 to Day 2. Mouse body weight was monitored daily for 4 days. B Body weight changes in HPIV3 infection. Graphical representation illustrating the body weight changes of mice in different groups over the course of the experiment. C and D Viral RNA copies, the infectious titers and the lung histopathology. At Day 3, animals were euthanized, lung tissues were collected, homogenized. Viral RNA level was measured with quantitative RT-qPCR and virion load was titered by TCID50 assay (C). Histopathology of lung tissues was assessed through hematoxylin and eosin (H&E) staining (D). E Experimental design for RSV infection. 6–8-week nude BALB/c mice were intranasally challenged with 3 × 106 PFU of RSV and treated with PBS or YT-DRI (2 mg/kg) via intraperitoneal injection. Treatment occurred once per day from Day 0 to Day 3. Daily monitoring of body weight was conducted for 5 days. F Body…