人类免疫缺陷病毒1型整合酶可在体内单独表达整合Moloney小鼠白血病病毒基因组。

W J Peng, J T Pan, M C Lai, C F Chiu, T H Lin
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引用次数: 0

摘要

利用表达的人类免疫缺陷病毒1型(HIV-1)整合酶(in)蛋白和携带传染性Moloney小鼠白血病病毒(MuLV)原病毒基因组拷贝的质粒作为底物,提出了一种体内整合试验。从载体pINSD中提取HIV-1 IN基因,克隆到载体pXT1中,得到pXT1-IN。采用定点诱变的方法,将传染性MuLV原病毒载体pMLV- k上一对U3和U5附着(att)序列上的两个和三个环结核苷酸改变为相应的HIV-1 att序列,生成载体pMLV*(U3U5)。将载体pMLV- k和pMLV*(U3U5)的部分MuLV IN序列删除,生成载体pMLV*(U3U5) delta IN和pMLV*(U3U5) delta IN。通过对释放和收集的病毒粒子进行非放射性逆转录酶(RT)测定,检测这些野生型和部分缺失或突变的MuLV IN在转染细胞中的整合情况。单独转染载体pMLV δ IN的NIH/3T3细胞未检测到RT活性。然而,转染载体pMLV δ IN或pMLV*(U3U5) δ IN的HIV-1 IN表达细胞有一定的RT活性。这表明在缺乏其他HIV-1蛋白表达的情况下,MuLV原病毒基因组被表达的HIV-1 in蛋白整合。以部分缺失的MuLV IN序列为靶点,对转染细胞提取的基因组DNA进行聚合酶链反应分析,进一步证实了这些MuLV原病毒基因组的整合。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
The genome of Moloney murine leukemia virus can be integrated by the integrase of human immunodeficiency virus type 1 expressed alone in vivo.

An in vivo integration assay using the expressed human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein and plasmids carrying a copy of the infectious Moloney murine leukemia virus (MuLV) provirus genome as substrates is presented. The HIV-1 IN gene was taken from vector pINSD and cloned into vector pXT1 to give pXT1-IN. Two and three nucleotides from the circle junction on one pair of U3 and U5 attachment (att) sequences on an infectious MuLV provirus vector pMLV-K were changed by means of site-directed mutagenesis to that of the corresponding HIV-1 att sequences to generate vector pMLV*(U3U5). The MuLV IN sequence was partially deleted for vectors pMLV-K and pMLV*(U3U5) to generate vectors pMLV delta IN and pMLV*(U3U5) delta IN. Integration of these wild type and MuLV IN partially deleted or att mutated MuLV provirus vectors in the transfected cells by the expressed HIV-1 IN was monitored by means of a non-radioactive reverse transcriptase (RT) assay for released and collected virions. No RT activity was detected for the NIH/3T3 cell singly transfected with vector pMLV delta IN. However some RT activities were observed for the HIV-1 IN expressing cell transfected either with vectors pMLV delta IN or pMLV*(U3U5) delta IN. This indicated that in the absence of other HIV-1 proteins expressed the MuLV provirus genome was integrated by the expressed HIV-1 IN protein. The integration of these MuLV provirus genomes was further confirmed by polymerase chain reaction analysis on the genomic DNA extracted from the transfected cells using the MuLV IN sequence remained from partial deletion as a target.

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