Titolo S, Pelletier A, Sauvé F, Brault K, Wardrop E, White P W, Amin A, Cordingley M G, Archambault J
Department of Biological Sciences, Bio-Méga Research Division, Boehringer Ingelheim (Canada) Ltd., Laval, Canada H7S 2G5.
J Virol. 1999 Jul;73(7):5282-93. doi: 10.1128/JVI.73.7.5282-5293.1999.
Replication of the genome of human papillomaviruses (HPV) is initiated by the recruitment of the viral E1 helicase to the origin of DNA replication by the viral E2 protein, which binds specifically to the origin. We determined, for HPV type 11 (HPV-11), that the C-terminal 296 amino acids of E1 are sufficient for interaction with the transactivation domain of E2 in the yeast two-hybrid system and in vitro. This region of E1 encompasses the ATP-binding domain. Here we have examined the role of this ATP-binding domain, and of ATP, on E2-dependent binding of E1 to the origin. Several amino acid substitutions in the phosphate-binding loop (P loop), which is implicated in binding the triphosphate moiety of ATP, abolished E2 binding, indicating that the structural integrity of this domain is essential for the interaction. The structural constraints imposed on the E1 P loop may differ between HPV-11 and bovine papillomavirus type 1 (BPV-1), since the P479S substitution that inactivates BPV-1 E1 is tolerated in the HPV-11 enzyme. Other substitutions in the E1 P loop, or in two other conserved motifs of the ATP-binding domain, were tolerated, indicating that ATP binding is not essential for interaction with E2. Nevertheless, ATP-Mg stimulated the E2-dependent binding of E1 to the origin in vitro. This stimulation was maximal at the physiological temperature (37 degrees C) and did not require ATP hydrolysis. In contrast, ATP-Mg did not stimulate the E2-dependent binding to the origin of an E1 protein containing only the C-terminal domain (353 to 649) or that of mutant E1 proteins with alterations in the DNA-binding domain. These results are discussed in light of a model in which the E1 ATP-binding domain is required for formation of the E2-binding surface and can, upon the binding of ATP, facilitate and/or stabilize the interaction of E1 with the origin.
人乳头瘤病毒（HPV）基因组的复制是由病毒E2蛋白将病毒E1解旋酶招募至DNA复制起点起始的，E2蛋白能特异性结合该起点。我们确定，对于11型HPV（HPV - 11），E1蛋白C端的296个氨基酸在酵母双杂交系统及体外实验中足以与E2的反式激活结构域相互作用。E1的这一区域包含ATP结合结构域。在此我们研究了该ATP结合结构域以及ATP在E1与起点的E2依赖性结合中的作用。磷酸结合环（P环）中几个与ATP三磷酸基团结合有关的氨基酸替换消除了E2结合，表明该结构域的结构完整性对于这种相互作用至关重要。HPV - 11和1型牛乳头瘤病毒（BPV - 1）对E1 P环施加的结构限制可能不同，因为使BPV - 1 E1失活的P479S替换在HPV - 11酶中可被耐受。E1 P环或ATP结合结构域其他两个保守基序中的其他替换也可被耐受，表明ATP结合对于与E2的相互作用并非必不可少。然而，ATP - Mg在体外刺激了E1与起点的E2依赖性结合。这种刺激在生理温度（37℃）时最大，且不需要ATP水解。相比之下，ATP - Mg并未刺激仅含C端结构域（353至649）的E1蛋白或DNA结合结构域发生改变的突变E1蛋白与起点的E2依赖性结合。根据一个模型对这些结果进行了讨论，在该模型中，E1 ATP结合结构域是形成E2结合表面所必需的，并且在ATP结合后可促进和/或稳定E1与起点的相互作用。
