Identification of trans-dominant HIV-1 rev protein mutants by direct transfer of bacterially produced proteins into human cells.
Identification of trans-dominant HIV-1 rev protein mutants by direct transfer of bacterially produced proteins into human cells.
复制标题
通过将细菌产生的蛋白质直接转移到人体细胞中来鉴定反式显性 HIV-1 rev 蛋白突变体。
DOI：
10.1093/nar/18.8.2037
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发表时间：
1990
影响因子：
14.9
通讯作者：
Pavlakis,GN
中科院分区：
文献类型：
--
作者：
Mermer,B;Felber,BK;Campbell,M;Pavlakis,GN
一个合成的rev基因含有取代，引入独特的限制性位点，但不改变推导的氨基酸序列被用作载体构建突变的rev。Rev结构域中的插入或取代突变导致蛋白质能够抑制Rev蛋白的反式功能。在含有Rev−突变前病毒的细胞系HLfB中监测Rev功能。HLfB细胞需要rev的存在才能产生病毒，这可以通过p24gag的免疫印迹检测来方便地监测。在细菌中表达并通过原生质体融合递送到HLfB细胞中后鉴定反式显性突变体。此外，通过共转染后突变蛋白在HLfB细胞中的表达来验证反式显性表型。这些研究确定了rev的氨基酸残基81和88之间的区域，其中不同的突变导致能够抑制Rev功能的蛋白质。
A syntheticrevgene containing substitutions which introduced unique restriction sites but did not alter the deduced amino acid sequence was used as a vehicle to construct mutations inrev. Insertion or substitution mutations within a domain ofRevresulted in proteins able to inhibit the function ofRevprotein in trans.Revfunction was monitored in a cell line, HLfB, which contained a rev−mutant provirus. HLfB cells require the presence ofrevfor virus production, which was conveniently monitored by immunoblot detection of p24gag. Trans-dominant mutants were identified after expression in bacteria and delivery into HLfB cells by protoplast fusion. In addition, the trans-dominant phenotype was verified by expression of the mutant proteins in HLfB cells after cotransfection. These studies define a region between amino acid residues 81 and 88 ofrev, in which different mutations result in proteins capable of inhibiting Rev function.