Adam J L, Briggs M W, McCance D J
Department of Microbiology and Immunology, University of Rochester, Rochester, New York, 14642, USA.
Virology. 2000 Jul 5;272(2):315-25. doi: 10.1006/viro.2000.0376.
The E5 gene of human papillomavirus type 16 encodes a highly hydrophobic membrane protein previously shown to inhibit endosomal acidification, presumably by binding to the 16-kDa pore-forming subunit of the vacuolar H(+)-ATPase (v-ATPase). The role of this interaction in the disruption of v-ATPase activity was explored through extensive mutagenesis of E5 to identify residues that mediate binding to the 16-kDa subunit. Coimmunoprecipitations revealed that the hydrophobic span between residues 41 and 54 is primarily responsible for this interaction and can be replaced with random hydrophobic amino acids. Studies using mutated 16-kDa proteins indicated that the fourth transmembrane domain of the pore subunit mediates binding to E5. Analysis of the E5 mutants in a yeast expression system revealed that several mutants that retained the capacity to bind to the 16-kDa subunit in COS-1 cells failed to disrupt vacuolar acidification. These data argue that E5 binding to the pore subunit is not sufficient for the associated activity of disruption of v-ATPase function.
人乳头瘤病毒16型的E5基因编码一种高度疏水的膜蛋白，此前研究表明该蛋白可能通过与液泡H(+)-ATP酶（v-ATPase）的16-kDa成孔亚基结合来抑制内体酸化。通过对E5进行广泛诱变以鉴定介导与16-kDa亚基结合的残基，从而探究这种相互作用在破坏v-ATPase活性中的作用。免疫共沉淀显示，41至54位残基之间的疏水区域主要负责这种相互作用，并且可以被随机疏水氨基酸取代。使用突变的16-kDa蛋白进行的研究表明，孔亚基的第四个跨膜结构域介导与E5的结合。在酵母表达系统中对E5突变体的分析表明，一些在COS-1细胞中保留了与16-kDa亚基结合能力的突变体未能破坏液泡酸化。这些数据表明，E5与孔亚基的结合不足以破坏v-ATPase功能的相关活性。
