IN-VITRO RESOLUTION OF THE DIMER BRIDGE OF THE MINUTE VIRUS OF MICE (MVM) GENOME SUPPORTS THE MODIFIED ROLLING HAIRPIN MODEL FOR MVM REPLICATION

Previous characterization of the terminal sequences of the minute viru s of mice (MVM) genome demonstrated that the right hand palindrome con tains two sequences, each the inverted complement of the other. Howeve r, the left hand palindrome was shown to exist as a unique sequence [A stell at al., J. Virol. 54: 179-185 (1985)]. The modified rolling hair pin (MPH) model for MVM replication provided an explanation of 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 . This report describes in vitro resolution of the wild-type dimer bri dge sequence of MVM using recombinant (baculovirus) expressed NS-1 and a replication extract from LA9 cells. The resolution products are con sistent with those predicted by the MRH model, providing support for t his replication mechanism. In addition, mutant dimer bridge clones wer e constructed and used in the resolution assay. The mutant structures included removal of the asymmetry in the hairpin stem, inversion of th e sequence at the initiating nick site, and a 2-bp deletion within one stem of the dimer bridge. In all cases, the mutant dimer bridge struc tures are resolved; however, the resolution pattern observed with the mutant dimer bridge compared with the wild-type dimer bridge is shifte d toward symmetrical resolution. These results suggest that sequences within the left hand hairpin (and hence dimer bridge sequence) are res ponsible for asymmetric resolution and conservation of the unique sequ ence within the left hand palindrome of the MVM genome. (C) 1994 Acade mic Press, Inc.