Abietic acid targets RAN to restrict coxsackievirus B3 infection

Figures

• 

  FIGURE 1. Abietic acid (AA) exhibits potent antiviral activity against CVB3 in vitro. (A) The chemical structure of AA. (B) The indicated concentrations of AA were employed for the treatment of HeLa cells over a period of 48 h, and CCK-8 assay was performed to measure cell viability. (C) HeLa cells were infected with CVB3 (MOI = 1) and then treated with increasing concentrations of AA for 24 h. RT-qPCR was utilized to determine viral genome copy numbers. (D) HeLa cells underwent infection using CVB3 (MOI = 1) and treated via 25 μM AA over a period of 24 h. Morphological variations were detected using a light microscope. Scale bar = 10 μm. (E) HeLa cells were inoculated with CVB3 at an MOI of 1, followed by treatment with indicated concentrations of AA over a period of 24 h. The protein expression level of CVB3 3D was analyzed via Western blot analysis, with GAPDH used as the loading control. (F) Cells and culture supernatants from E were gathered, and viral titers were identified using TCID50 assay. (G) HeLa cells were inoculated with EGFP-CVB3 at an MOI of 5, followed by treatment with the indicated concentrations of AA over a period of 24 h. EGFP fluorescence was visualized utilizing a…

• 

  FIGURE 2. AA blocks CVB3 entry and replication. (A) Schematic illustration of the time-of-drug-addition assay. (B) CVB3 was pre-incubated using 25 μM AA over a period of 1 h ahead of infection of HeLa cells. Viral genome copy numbers were assessed using RT-qPCR. (C) HeLa cells underwent pretreatment using 25 μM AA for over a period of 1 h, followed by washing, and then infection with CVB3 (MOI = 1). Viral genome copy numbers were determined by RT-qPCR. (D) HeLa cells underwent infection with CVB3 (MOI = 1) in the presence of 25 μM AA at 4 °C for 1 h. Unbound virus was removed by washing, and viral genome copy numbers were determined by RT-qPCR. (E) HeLa cells underwent infection with CVB3 (MOI = 1) for 1 h, then treated with 25 μM AA for 24 h. Viral genome copy numbers were calculated using RT-qPCR. (F) Viral growth curve. Values are presented as mean ± SD. ns, not significant; *P < 0.05, ***P < 0.001.

• 

  FIGURE 3. AA directly targets RAN and downregulates its expression to inhibit CVB3 replication. (A) HeLa cells experienced infection with CVB3 at indicated MOIs over a period of 24 h. RAN mRNA levels were measured by RT-qPCR, and protein levels of RAN and viral 3D were identified via Western blot. GAPDH served as a loading control. (B) HeLa cells underwent transfection with siNC or siRAN for 48 h, then infected with CVB3 (MOI = 1) for 24 h. Viral genome copy numbers were measured using RT-qPCR, and protein levels of RAN and viral 3D were identified via Western blot. (C) HeLa cells underwent transfection with pLenti-vector or increasing doses (0.5, 1, 2 μg) of pLenti-RAN over a period of 48 h, and then were infected infected with CVB3 (MOI = 1) for 24 h. Viral genome copy numbers were measured by RT-qPCR, and protein levels of HA, RAN, and viral 3D were detected by Western blotting. (D) HeLa cells underwent infection with CVB3 (MOI = 1) over a period of 2 h, followed by treatment with 25 μM AA for 24 h. RAN mRNA levels were measured by RT-qPCR, and protein levels of RAN and viral 3D were evaluated via Western blotting. (E) RAN knockdown abrogates AA’s antiviral effect. HeLa cells underwent…

• 

  FIGURE 4. AA restores STAT1/2 nuclear import in a RAN-dependent manner. (A) HeLa cells underwent infection with CVB3 (MOI = 1) and treatment with 25 μM AA for 24 h. Nuclear and cytoplasmic fractions were isolated, and the distribution of p-STAT2, p-STAT1, STAT1, and STAT2 was analyzed by Western blotting. Histone H3 and GAPDH served as nuclear and cytoplasmic markers, respectively. (B) HeLa cells experienced treatment with 25 μM AA, IFN-α, or their combination. Nuclear fractions were isolated, and p-STAT1/2 levels were detected by Western blot. (C) HeLa cells experienced treatment with 25 μM AA, IFN-α, or their combination over a period of 24 h. ISGs mRNA levels were measured via RT-qPCR. (D) Lysates from HeLa cells overexpressing HA-RAN were immunoprecipitated with anti-HA antibody. The immunocomplexes were analyzed by Western blot using antibodies against p-STAT2, p-STAT1, and HA. Input and IgG controls are presented. Values are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

• 

  FIGURE 5. In vivo antiviral efficacy of AA against CVB3-induced myocarditis. (A) Schematic illustration of the in vivo experimental design. (B) Mice were weighed daily, and body weights were normalized to initial values. (C) Survival curves of mice in each group over 7 days post-infection. (D) Heart tissues were collected at 7 dpi, and mRNA levels of IL-6, TNF-α, and IL-1β were measured by RT-qPCR. (E) Heart tissues were collected at 7 dpi, and CVB3 genome copy numbers were detected via RT-qPCR. (F) Serum was collected at 7 dpi, and CK-MB and LDH activities were measured. (G) Heart tissue lysates were prepared at 7 dpi, and viral 3D protein expression was analyzed using Western blotting. (H) Heart sections were stained utilizing HE at 7 dpi and examined under a light microscope. Scale bar = 10 μm. Values are expressed as mean ± SD. *P < 0.01, ***P < 0.001.

• 

  FIGURE 6. Schematic diagram of the anti-CVB3 mechanism of AA.