Liu J S, Kuo S R, Broker T R, Chow L T
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642, USA.
J Biol Chem. 1995 Nov 10;270(45):27283-91. doi: 10.1074/jbc.270.45.27283.
We examined the functions of human papillomavirus type 11 (HPV-11) E1 and E2 proteins purified from Sf9 cells infected with recombinant baculoviruses in cell-free HPV-11 origin (ori) replication. The E1 protein binds specifically to a wild type but not to a mutated sequence in the ori spanning nucleotide position 1. It also has a relatively strong affinity for nonspecific DNA. A neutralizing antiserum directed against the amino-terminal one-third of the E1 protein totally abolishes initiation and elongation, suggesting that it functions as an initiator and a helicase at the replication fork. An antiserum against the carboxyl-terminal portion of E1 protein abolished replication only when added prior to initiation. Thus this portion of E1 is hidden in the replication complexes. The HPV-11 E2 protein appears not to be essential for elongation, but it must be present in the preinitiation complex for the E1 protein to recruit host DNA replication machinery to the ori. E2 antiserum added after preincubation in the absence of the cell extracts totally abolished replication. An identical conclusion is also reached for the bovine papillomavirus type 1 E2 protein. Finally, the HPV-11 E2C protein lacking the transacting domain of the full-length E2 protein partially inhibits E2-dependent ori replication.
我们检测了从感染重组杆状病毒的Sf9细胞中纯化的人乳头瘤病毒11型（HPV-11）E1和E2蛋白在无细胞HPV-11复制起点（ori）复制中的功能。E1蛋白特异性结合ori中跨越核苷酸位置1的野生型序列，而不与突变序列结合。它对非特异性DNA也有相对较强的亲和力。一种针对E1蛋白氨基末端三分之一的中和抗血清完全消除了起始和延伸，这表明它在复制叉处作为引发剂和解旋酶发挥作用。一种针对E1蛋白羧基末端部分的抗血清仅在起始前添加时才会消除复制。因此，E1的这部分隐藏在复制复合物中。HPV-11 E2蛋白似乎对延伸不是必需的，但它必须存在于起始前复合物中，以便E1蛋白将宿主DNA复制机制招募到ori。在无细胞提取物的情况下预孵育后添加的E2抗血清完全消除了复制。对于牛乳头瘤病毒1型E2蛋白也得出了相同的结论。最后，缺乏全长E2蛋白反式作用结构域的HPV-11 E2C蛋白部分抑制了E2依赖的ori复制。
