Lee K Y, Broker T R, Chow L T
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Virol. 1998 Jun;72(6):4911-7. doi: 10.1128/JVI.72.6.4911-4917.1998.
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 E1 and E2 gene products expressed as fusion proteins in Escherichia coli. The transcription factor YY1 has been shown to regulate RNA transcription by binding to a sequence overlapping the putative E1 protein binding site in the HPV-18 ori. We show that exogenously added YY1 fusion protein inhibited HPV-18 ori replication. Cotransfection of YY1 expression vectors also inhibited transient replication in 293 cells. However, inhibition did not appear to be mediated by binding to its cognate site in the ori as YY1 also inhibited the replication of the HPV-11 ori, which does not have a known or suspected YY1 binding site. Moreover, inhibition was not alleviated by the inclusion of YY1 binding oligonucleotides in the replication reaction mixtures. Rather, we demonstrated a direct interaction between purified fusion E2 protein and fusion YY1 protein by the pull-down assay and a partial restoration of replication activity by an elevated E2 protein concentration. These results suggest that YY1 can inhibit HPV ori replication by interfering with E2 protein functions.
我们通过使用在大肠杆菌中作为融合蛋白表达的纯化人乳头瘤病毒18型（HPV - 18）复制起点（ori）的DNA，建立了无细胞复制体系。转录因子YY1已被证明通过与HPV - 18 ori中与假定的E1蛋白结合位点重叠的序列结合来调节RNA转录。我们发现，外源添加的YY1融合蛋白抑制了HPV - 18 ori的复制。YY1表达载体的共转染也抑制了293细胞中的瞬时复制。然而，抑制作用似乎不是通过与ori中的同源位点结合介导的，因为YY1也抑制了HPV - 11 ori的复制，而HPV - 11 ori没有已知或疑似的YY1结合位点。此外，在复制反应混合物中加入YY1结合寡核苷酸并不能减轻抑制作用。相反，我们通过下拉实验证明了纯化的融合E2蛋白与融合YY1蛋白之间的直接相互作用，并且通过提高E2蛋白浓度部分恢复了复制活性。这些结果表明，YY1可以通过干扰E2蛋白功能来抑制HPV ori的复制。
