Journal: Journal of advanced research

Article Title: Antimycin A inhibits alpha-herpesvirus replication by disrupting the formation of pyrimidinosomes.

doi: 10.1016/j.jare.2025.05.016

Figure Lengend Snippet: Fig. 4. Antimycin A exhibits extensive antiviral activity against alpha-herpesvirus. Antimycin A effectively inhibited HSV-1 (A-C) and HSV-2 (G-H) infections in Vero E6 cells, PRV (D-E) infection in PK-15 cells, and EHV-1 (J-K) infection in RK13 cells. Vero-E6 cells, PK-15 cells, and RK13 cells were pretreated for 12 h with increasing concentrations of Antimycin A and then infected with HSV-1 (A-C), PRV (D-E), HSV-2 (G-I), and EHV-1 (J-K) at MOIs of 0.5, 0.1, 0.5, and 0.5, respectively. At 24 hpi, cells were fixed and analyzed by fluorescence imaging. (A, D, G and J) Infection levels were quantified using a fluorescent microplate reader (black curve), while cell viability was measured using the CCK-8 Assay (orange curve). The CC50 for each compound was calculated via a four-parameter logistic nonlinear regression model in GraphPad Prism. Dotted lines indicate 50 % inhibition. Data represent the means ± SEM from n = 3 independent experiments of infectious virions, normalized to DMSO-treated wells. The IC50 values for HSV-1, PRV, HSV-2, and EHV-1 were determined by nonlinear regression analysis. (B, E, H and K) eGFP expression in infected cells, either untreated (0 lM) or treated with various concentrations (0.0015–5 lM) of Antimycin A, was visualized by fluorescence microscopy at the same time point. Representative images are shown. Bars, 300 lm. Magnification, ×10. (C, F and I) Western blot analysis was performed to quantify infection in cells infected with HSV-1, PRV, or HSV-2. For HSV-1, infection was assessed using ICP4, VP16, and gD as markers. For PRV, infection levels were quantified by immunoblotting for UL54. For HSV-2, infection was quantified by immunoblotting for VP16. b-actin was used as the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in this study included goat BHV-1 antisera, mouse anti-PRV UL47 monoclonal antibody (MAb), mouse anti-HSV-1 ICP4 monoclonal antibody (Santa Cruz, sc69809), mouse anti-HSV-1 VP16 monoclonal antibody (Santa Cruz, sc7545), mouse anti-HSV-1 gD monoclonal antibody (Santa Cruz, sc21719), and mouse anti-b-actin MAb (Proteintech, 66009–1-lg).

Techniques: Activity Assay, Infection, Imaging, CCK-8 Assay, Inhibition, Expressing, Microscopy, Western Blot, Control

Journal: Journal of advanced research

Article Title: Antimycin A inhibits alpha-herpesvirus replication by disrupting the formation of pyrimidinosomes.

