Construction and Characterization of an Infectious Murine Gammaherpesivrus-68 Bacterial Artificial Chromosome
Figure 5
ORF11 is not required for MHV-68 to resist IFN-α. (a) ORF11 inhibits the IFN-mediated induction of the ISRE reporter activity. 293T cells were transfected with the ISRE-driven firefly luciferase reporter and the protein expression plasmid of a viral ORF indicated at the bottom of the graph. For normalization, a Renilla luciferase construct driven by the housekeeping PGK promoter was included in each transfection. One day after transfection, the cells were either mock treated or treated with units/ml of IFN-α and 24 hrs later, the total lysates were harvested for Dual Luciferase assays. For another experiment, the cells were treated with 105 units/ml of IFN-α. The fold of induction was derived by dividing the normalized valuses of treated samples with those of mock treated samples. (b) Restriction enzyme analysis of the genomes of pORF11S(BAC)v and ORF11S(loxP). Each virion DNA sample was digested with either combination of SacI and PacI, EcoRI or HindIII. Lane 1 is virion DNA of wild-type MHV-68; lane 2 is virion DNA of pMHV68(BAC)v; lane 3 is virion DNA of MHV68(loxP); lane 4 is virion DNA of pORF11S(BAC) and lane 5 is virion DNA of ORF11S(loxP). The wild-type fragments marked with solid squares were eliminated in lanes 4 and 5 due to the insertion of stop codons. The a and b fragments were generated as a result of the insertion of the stop codons. The c fragments were resulted from the removal of the BAC sequence. (c) Lack of ORF11 does not increase the sensitivity of MHV-68 to IFN-α. NIH3T3 cells were pretreated with 100 units/ml of IFN-α for 16 hrs and then infected with MHV-68(LoxP) or ORF11S(LoxP) at an MOI of 0.01. The whole cultures were harvested at various times post-infections for plaque assays to determine the viral titers.
