MINUTE VIRUS OF MICE TRANSCRIPTIONAL ACTIVATOR PROTEIN NS1 BINDS DIRECTLY TO THE TRANSACTIVATION REGION OF THE VIRAL P38 PROMOTER IN A STRICTLY ATP-DEPENDENT MANNER

The NS1 polypeptide of minute virus of mice (MVM) is a potent transcri ptional activator of the MVM P38 promoter, The minimum region of this promoter required for transactivation has been identified and termed t he transactivation region (tar), However, the function of tar and the biochemical steps involved in NS1-mediated transactivation are not wel l understood, Here we provide evidence that NS1 binds directly and spe cifically to tar in a strictly ATP-dependent manner. A DNA fragment co ntaining tar was specifically coimmunoprecipitated with purified bacul ovirus-expressed MVM NS1, using antibodies directed against NS1. amino - or carboxy-terminal peptides, Using this immunoprecipitation assay, we found that the NS1-tar interaction was enhanced approximately 10-fo ld by ATP, but subsequent incubation at elevated temperatures in the p resence, but not the absence, of MgCl2 caused rapid loss of tar bindin g. This finding suggests that the tar-NS1 complex has a short half-lif e under assay conditions which favor ATP hydrolysis, Specific binding was efficiently inhibited by self-ligated oligonucleotides containing the core DNA sequence (ACCA)(3), but the same nonligated 20- and 21-me r oligonucleotides were'unable to compete effectively, indicating that NS1 only binds to its cognate site when this site is presented on DNA fragments of sufficient size, DNase I footprinting experiments perfor med in the presence of gamma S-ATP revealed that NS1 protects a 43-bp sequence extending asymmetrically from the (ACCA)(2) sequence toward t he TATA box of the promoter. NS1 footprints obtained at other sites in the MVM genome were similarly large and asymmetric, all extending app roximately 31 bp 5' from the core (ACCA)(2-3) sequence. Surprisingly, no footprints were obtained in the absence of gamma S-ATP even under l ow-stringency binding conditions, However, ATP could be omitted from t he reactions if NS1 was first incubated with antibodies directed again st its 16-amino-acid carboxy-terminal peptide. Since these antibodies probably create intermolecular cross-links, this finding suggests that NS1 may only bind its cognate site efficiently, or perhaps at all, if the transactivator is first induced to form oligomers. From these dat a, we hypothesize that ATP binding may also induce NS1 to oligomerize and that such assembly is required before the protein can bind effecti vely to the far sequence, The functional implications of the NSl-tar i nteraction will be discussed.