Daly T J, Rennert P, Lynch P, Barry J K, Dundas M, Rusche J R, Doten R C, Auer M, Farrington G K
Repligen Corporation, Cambridge, Massachusetts 02139.
Biochemistry. 1993 Aug 31;32(34):8945-54. doi: 10.1021/bi00085a028.
Perturbations within the transactivation and carboxy-terminal domains of HIV-1 Rev were examined for effects on Rev responsive element (RRE) binding activities in vitro and biological activity in vivo. Binding affinities, specificities, and multimerization of the transactivation mutants M10 and Rev/Rex M10-16 on the RRE were equivalent to wild-type Rev. Substitution of the Rex transactivation domain within Rev resulted in the incorporation of an internal methionine residue which, when cleaved with CNBr and subsequently purified, produced a protein species (CNBr-Rev) unable to fully multimerize on the RRE. Instead, two discrete protein-dependent species were generated in the gel shift assay. Furthermore, CNBr-Rev was observed to bind to the RRE with high specificity and an equilibrium binding constant of 6 x 10(-10) M. A C-terminal Rev deletion mutant (Rev M9 delta 14) lacking amino acids 68-112 displayed identical RRE binding characteristics to the CNBr-Rev protein. This protein, which lacks both the activation and the C-terminal domains, was biologically inactive but maintained the ability to discriminate the RRE from nonspecific RNA. Deletion of amino acids 92-112 resulted in a Rev mutant (Rev M11 delta 14) which bound to the RRE with wild-type affinity and high specificity. This purified mutant was observed to be aberrant in multimerization activity on the RRE with reduced multimerization apparent in the gel shift assay. However, Rev M11 delta 14 possessed biological activity equivalent to wild-type Rev in a cell-based p24 ELISA assay. These results suggest that polymerization on the RRE is dispensable for Rev activity and that two monomeric Rev proteins bound to the RRE are sufficient for biological activity. Furthermore, in vivo experiments using the Rev/Rex chimeric mutant and the M10 transdominant mutant as well as in vitro dissociation rate studies with Rev M11 delta 14 and Rev M9 delta 14 suggest that the M9 through M11 domain of the protein may be involved in RRE-dependent specific Rev dimerization.
研究了HIV-1 Rev反式激活结构域和羧基末端结构域内的扰动对体外Rev反应元件（RRE）结合活性和体内生物学活性的影响。反式激活突变体M10和Rev/Rex M10-16在RRE上的结合亲和力、特异性和多聚化与野生型Rev相当。Rev内Rex反式激活结构域的替换导致掺入一个内部甲硫氨酸残基，用溴化氰切割并随后纯化后，产生一种蛋白质（溴化氰-Rev），其无法在RRE上完全多聚化。相反，在凝胶迁移试验中产生了两种离散的蛋白质依赖性物种。此外，观察到溴化氰-Rev以高特异性结合RRE，平衡结合常数为6×10^(-10) M。缺少氨基酸68-112的C末端Rev缺失突变体（Rev M9 delta 14）对RRE的结合特性与溴化氰-Rev蛋白相同。这种既缺少激活结构域又缺少C末端结构域的蛋白质没有生物学活性，但仍保持区分RRE和非特异性RNA的能力。缺失氨基酸92-112导致一个Rev突变体（Rev M11 delta 14），它以野生型亲和力和高特异性结合RRE。观察到这种纯化的突变体在RRE上的多聚化活性异常，在凝胶迁移试验中多聚化减少。然而，在基于细胞的p24 ELISA试验中，Rev M11 delta 14具有与野生型Rev相当的生物学活性。这些结果表明，在RRE上的聚合对于Rev活性是可有可无的，并且两个结合到RRE的单体Rev蛋白足以产生生物学活性。此外，使用Rev/Rex嵌合突变体和M10反式显性突变体的体内实验以及对Rev M11 delta 14和Rev M9 delta 14的体外解离速率研究表明，该蛋白质M9至M11结构域可能参与RRE依赖性的特异性Rev二聚化。
