Figure 2.

Vaccinia virus B18R gene product enhances vesicular stomatitis virus expression in HT29 cancer cell lines. (a) HT29 were treated or not with VVDD-eGFP or VVΔB18R-eGFP [0.1 multiplicity of infection (MOI)] for 2 hours. Supernatant of each condition was filtered at 0.22 µm. Filtrated media was added to naive HT29. A VSVΔ51-DsRed (0.0001 MOI) infection was performed or not for 45 minutes. Enhanced green fluorescent protein (eGFP) and DsRed were detected 48 hours after infection. One of three independent experiments. (b) VSV titers were determined. Mean ± SEM from three independent experiments. (c) HT29 were treated or not with VVDD-eGFP or VVΔB18R-eGFP (0.1 MOI) for 2 hours alone, or in combination with VSVΔ51-DsRed (0.0001 MOI) for 45 minutes. eGFP and DsRed were detected 48 hours after. One of three independent experiments. (d) Viral titers were determined after infection as in c. Black and white bars: VV and VSV. Mean ± SEM from three independent experiments. (e) Isobologram: 786-O cells were treated with serial dilutions of B18R gene product followed by VSV and processed as Figure 1d. CI: Combination index. F_a: fraction of cells affected. Error bars: mean ± SEM. One of two independent experiments in quadruplicate. (f) HT29 were pretreated or not with IFN-α (30 IU) and treated or not with VVDD or VVΔB18R-eGFP (0.1 MOI) for 2 hours. In the last 45 minutes, VSVΔ51-DsRed (0.0001 MOI) was added in combination or not with recombinant B18R (0.01 µg). eGFP and red fluorescent protein (RFP) were detected 48 hours after. One of two independent experiments. (g) HT29 were pretreated or not with various concentration of recombinant B18R (0.1–10⁻⁶ µg) protein during 2 hours and treated with VVDD-eGFP or VVΔB18R-eGFP (0.1 MOI) for 2 hours. VSVΔ51-DsRed (0.0001 MOI) was added with or not various concentration of IFN-α (5–30 IU). Supernatants were collected for VSVΔ51 titration 48 hours later. 1: VVDB18R + VSV, 2: VVDB18r + B18R + VSV. One experiment is presented.