牛免疫缺陷病毒反式激活因子作用机理的研究

STUDY OF THE MECHANISM OF BOVINE IMMUNODEFICIENCYVIRUS TAT PROTEIN TRANSACTIVATION

牛免疫缺陷病毒（Ｂｏｖｉｎｅｉｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙｖｉｒｕｓ，ＢＩＶ）基因组编码的反式激活因子（ｔｒａｎｓａｃｔｉｖａｔｏｒ，Ｔａｔ）可以提高ＢＩＶ长末端重复区（ｌｏｎｇｔｅｒｍｉｎａｌｒｅｐｅａｔ，ＬＴＲ）启动子的表达。为研究ＢＩＶＴａｔ蛋白的作用机理，分别从ＢＩＶＬＴＲ５′端及３′端构建不同的缺失，从荧光素酶基因（ｌｕｃｉｆｅｒａｓｅ，Ｌｕｃ）为报道基因，通过共转染小牛肺细胞检测ＢＩＶＬＴＲ中与Ｔａｔ蛋白反式激活作用相关的序列。转染结果表明ＢＩＶＬＴＲ启动子始于－５２位的上游区不显著影响Ｔａｔ蛋白的激活作用，转录起始点下游的序列与Ｔａｔ蛋白的激活作用密切相关；并且－２－＋３２间的序列为ＢＩＶＴａｔ蛋白靶序列的一部分。将ＢＩＶＬＴＲ启动子下游序列（－２－＋２０４）亚克隆到ＨＩＶ－１ＬＴＲ启动子－５位之后，ＢＩＶＴａｔ蛋白可极大地提高该杂合启动子表达，进一步说明ＢＩＶＴａｔ蛋白的靶序列存在于－２－＋２０４的范围内；同时将ＢＩＶＬＴＲ启动子上游序列（－４１０－－３）接于ＨＩＶ－１ＬＴＲ下游序列（－２－＋７８）之前，ＨＩＶ－１Ｔａｔ蛋白可以激活该杂合启动子表达，说明ＢＩＶＬＴＲ上游序列可以支持ＨＩＶ－?

he bovine immunodeficiency virus(BIV)transactivator(Tat)protein can enhance the expression of BIV long terminal repeat(LTR)promoter.To study the mechanism of transactivation,deletions of BIV LTR were constructed from either 5’or 3’end,and luciferase gene wasemployed as the reporter gene.By cotransfection of fetal bovine lung cells,sequences in BIVLTR responsive to Tat protein transactivation were identified.The results indicated thatsequence upstream from -52 had little effect on transactivation.On the other hand deletion ofthe downstream sequence resulted in non-transactivation of the truncated promoter by Tatprotein,and sequence-2-+32 was characterized as part of the target sequence of Tatprotein.Moreover,BIV Tat protein could highly augment the expression of the hybridpromoter composed of BIV LTR downstream sequence (-2-+204)and HIV-1 upstream sequence(-135--5),which further confirmed that the target sequence of BIV Tat is located in BIVLTR downstream sequence(-2-+204)Inthe meantime,when BIV LTR upstream sequence(-410--3) was ligated with HIV-1 downstream sequence(-4-+78),HIV-1 Tat protein could transactivate the expression of the hybrid promoter,which suggested that BIV LTR upstream sequence(-410--2)could support the transactivation of HIV-1 Tat proteinthrough its target sequence.