A novel replicative form DNA of Aleutian disease virus: the covalently closed linear DNA of the parvoviruses.

The analysis of replicative form (RF) DNA of Aleutian disease virus (ADV) by alkaline gel electrophoresis revealed that all RF DNA species segregate into DNA single strands which represent integral multiples of a genome equivalent. This demonstrates that as with other autonomous parvoviruses, the virion and complementary DNA strands are frequently linked by hairpin structures and that also, nicks are present at subterminal sites. Approximately 50% of the 5'-terminal hairpins contain a subterminal nick whereas no nick is detectable in the 3'-terminal hairpin. This finding together with the presence of nicks in the 3' palindrome sequence of the dimer RF DNA (D RF DNA) bridge fragment is the first experimental proof for the so far hypothetical substrate specificity of a nickase. A novel DNA structure was identified in the monomer (M) RF DNA population. This molecule, designated 'monomer covalently closed linear RF DNA' (Mccl RF DNA), consists of a continuous, self-complementary, circular polynucleotide chain of twice the genome length. It was directly visualized by electron microscopy that denatured ADV M RF DNA is a single-stranded circular molecule of twice the genome length with covalently closed terminal hairpins on either end. Alkaline gradient centrifugations, enzymic assays and electrophoretic techniques confirmed the proposed structure. Moreover, evidence was obtained that the D RF DNA species contains an analogous Dccl RF DNA. It is suggested that the newly described Mccl RF DNA form is an important intermediate common to the DNA replication of all autonomously replicating parvoviruses.