doi: 10.1016/j.jare.2025.05.016

Figure Lengend Snippet: Fig. 4. Antimycin A exhibits extensive antiviral activity against alpha-herpesvirus. Antimycin A effectively inhibited HSV-1 (A-C) and HSV-2 (G-H) infections in Vero E6 cells, PRV (D-E) infection in PK-15 cells, and EHV-1 (J-K) infection in RK13 cells. Vero-E6 cells, PK-15 cells, and RK13 cells were pretreated for 12 h with increasing concentrations of Antimycin A and then infected with HSV-1 (A-C), PRV (D-E), HSV-2 (G-I), and EHV-1 (J-K) at MOIs of 0.5, 0.1, 0.5, and 0.5, respectively. At 24 hpi, cells were fixed and analyzed by fluorescence imaging. (A, D, G and J) Infection levels were quantified using a fluorescent microplate reader (black curve), while cell viability was measured using the CCK-8 Assay (orange curve). The CC50 for each compound was calculated via a four-parameter logistic nonlinear regression model in GraphPad Prism. Dotted lines indicate 50 % inhibition. Data represent the means ± SEM from n = 3 independent experiments of infectious virions, normalized to DMSO-treated wells. The IC50 values for HSV-1, PRV, HSV-2, and EHV-1 were determined by nonlinear regression analysis. (B, E, H and K) eGFP expression in infected cells, either untreated (0 lM) or treated with various concentrations (0.0015–5 lM) of Antimycin A, was visualized by fluorescence microscopy at the same time point. Representative images are shown. Bars, 300 lm. Magnification, ×10. (C, F and I) Western blot analysis was performed to quantify infection in cells infected with HSV-1, PRV, or HSV-2. For HSV-1, infection was assessed using ICP4, VP16, and gD as markers. For PRV, infection levels were quantified by immunoblotting for UL54. For HSV-2, infection was quantified by immunoblotting for VP16. b-actin was used as the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in this study included goat BHV-1 antisera, mouse anti-PRV UL47 monoclonal antibody (MAb), mouse anti-HSV-1 ICP4 monoclonal antibody (Santa Cruz, sc69809), mouse anti-HSV-1 VP16 monoclonal antibody (Santa Cruz, sc7545), mouse anti-HSV-1 gD monoclonal antibody (Santa Cruz, sc21719), and mouse anti-b-actin MAb (Proteintech, 66009–1-lg).

Techniques: Activity Assay, Infection, Imaging, CCK-8 Assay, Inhibition, Expressing, Microscopy, Western Blot, Control

Journal: Frontiers in Microbiology

Article Title: Herpes simplex virus 1 glycoprotein C promotes virus penetration from endosomes during entry, independent of interaction with heparan sulfate

doi: 10.3389/fmicb.2025.1549349

Figure Lengend Snippet: Impact of gC on the basolateral route of HSV-1 infection of polarized cells and on accumulation of entering HSV-1 particles in endosomes. (A) HaCaT cells were seeded at low density on glass coverslips. Islets formed by 4 days of culture. Cultures were left untreated or treated with 50 mM ammonium chloride for 20 min at 37°C, and were infected with HSV-1 ΔgC or gCR (MOI ~ 0.5) for 4 h. Cultures were fixed and stained with MAb P1101 to ICP4 (red), and counterstained with DAPI. Images are representative of at least three independent experiments. (B) HSV-1 ∆gC or gCR was added to CHO-HVEM cells (MOI 150–500). At 30 min. p.i., cells were fixed and processed for TEM analysis. Representative images are shown. Black arrowheads indicate intracellular, enveloped HSV particles. (C) Endosomes containing at least two virions were enumerated. * p -value < 0.05, Student’s t test.

Article Snippet: Infected cells were detected by immunofluorescence with anti-HSV-1 ICP4 mouse monoclonal antibody P1101 (Virusys) followed by goat anti-mouse IgG labeled with Alexa Fluor 488 (Invitrogen).

Techniques: Infection, Staining

Journal: Frontiers in Microbiology

Article Title: Herpes simplex virus 1 glycoprotein C promotes virus penetration from endosomes during entry, independent of interaction with heparan sulfate

doi: 10.3389/fmicb.2025.1549349

Figure Lengend Snippet: Role of gC in endocytic internalization of HSV-1 from the plasma membrane. HSV-1 ΔgC or gCR was added to CHO-HVEM cells on ice at 4°C for 1 h. After shift to 37°C, extracellular virus was inactivated with sodium citrate at the indicated times p.i. At 6 h p.i., entry was detected by quantitating HSV-1 ICP4-positive cells by immunofluorescence microscopy. Mean and standard deviation of three independent experiments are shown.

Article Snippet: Infected cells were detected by immunofluorescence with anti-HSV-1 ICP4 mouse monoclonal antibody P1101 (Virusys) followed by goat anti-mouse IgG labeled with Alexa Fluor 488 (Invitrogen).

Techniques: Clinical Proteomics, Membrane, Virus, Immunofluorescence, Microscopy, Standard Deviation