A physical map of the viral genome for infectious pancreatic necrosis virus Sp: analysis of cell-free translation products derived from viral cDNA clones.

Abstract

The two segments of double-stranded RNA from infectious pancreatic necrosis virus Sp were cloned into the plasmid vector pUC8. Two sets of overlapping clones were identified by restriction enzyme and Southern blot analyses. Each of these sets was shown by Northern blot analysis to be exclusively related to either segment A or B of the genomic RNA. The entire lengths of the cloned segments were estimated to be 2.9 and 2.6 kilobases, respectively. Sequences from the two segments of viral cDNA were subcloned into the bacteriophage T7 RNA polymerase vectors pT71 and pT72. The activity of the single-stranded RNAs transcribed from these subclones in a rabbit reticulocyte lysate translation system provided information on the polarity of and the protein products coded for by each subclone. The four proteins encoded by the genome of infectious pancreatic necrosis virus were identified among the translation products of the individual cloned segments by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By constructing plasmids containing deletions in the sequences from either the 5' or 3' end of segment A, we were able to construct a physical map for the larger segment of double-stranded RNA. The proteins derived from these plasmids indicated that the linear gene order for viral proteins encoded in segment A is beta, gamma 2, and gamma 1.