Specific initiation of replication at the right-end telomere of the closed species of minute virus of mice replicative-form DNA

Abstract

We have developed an in vitro system that supports the replication of natural DNA templates of the autonomous parvovirus minute virus of mice (MVM). MVM virion DNA, a single-stranded molecule bracketed by short, terminal, self-complementary sequences, is converted into double-stranded replicative-form (RF) DNA when incubated in mouse A9 fibroblast extract. The 3 end of the newly synthesized complementary strand is ligated to the right-end hairpin of the virion strand, resulting in the formation of a covalently closed RF (cRF) molecule as the major conversion product. cRF DNA is not further replicated in A9 cell extract alone. On addition of purified MVM nonstructural protein NS1 expressed from recombinant baculoviruses or vaccinia viruses, cRF DNA is processed into a right-end (5 end of the virion strand) extended form (5eRF). This is indicative of NS1-dependent nicking of the right-end hairpin at a distinct position, followed by unfolding of the hairpin and copying of the terminal sequence. In contrast, no resolution of the left-end hairpin can be detected in the presence of NS1. In the course of the right-end nicking reaction, NS1 gets covalently attached to the right-end telomere of the DNA product, as shown by immunoprecipitation with NS1-specific antibodies. The 5eRF product is the target for additional rounds of NS1-induced nicking and displacement synthesis at the right end, arguing against the requirement of the hairpin structure for recognition of the DNA substrate by NS1. Further processing of the 5eRF 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 of the whole molecule by displacement synthesis initiated at the right-end telomere. Formation of dRF DNA is highly stimulated by NS1. The experimental results presented in this report support various assumptions of current models of parvovirus DNA replication and provide new insights into the replication functions of the NS1 protein.

Neoplastic Transformation-Associated Stimulation of the In Vitro Resolution of Concatemer Junction Fragments from Minute Virus of Mice DNA

“…1. The single-stranded virion DNA gets converted into a closed, double-stranded, monomeric replicative form (cRF) by extension of the 3Јterminal hairpin (left-hand terminus) and ligation of the growing strand to the folded-back 5Ј terminus (right-hand terminus) (step 1) (3,16). Further processing of cRF DNA requires the activity of the major viral nonstructural protein NS1.…”

“…NS1 is involved in the formation of a strand-and sequence-specific nick at the cRF 5Ј telomere, followed by initiation of displacement synthesis and terminal extension, giving rise to an extended molecule (5Ј-terminally extended monomeric replicative form [5ЈeRF]) (step 2) (3,15,46). Hairpin refolding at the extended terminus, supported by host cell nuclear factors (3,13,14,47) and efficiently stimulated by NS1 (47), creates a so-called rabbit-ear structure (5Ј-rabbit-eared monomeric replicative form [5ЈreRF]) (step 3). This structure provides a primer for strand displacement synthesis and dimeric replicative-form (dRF) formation (step 4) (3,5,19,46,47).…”

“…Hairpin refolding at the extended terminus, supported by host cell nuclear factors (3,13,14,47) and efficiently stimulated by NS1 (47), creates a so-called rabbit-ear structure (5Ј-rabbit-eared monomeric replicative form [5ЈreRF]) (step 3). This structure provides a primer for strand displacement synthesis and dimeric replicative-form (dRF) formation (step 4) (3,5,19,46,47). Subsequent NS1-dependent resolution of concatemer junctions results in the formation of two types of monomeric replicativeform (RF) DNA, with covalently closed (5ЈeRF) or extended (3Ј-5ЈeRF) left-hand termini, respectively (steps 5a and 5b) (17,18,20,21).…”

“…Natural MVM mRF DNA templates, i.e., cRF and 5ЈeRF ( Fig. 1), were obtained from MVM-infected A9 cells by Hirt extraction, centrifugation through 5 to 30% neutral sucrose gradients, and further purification on 10 to 30% alkaline sucrose gradients (3,39).…”

“…For the analysis of short terminal fragments, replication products were digested with PshAI and fractionated by native 5% polyacrylamide gel electrophoresis. PshAI cleaves MVM DNA at nucleotide 4916, giving rise to right-end fragments of 130 or 254 bp, respectively, depending on whether the telomere is in the hairpin or extended configuration (3,46). While the hairpin fragment is only weakly labeled due to nonspecific repair synthesis, the extended species becomes heavily labeled as the result of the nicking and extension reactions leading to the copying of the terminal hairpin sequence.…”