Investigating the Potential Role of Capsaicin in Facilitating the Spread of Coxsackievirus B3 via Extracellular Vesicles

Figures

• 

  Figure 1. Capsaicin enhances CVB3 infection. Human cervical cancer cells HeLa were treated with 10 µM Capsaicin for 15 min and infected with EGFP-CVB3 at multiplicity of infection (MOI) of 0.001 for 48 h. (A). Fluorescence microscopy compares viral EGFP expression between vehicle (DMSO-treated) and capsaicin-treated cells. Phase contrast images are shown below respective fluorescence image fields. Scale bars represent 100 µm. (B). Flow cytometry of infected cells revealed significantly increased EGFP-positivity in capsaicin-treated cells as compared to controls. (C). Plaque assay quantification of infectious virus in extracellular media. (D). Western blot on cell lysates from (A) showing VP1 viral capsid protein. Ponceau S is shown below. (VEH: Vehicle; CAP: Capsaicin). (E). Densitometric quantification of Western blot in (B) (** p < 0.01; Student’s t-test, n = 3, values indicate mean ± standard deviation (SD)).

• 

  Figure 2. Capsaicin induces viral release in EVs. HeLa cells were treated with 10 µM Capsaicin for 15 min and infected with EGFP-CVB3 at an MOI of 0.001 for 48 h. (A). Western blot on isolated EVs showing VP1 viral capsid protein. Ponceau S is shown below. (VEH: Vehicle; CAP: Capsaicin) (B). Densitometric quantification of Western blot in (A) (* p < 0.05; Student’s t-test, n = 3). (C). Plaque assay quantification of viral titers in EV (* p < 0.05; Student’s t-test, n = 3) (D). Plaque assay of remaining free virus in the supernatant following EV isolation from capsaicin and DMSO-treated cells (p = 0.193; Student’s t-test, n = 3, values indicate mean ± SD).

• 

  Figure 3. Capsaicin increases mitochondrial content in EVs. HeLa cells were treated with 10 µM Capsaicin for 15 min and infected with EGFP-CVB3 at multiplicity of infection (MOI) of 0.001 for 48 h. Western blot on EVs. (A) and whole cell lysates (D) detecting TOM70 and phospho-DRP-1 (Ser 616). Ponceau S is shown below. (VEH: Vehicle; CAP: Capsaicin). Densitometric quantification of Western blots for EVs (B,C) and cell lysates (E,F). (** p < 0.01; **** p < 0.0001; Student’s t-test, n = 3, values indicate mean ± SD).

• 

  Figure 4. Capsaicin induced EVs are enriched with HSP70. HeLa cells were treated with 10 µM Capsaicin for 15 min and infected with EGFP-CVB3 at MOI of 0.001 for 48 h. (A). Western blot on EVs and (C) whole cell lysates detecting HSP70. Ponceau S is shown below. (VEH: Vehicle; CAP: Capsaicin) (B,D). Densitometric quantification of Western blot in (A,C). (*** p < 0.001, **** p < 0.0001; Student’s t-test, n = 3, values indicate mean ± SD).

• 

  Figure 5. Silencing HSPA1A inhibits CVB3 infection. (A). HeLa cells were transfected with siRNA targeting HSP70 (siHSPA1A) or scrambled RNA (siSCRAMBLE) for 48 h and subsequently infected with EGFP-CVB3 at MOI 0.001 for 24 h. (A) Fluorescence microscopy comparing viral EGFP expression between cells transfected with either siSCRAMBLE or siHSPA1A. Corresponding phase contrast images are shown below the fluorescence images. Scale bars represent 150 µm. (B). Western blot on cell lysates from (A) detecting HSP70 and VP1 viral protein levels. Ponceau S is shown below. (SCR: SCRAMBLE; HSP: HSPA1A) (C,D). Densitometric quantification of Western blots in (B). (E). Plaque assay quantification of infectious virus in media from siSCRAMBLE and siHSPA1A transfected cells. (* p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t-test, n = 3, values indicate mean ± SD).

