Bouhamdan M, Benichou S, Rey F, Navarro J M, Agostini I, Spire B, Camonis J, Slupphaug G, Vigne R, Benarous R, Sire J
INSERM U372, 13276 Marseille, France.
J Virol. 1996 Feb;70(2):697-704. doi: 10.1128/JVI.70.2.697-704.1996.
The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C- and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr.
辅助基因产物Vpr在人类免疫缺陷病毒1型感染过程中的作用仍不清楚。我们利用酵母双杂交系统来鉴定与Vpr相互作用并可能涉及其功能的细胞蛋白。已分离出一个编码人尿嘧啶DNA糖基化酶(UNG)的cDNA克隆,UNG是一种参与去除DNA中尿嘧啶的DNA修复酶。通过使用翻译后的放射性标记Vpr和作为谷胱甘肽S-转移酶融合蛋白表达的UNG重组蛋白进行体外蛋白质-蛋白质结合试验,证明了Vpr与UNG之间的相互作用。相反,已证明纯化的UNG与作为谷胱甘肽S-转移酶融合蛋白表达的Vpr重组蛋白相互作用。免疫共沉淀实验证实,Vpr和UNG在表达Vpr的细胞内相互关联。通过使用一组C端和N端缺失的Vpr突变体,我们确定Vpr的核心蛋白(氨基酸15至77)参与与UNG的相互作用。我们还通过体外实验证明,UNG与Vpr相互作用后仍保留其酶活性。