TRANSCRIPTION FACTOR YY1 REPRESSES CELL-FREE REPLICATION FROM HUMAN-PAPILLOMAVIRUS ORIGINS

We have established cell-free replication for the human papillomavirus type 18 (HPV-18) origin of replication (ori)-containing DNA by using purified HPV-18 El and E2 gene products expressed as fusion proteins i n Escherichia coli. The transcription factor YY1 has been shown to reg ulate RNA transcription by binding to a sequence overlapping the putat ive El protein binding site in the HPV-18 ori. We show that exogenousl y added YY1 fusion protein inhibited HPV-18 ori replication. Cotransfe ction of YY1 expression vectors also inhibited transient replication i n 293 cells. However, inhibition did not appear to be mediated by bind ing to its cognate site in the ori as YY1 also inhibited the replicati on of the HPV-11 ori, which does not have a known or suspected YY1 bin ding site. Moreover, inhibition was not alleviated by the inclusion of YY1 binding oligonucleotides in the replication reaction mixtures. Ra ther, we demonstrated a direct interaction between purified fusion E2 protein and fusion YY1 protein by the pull-down assay and a partial re storation of replication activity by an elevated E2 protein concentrat ion. These results suggest that YY1 can inhibit HPV ori replication by interfering with E2 protein functions.