• 

  Figure 6. HSP70 inhibitor VER-155008 inhibits CVB3 infection. HeLa cells were treated with 20 µM VER-155008 for 1 h and infected with EGFP-CVB3 at multiplicity of infection (MOI) of 0.001 for 24 h. (A). Fluorescence microscopy compares viral EGFP expression between vehicle (DMSO-treated) and VER-treated cells. Phase contrast images are shown below respective fluorescence image fields. Scale bars represent 100 µm. (B). Western blot on cell lysates from (A) showing viral capsid protein VP1. Ponceau S is shown below. (VEH: Vehicle; VER: VER-155008). (C). Densitometric quantification of Western blot in (B). (D). Plaque assay quantification of infectious virus in media from cells in (A). (** p < 0.01, *** p < 0.001; Student’s t-test, n = 3, values indicate mean ± SD).

• 

  Figure 7. Capsazepine inhibits CVB3 infection. HeLa cells were treated with 40 µM capsazepine for 4 h and infected with EGFP-CVB3 at multiplicity of infection (MOI) of 0.001 for 48 h. (A). Fluorescence microscopy compares viral EGFP expression between vehicle (DMSO-treated) and capsazepine-treated cells. Phase contrast images are shown below respective fluorescence image fields. Scale bars represent 100 µm. (B). Western blot on cell lysates from (A) showing VP1 viral capsid protein. Ponceau S is shown below. (VEH: Vehicle; CZP: Capsazepine). (C). Densitometric quantification of Western blot in (B,D). Plaque assay quantification of infectious virus in media from cells in (A). (* p < 0.05; Student’s t-test, n = 3, values indicate mean ± SD).

• 

  Figure 8. SB-366791 reduces pancreatic viral titers and tissue destruction in CVB3-infected mice. (A). Experimental timeline showing 10-week-old C57BL/6 male mice were treated with 1 mg/kg SB-366791 or equivalent volume vehicle each day for 3 days via intraperitoneal (IP) administration. On the second day of treatment, mice were infected IP with EGFP-CVB3 at a concentration of 107 PFU/mouse. A total of 2 days post-infection, mice were sacrificed, and pancreata were collected. (B). Pancreatic viral titers as measured by plaque assays on pancreatic homogenates. (* p < 0.05; Student’s t-test, n = 4). (C). Representative microscopic images of the pancreas stained with hematoxylin and eosin (H&E, left panels; scale bar: 100 µm) or QuPath images (right panels; scale bar: 100 µm) for automatic quantification of cell types and stroma of same images shown in the left panels. (D). Percentage of each cell type feature detected in vehicle (upper chart) and SB-366791 (lower chart) treated infected mice. The percentage of each cell type was determined in QuPath by dividing the number of cells assigned to each classified subtype by the total number of cells detected within the corresponding tissue…

• 

  Figure 9. Capsaicin has a significant impact on CVB3 infection dynamics. (A). Capsaicin-mediated TRPV1 activation induces mitochondrial fission, which leads to mitophagy. CVB3 hijacks these mitophagosomes to form mitophagosome-derived EVs, within which the virus multiplies. These EVs then facilitate viral dissemination, serving as a vehicle for CVB3 release. (B). In addition, these capsaicin-treated EVs also showed higher levels of HSP70. Earlier literature suggests that TRPV1 plays a crucial role in controlling the cellular heat shock response via activation of Heat shock Factor-1 (HSF-1) [35]. HSF-1 then trimerizes and translocate to the nucleus where it binds to HSPA1A promotor, resulting in translation of the protein HSP70 [82]. In our study, we observed that inhibiting HSP70 significantly impaired CVB3 release via EVs, indicating that HSP70 is crucial for viral egress. (C). Previous studies have shown a prominent role of HSP70 in mitophagy, particularly in HEK293 cells, but whether HSP70 interacts with mitochondrial proteins or primes cells for mitophagy in the context of CVB3 infection remains to be fully explored and needs further investigation.