MUTATIONAL ANALYSIS OF ADENOASSOCIATED VIRUS REP PROTEIN-MEDIATED INHIBITION OF HETEROLOGOUS AND HOMOLOGOUS PROMOTERS

The four Rep proteins encoded by adeno-associated virus type 2 (AAV-2) inhibit transcription of their own promoters and of several heterolog ous promoters. To gain insight into the molecular mechanism of Rep-med iated transcription repression, we studied the effects of the four Rep proteins on the accumulation of mRNA transcribed from the human papil lomavirus type 18 upstream regulatory region HPV18 URR, the human immu nodeficiency virus long terminal repeat, and the AAV-2 p5 and plB prom oters by transient transfection experiments in HeLa cells. We observed a distinct contribution of the C- and N-terminal sequences in which t he four Rep proteins (Rep78, Rep68, Rep52, and Rep40) differ from each other. While Rep78 showed a more than 10-fold inhibition of the four promoters studied, transcriptional repression mediated by Rep68 and Re p52 was reduced and nearly completely abolished for Rep40, The contrib ution of the C terminus of Rep78 was reduced with respect to the inhib ition of the AAV-2 p5 and p19 promoters. Point mutations and deletions showed that a C-terminal zinc binding motif is required for zinc bind ing in vitro but plays no obvious role in the inhibition of homologous and heterologous promoters. Overall, inhibition of the four different promoters was dependent on the identical Rep protein domains,vith the exception of the AAV-2 p5 promoter, Expression of the AAV-2 p5 promot er was inhibited by a Rep78 protein with a mutation in the nucleotide binding motif, whereas expression of the AAV-2 p19 promoter, the human immunodeficieucy virus long terminal repeat, and the HPV18 URR was no t, Mutational analysis of the HPV18 URR showed that several, but not a single, cis regulatory elements are involved in the inhibition proces s. This finding suggests that transcriptional repression is mediated b y protein-protein interactions of the Rep proteins either with multipl e transcription factors or,vith target proteins of sequence-specific t ranscription factors of the basal transcription machinery.