ANALYSIS OF THE INTERNAL REPLICATION SEQUENCE INDICATES THAT THERE ARE 3 ELEMENTS REQUIRED FOR EFFICIENT REPLICATION OF MINUTE VIRUS OF MICE MINIGENOMES

Prior analysis of minigenomes of minute virus of mice carried out by o ur laboratory indicated that sequences within the region of nucleotide s 4489 to 4695, inboard of the 5' palindrome, are required for efficie nt DNA replication of the virus and are the site of specific interacti ons with unidentified factors present in a host cell nuclear extract ( P. Tam and C. R. Astell, Virology 193:812-824, 1993; P. Tam and C. R. Astell, J. Virology 68:2840-2848, 1994), In order to examine this regi on in finer detail, a comprehensive library of linker-scanning mutants spanning the region was tested for the ability to support replication of minigenome constructs and for the ability to interact with host ce ll factors, Three short discrete sequence elements critical for replic ation competence were observed. Binding of host cell nuclear factors w as localized to four sites, with two major complexes each appearing to have two binding sites within the region, All factor binding sites we re found to be directly adjacent to or overlapping with sequence eleme nts contributing to replication competence, and evidence suggesting a correlation between factor binding and minigenome replication is prese nted, A possible model is proposed for function of a viral origin with in the region of the internal replication sequence which addresses the still-unresolved problem of how parvoviruses overcome the thermodynam ic energy barrier involved in the rearrangement of the 5'-terminal pal indrome from an extended farm to a hairpin conformation.