Biochemical and molecular characterizations of a novel DNA repair enzyme, CPD-photolyase, and late promoters of fowlpox virus.

摘要

To understand stability and persistence of fowlpox virus (FPV), we recently characterized an FPV-encoded DNA repair enzyme, CPD-photolyase. The DNA repair activity of the FPV-encoded protein was demonstrated by its ability to functionally complement and rescue photolyase-deficient bacteria. When the photolyase deficient FPV (Phr− FPV) was exposed to UV light, the mutant was at least 100 times more sensitive than the unaltered parental virus. A subsequent pathogenesis study demonstrated that the parental FPV replicated more efficiently than the Phr− FPV. When virions isolated from the infected scabs were exposed to UV irradiation, the infectivity of UV damaged parental FPV was rescued by CPD-photolyase-mediated-photoreactivation pathway by at least 50%. In contrast, the viability of Phr− FPV was unaltered by exposure to white light. Even though the Phr− FPV replicated less efficiently in chickens, it gave a complete protection to a subsequent challenge infection. Thus the use of light-sensitive mutant virus as an alternate vaccine is suggested.; To further exploit FPV as a vector, characterization of strong promoters were attempted using a lacZ gene based reporter assay. The transcriptional element of ORF P190 appeared to be the strongest. In contrast, the promoter associated with the ORF, P180, was the weakest. Interestingly, a novel bi-directional promoter controlling the expression of ORFs 130 and 131 appeared to be active whose expression was one log fold less than that of the P190 promoter. Subsequently when the critical positions in the spacer and the thymidine after the start codon were substituted with those present in P190, the activity of the weakest promoter was increased to as high as thirty times more than the unaltered promoter, P180. When similar substitutions were made in the strongest promoter, P190, its activity was reduced to 50%. Thus the present study underscores the significance of nucleotides flanking the transcriptional and translational start sites. The promoters identified from this study, P190, P191, P174, as well as the genetically manipulated promoter, P180SG and the bi-directional promoter, P130 and P131 can be used for the expression of foreign genes by FPV vectors.