SPECIFIC INITIATION OF REPLICATION AT THE RIGHT-END TELOMERE OF THE CLOSED SPECIES OF MINUTE VIRUS OF MICE REPLICATIVE-FORM DNA

We have developed an in vitro system that supports the replication of natural DNA templates of the autonomous parvovirus minute virus of mic e (MVM). MVM virion DNA, a single-stranded molecule bracketed by short , terminal, self-complementary sequences, is converted into double-str anded replicative-form (RF) DNA when incubated in mouse A9 fibroblast extract. The 3' end of the newly synthesized complementary strand is l igated to the right-end hairpin of the virion strand, resulting in the formation of a covalently closed RF (cRF) molecule as the major conve rsion product, cRF DNA is not further replicated in A9 cell extract al one. On addition of purified MVM nonstructural protein NS1 expressed f rom recombinant baculoviruses or vaccinia viruses, cRF DNA is processe d into a right-end (5' end of the virion strand) extended form (5'eRF) . This is indicative of NS1-dependent nicking of the right-end hairpin at a distinct position, followed by unfolding of the hairpin and copy ing of the terminal sequence. In contrast, no resolution of the left-e nd hairpin can he detected in the presence of NS1. In the course of th e right-end nicking reaction, NS1 gets covalently attached to the righ t-end telomere of the DNA product, as shown by immunoprecipitation wit h NS1-specific antibodies. The 5'eRF product is the target for additio nal rounds of NS1-induced nicking and displacement synthesis at the ri ght end, arguing against the requirement of the hairpin structure for recognition of the DNA substrate by NS1. Further processing of the 5'e RF template in vitro leads to the formation of dimeric RF (dRF) DNA in a left-to-left-end configuration, presumably as a result of copying o f the whole molecule by displacement synthesis initiated at the right- end telomere. Formation of dRF DNA is highly stimulated by NS1. The ex perimental results presented in this report support various assumption s of current models of parvovirus DNA replication and provide new insi ghts into the replication functions of the NS1 protein.