Russell J, Botchan M R
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Virol. 1995 Feb;69(2):651-60. doi: 10.1128/JVI.69.2.651-660.1995.
Papillomavirus DNA replication requires the viral trans-acting factors E1 and E2 in addition to the host cell's general replication machinery. The origins of DNA replication in bovine and human papillomavirus genomes have been localized to a specific part of the upstream regulatory region (URR) which includes recognition sites for E1 and E2 proteins. To fine map cis-acting elements influencing human papillomavirus type 11 (HPV-11) DNA replication and to determine the relative contributions of such sites, we engineered consecutive linker substitution mutations across a region of 158 bp in the HPV-11 origin and tested mutant origins for replication function in a cell-based transient replication assay. Our results both confirm and extend the findings of others. E2 binding sites are the major cis components of HPV-11 DNA replication, and there is evidence for synergy between these sites. Differential capacity of the three E2 binding sites within the origin to affect replication may be attributed, at least in part, to context. At least one E2 binding site is essential for replication. The imperfect AT-rich palindrome of the E1 helicase binding site is not essential since replication occurs even in the absence of this sequence. However, replication is enhanced by the presence of the palindromic sequence in the HPV-11 origin. Sequence components adjacent to the E1 and E2 binding sites, comprising AT-rich and purine-rich elements and the consensus TATA box sequence, probably contribute to the overall efficiency of replication, though they are nonessential. None of the other cis elements of the HPV-11 origin region analyzed seems to influence replication significantly in the system described. The HPV-11 origin of DNA replication therefore differs from those of the other papovaviruses, simian virus 40 and polyomavirus, inasmuch as an intact helicase binding site and adjacent AT-rich components, while influential, are not absolutely essential.
乳头瘤病毒DNA复制除了需要宿主细胞的一般复制机制外，还需要病毒反式作用因子E1和E2。牛乳头瘤病毒和人乳头瘤病毒基因组中的DNA复制起点已定位到上游调控区（URR）的特定部分，该区域包括E1和E2蛋白的识别位点。为了精细定位影响人乳头瘤病毒11型（HPV - 11）DNA复制的顺式作用元件并确定这些位点的相对贡献，我们在HPV - 11复制起点的158 bp区域设计了连续的接头取代突变，并在基于细胞的瞬时复制试验中测试了突变起点的复制功能。我们的结果证实并扩展了其他人的发现。E2结合位点是HPV - 11 DNA复制的主要顺式成分，并且有证据表明这些位点之间存在协同作用。复制起点内三个E2结合位点影响复制的能力差异可能至少部分归因于其环境。至少有一个E2结合位点对复制至关重要。E1解旋酶结合位点的不完全富含AT的回文序列并非必不可少，因为即使没有该序列也能发生复制。然而，HPV - 11复制起点中回文序列的存在会增强复制。与E1和E2结合位点相邻的序列成分，包括富含AT和富含嘌呤的元件以及共有TATA盒序列，可能有助于复制的整体效率，尽管它们并非必不可少。在所描述的系统中，分析的HPV - 11复制起点区域的其他顺式元件似乎都不会对复制产生显著影响。因此，HPV - 11的DNA复制起点与其他乳头多瘤空泡病毒（猴病毒40和多瘤病毒）的复制起点不同，因为完整的解旋酶结合位点和相邻的富含AT的成分虽然有影响，但并非绝对必要。
