A murine host cell factor required for nicking of the dimer bridge of MVM recognizes two CG nucleotides displaced by 10 basepairs.
During replication of the minute virus of mice (MVM) genome, a dimer replicative form (RF) intermediate is resolved into two monomer RF molecules in such a way as to retain a unique sequence within the left hand hairpin terminus of the viral genome. Although the proposed mechanism for resolution of the dimer RF remains uncertain, it likely involves site-specific nicking of the dimer bridge. The RF contains two double-stranded copies of the viral genome joined by the extended 3' hairpin. Minor sequence asymmetries within the 3' hairpin allow the two halves of the dimer bridge to be distinguished. The A half contains the sequence [sequence: see text], whereas the B half contains the sequence [sequence: see text]. Using an in vitro assay, we show that only the B half of the MVM dimer bridge is nicked site-specifically when incubated with crude NS-1 protein (expressed in insect cells) and mouse LA9 cellular extract. When highly purified NS-1, the major nonstructural protein of MVM, is used in this nicking reaction, there is an absolute requirement for the LA9 cellular extract, suggesting a cellular factor (or factors) is (are) required. A series of mutations were created in the putative host factor binding region (HFBR) on the B half of the MVM dimer bridge adjacent to the NS-1 binding site. Nicking assays of these B half mutants showed that two CG motifs displaced by 10 nucleotides are important for nicking. Gel mobility shift assays demonstrated that a host factor(s) can bind to the HFBR of the B half of the dimer bridge and efficient binding depends on the presence of both CG motifs. Competitor DNA containing the wild-type HFBR sequence is able to specifically inhibit nicking of the B half, indicating that the host factor(s) bound to the HFBR is(are) essential for site-specific nicking to occur.
Liu
Q
Q
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, Canada.
Astell
C R
CR
D000818:Animals; D001482:Base Composition; D001685:Biological Factors / Q000378:metabolism*; D002460:Cell Line; D019281:Dimerization; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D051379:Mice; D008915:Minute Virus of Mice / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D018411:Spodoptera / Q000166:cytology; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D001685:Biological Factors; D015226:Dinucleoside Phosphates; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C081367:NS1 protein, minute virus of mice; C015772:CpG dinucleotide