Molecular cloning and analysis of the genome of bovine parvovirus

The genome of bovine parvovirus (BPV) has been cloned by blunt end ligation of double-stranded virion DNA into the plasmid pUC8. The resulting genomic clones were infectious after transfection into bovine fetal lung (BFL) cells. Sequencing of the plasmids demonstrated that deletions were common at both ends of the cloned BPV genome. Deletions of up to 34 bases at the 3’ end lowered but did not abolish infectivity, while a deletion of 52 bases eliminated infectivity, End label analysis demonstrated the repair of deletions of up to 34 bases at the 3’ end or 35 bases at the 5’ end to the wild type length. Mutually inverted sequence orientations of the palindromic termini, known as the flip and flop forms, can occur during replication of parvovirus DNA. Cloning of BPV terminal sequences permitted the identification of the 3’ flop sequence inversion as a natural component of BPV DNA. This is the first report of sequence inversions within the 3’ end of an autonomous parvovirus. Clones with the 3’ flop or flip conformations were equally infectious. Wild type virion DNA was shown to have predominantly the 3’ flip conformation but a significant amount of 3’ flop was also detected. At the 5’ end, both the flip and flop sequence conformations were identified in nearly equal amounts. The progeny virion DNA from transfection of genomic clones had the same ratio of flip to flop as did wild type at both the 3’ and 5’ ends, regardless of the starting terminal conformations of the genomic clone. These data suggest that, while sequence inversion occurs at both termini during BPV DNA replication, some mechanism exists for the preferential replication of the 3’ flip conformation. Replicative form DNA from BPV infected cells had the same ratio of flip and flop at each end and the same termini as virion DNA. A set of deletion and frameshift mutants affecting each of the coding regions of BPV was constructed using one of the genomic clones. None of these mutants was infectious when transfected into BFL cells, which demonstrates that all three of the major open reading frames are essential for the production of infectious virus.