A novel technique for in-vivo assay of viral regulatory regions in genomes of animal RNA viruses.
A novel in-vivo assay system for mapping and analyzing regulatory signals which promote transcription and expression of viral RNA genomes is described. The system was developed using infectious bursal disease virus (IBDV), a double-stranded RNA virus and Vero cells which are permissive for IBDV, as a model. The model system consisted of engineered modifications of an enhancer-less pGL3-Promoter vector such that deleted lengths of the 5' noncoding region of genome segment A of IBDV were positioned in either the plus-sense or the minus-sense orientation immediately downstream of the SV40 promoter and upstream of the luciferase (LUC) reporter gene. Transient transfections of these constructs in IBDV-infected and uninfected Vero cells resulted in endogenous generation of recombinant viral RNA-LUC containing the 5' terminal viral RNA sequences in either the plus-sense or the minus-sense orientation. LUC assays of the Vero cell lysates allowed the localization of promoter activity in the 5'-terminal 32 base pairs of genomic segment A of IBDV. Because the viral RNA transcripts produced in-vivo can be either plus-sense or minus-sense, the system can be used to assay for regulatory regions for transcription or replication for any animal RNA virus.
Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Canada.
M M
MM
Nagarajan
F S
FS
Kibenge
D000818:Animals; D002522:Chlorocebus aethiops; D002874:Chromosome Mapping / Q000379:methods*; D007243:Infectious bursal disease virus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012367:RNA, Viral; D014709:Vero Cells
D012367:RNA, Viral