Role of the major homology region in assembly of HIV-1 Gag.
Role of the major homology region in assembly of HIV-1 Gag.
复制标题
主要同源区域在 HIV-1 Gag 组装中的作用。
DOI：
10.1021/bi002040l
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Scarlata,S
中科院分区：
文献类型：
--
作者：
Provitera,P;Goff,A;Harenberg,A;Bouamr,F;Carter,C;Scarlata,S
主要同源区（MHR）是包括HIV-1在内的所有逆转录病毒的gaggene中高度保守的序列。它在组装中的作用尚不清楚，但该基序的缺失显著损害了膜结合和病毒颗粒的形成。为了开始描述这个缺陷，我们已经确定了这个区域对组装过程能量学的贡献。本特性荧光研究以tRNA和1-棕榈酰-2-油基磷脂酰丝氨酸大单层囊泡为模型，确定与膜和RNA结合相关的自由能变化。对于野生型蛋白，无论Gag先结合RNA还是先结合膜，其自由能的变化都在rt [600 cal/(mol·K)]以内。因此，Gag的初始结合可以与任何一种底物结合，但体内条件有利于与RNA的初始结合，可能是由于其较高的局部浓度。在建立了组装模式后，我们比较了gagwtw与缺失突变体GagΔMHR的结合能。与GagΔMHR相比，gagwt2蛋白与细胞膜结合的亲和力高2倍，与RNA的结合相似。Gag与RNA或膜的结合亲和力分别比GagΔMHRfor与膜或RNA的结合亲和力高~ 2−4倍。最重要的是，突变体在RNA或膜表面的自结合能力显著受损。MHR在促进蛋白-蛋白相互作用中的关键作用也体现在被感染细胞中切割产物数量的改变和膜结合、含rna的复制中间体的缺乏上。这些结果表明，Gag首先与RNA结合，然后组装成具有大膜结合面的多聚体复合物，从而促进随后的膜结合。MHR的缺失破坏了完成这一过程所需的蛋白质-蛋白质相互作用。
The major homology region (MHR) is a highly conserved sequence in thegaggene of all retroviruses, including HIV-1. Its role in assembly is unknown, but deletion of the motif significantly impairs membrane binding and viral particle formation. To begin characterizing this defect, we have determined the contribution of this region to the energetics of the assembly process. Intrinsic fluorescence studies were conducted to determine the change in free energy associated with membrane and RNA binding using tRNA and large unilamellar vesicles of 1-palmitoyl-2-oleoylphosphatidylserine as models. For the wild-type protein, the change in free energy was withinRT[600 cal/(mol·K)] whether Gag binds first to RNA or to the membrane. Thus, the initial binding of Gag can be to either substrate, but in vivo conditions favor initial association to RNA presumably due to its higher local concentration. After establishing the pattern of assembly, we compared the binding energy of GagWTversus the deletion mutant, GagΔMHR. GagWTbound to membranes with a 2-fold higher affinity than GagΔMHR, and the binding to RNA was similar for the two proteins. Gag prebound to RNA or to membrane exhibited ∼2−4-fold greater binding affinity than GagΔMHRfor binding the membrane or RNA, respectively. Most importantly, the mutant was significantly impaired in its ability to self-associate on RNA or on membrane surfaces. This key role of the MHR in promoting productive protein−protein interactions was also seen in altered amounts of cleavage products and the lack of membrane-bound, RNA-containing replication intermediates in infected cells. These results suggest that Gag first binds to RNA and then assembles into a multimeric complex with a large membrane-binding face that facilitates subsequent membrane binding. Deletion of the MHR disrupts the protein−protein interactions required to complete this process.