Figure 4. Generation of MV-eGFP-Pwt and its IFN-inducing properties in tumor and normal cells.

(a) Schematic representation of the full-length infectious molecular clone of attenuated MV-eGFP and the cloning strategy for generating MV-eGFP-Pwt, which expresses the wild-type P gene. The XbaI/SalI fragment, containing the Pwt gene, was digested from a plasmid encoding the full-length infectious clone of wild-type MV IC-B strain, p(+)MV323. The fragment was subcloned into a shuttle vector pSS8 using the corresponding restriction sites and inserted as a BsiWI fragment into the full-length p(+)MV-eGFP plasmid to generate p(+)MV-eGFP-Pwt. (b) Photographs of BJAB and ARH-77 cells infected with MV-eGFP, MV-eGFP-Pwt or MVwt323 (MOI 1.0) taken at 48 h after infection. (c–e) MV-eGFP-Pwt induced less IFN in comparison with MV-eGFP, but did not completely silence IFN biosynthesis compared to MVwt323. IFN-α or IFN-β levels in (c) BJAB lymphoma cells, (d) ARH-77 cells, or (e) peripheral blood mononuclear cells from three different healthy donors’ infected with MV viruses at an MOI of 1.0. Conditioned media were collected on ice 48 h later and IFN levels were measured by specific ELISA. IFN values were normalized for percentage of infected cells quantified by FACS analysis. Data are representative of three separate experiments with similar results.