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UBC Theses and Dissertations

Resolution of the dimer bridge of Minute Virus of Mice : roles of the viral NS-1 protein and a host cell factor Liu, Qingquan


Previous characterization of the terminal sequences of the Minute Virus of Mice (MVM) genome demonstrated that the right hand palindrome contains two sequences, each the inverted complement of the other. However, the left hand palindrome was shown to exist as a unique sequence (Astell et al, Virology 54:171-177, 1985). The Modified Rolling Hairpin Model (MRHM) for MVM replication provided an explanation for how the right hand palindrome could undergo hairpin transfer to generate two sequences while the left end palindrome within the dimer bridge could undergo asymmetric resolution and retain the unique left end sequence (Astell et al, ibid). The MRHM proposed that an initiating nick occurs on the parental viral strand of the A-half of the dimer bridge while a subsequent nick cuts the B-half of the dimer bridge (on the progeny viral strand). This thesis describes studies on the in vitro resolution of the wildtype dimer bridge sequence of MVM using recombinant (baculovirus) expressed NS-1, the major nonstructural protein of MVM, and a replication extract from mouse LA9 cells. The resolution products are consistent with those predicted by the MRHM, thus providing support for this replication mechanism. In addition, mutant dimer bridge clones were constructed and used in the resolution assay. The mutant structures included removal of the "bubble" asymmetry of the dimer bridge, inversion of the sequence which was proposed to include the initiating nick site, and a two bp deletion within the A-half between the "bubble" sequence and putative initiating nick site of the dimer bridge. The resolution patterns of these mutant dimer bridge structures suggest that the resolution procedure of the dimer bridge proposed in the MRHM is likely incorrect, although some sequences within the dimer bridge are likely responsible for asymmetric resolution and thus conservation of the unique sequence of the left hand hairpin of the MVM genome (Liu et a/., 1994). In parallel studies described by others, the B-half (but not the A-half) of the dimer bridge was shown to be nicked site-specifically by a HeLa cellular replication extract supplemented with a HeLa cell nuclear extract containing expressed NS-1 (Cotmore and Tattersall, EMBO J. 13:4145-4152, 1994). Furthermore, it was predicted that host factor [possibly the activating transcription factor (ATF)] is required for the NS-1 nicking. One reason for this prediction is that mutation of an important nucleotide (the first C) in the putative ATF binding site (CGTCA) greatly decreased the nicking efficiency of the B-half dimer bridge (Cotmore and Tattersall, ibid). Data presented in this thesis confirm that the B-half (but not the Ahalf) of the MVM dimer bridge is nicked site-specifically when incubated with crude NS-1 protein (expressed in insect cells) and mouse LA9 cellular replication extract. When highly purified NS-1 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 or HFBR of the B-half dimer bridge. Nicking assays of these B-half mutants showed that two CG motifs displaced by 10 nucleotides are important for nicking. Gel mobility shift assays demonstrate that a host factor(s) can bind to the HFBR and efficient binding depends on the presence of both CG motifs. Competitor DNA containing the wildtype 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. These data also suggest that it is highly unlikely that ATF is the host factor required for NS-1 nicking (Liu and Astell, Virology, 1996, in press.)

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