Journal: Antimicrobial Agents and Chemotherapy
Article Title: Griffithsin and Carrageenan Combination To Target Herpes Simplex Virus 2 and Human Papillomavirus
Figure Lengend Snippet: GRFT prevents HSV-2 and HPV postadsorption events. (A) HSV-2 G or HPV16 PsV and different concentrations of GRFT or medium (virus control) were preincubated for 0 h, 0.5 h, and 2 h at 37°C before being added to prechilled Vero cells (for HSV-2) or HeLa cells (for HPV16 PsV) and kept at 4°C for 2 h. The cells were washed 3 times before addition of the overlay and incubation at 48 h and 37°C in a 5% CO 2 atmosphere with 98% humidity. Finally, the cells were fixed and stained prior to counting the numbers of PFU. (B) The same as in panel A for HSV-2, but after 2 h of incubation at 4°C, the cells were switched to 37°C for an additional 2 h, followed by a 2-min treatment with citric acid buffer (pH 3.0). (C) Different concentrations of CG or GRFT were added at the indicated time points, with time zero representing the time of initial HPV16 PsV inoculation. The graph shows the percent reporter gene expression (mean ± SD) relative to that for the virus control (triplicate assays were performed per condition). (D) Western blot of purified HSV-2 lysate. Membranes were incubated overnight at 4°C with mouse anti-gD MAb DL11 (lane 1) or 1 μg/ml GRFT (lane 2). The membranes were washed and incubated for 1 h at room temperature with HRP-conjugated anti-mouse Ig (lane 1) or were incubated overnight at 4°C with rabbit anti-GRFT antibody (lane 2), before being washed and incubated with HRP-conjugated anti-rabbit immunoglobulin antibody (1 h at room temperature). (E) HSV-2-infected Vero cells were lysed, preincubated with GRFT, and immunoprecipitated with anti-GRFT antibody. Lanes 1, cell lysate without immunoprecipitation; lanes 2, HeLa cell lysate 24 h after HSV-2 infection with immunoprecipitation in the presence of GRFT and anti-GRFT antibody; lanes 3, same as lanes 2 but 48 h after HSV-2 infection; lanes 4 and 5, immunoprecipitation controls without and with GRFT, respectively. In the experiments described in the legends to panels D and E, the membranes were processed using an ECL Western blotting detection system to capture the bands on X-ray films. A representative blot of three repeats is shown. (F) Soluble GRFT, N-glycosylated (N-glyco), nonglycosylated gD, or a mixture of N-glycosylated and nonglycosylated gD-GRFT was preincubated before addition to Vero cells. HSV-2 G was then added, and infection was detected by an immunohistochemical assay after overnight incubation. Statistical analysis was performed using the Mann-Whitney U test ( P
Article Snippet: The membranes were washed and incubated with rabbit polyclonal anti-GRFT (Pacific Immunology, Ramona, CA) or anti-mouse immunoglobulin conjugated with horseradish peroxidase (HRP) (Promega, Madison, WI).
Techniques: Incubation, Staining, Expressing, Western Blot, Purification, Infection, Immunoprecipitation, Immunohistochemistry, MANN-WHITNEY