Yi Hyun Ah, Diaz-Rohrer Barbara, Saminathan Priyanka, Jacobs Amy
Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, The State University of New York , Buffalo, New York 14214, United States.
Biochemistry. 2015 Mar 3;54(8):1681-93. doi: 10.1021/bi501171r. Epub 2015 Feb 19.
The transmembrane subunit (gp41) of the HIV envelope protein complex (Env) mediates the viral fusion step of HIV entry. The membrane proximal external region (MPER), one of the functional domains of gp41, has been the focus of a great deal of research because it is a target for neutralizing antibodies. In this study, we examined 23 amino acid residues in the MPER (660-683) in both a CXCR4 coreceptor-utilizing strain (HXB2) and a CCR5-utilizing strain (JRFL) by alanine scanning mutagenesis. Despite the high degree of gp41 sequence conservation, the effects of alanine mutation in the MPER were different between the two strains. Most mutations in HXB2 had fusogenicity and protein expression levels not less than 50% of that of the wild type in the case of cell-cell fusion. However, ∼30% of the mutants in HXB2 showed a severe defect in fusogenicity in viral entry. Mutations in the MPER of strain JRFL had more dramatic effects than that in HXB2 in cell-cell fusion and viral entry. The fact that there are large differences in the effects of mutation between two strains suggests the potential for the interaction of the MPER with nonconserved sequences such as the fusion peptide and/or other NHR domains as well as potential long-range structural effects on the conformational changes that occur with the Env complex during membrane fusion.
人类免疫缺陷病毒包膜蛋白复合物（Env）的跨膜亚基（gp41）介导了HIV进入过程中的病毒融合步骤。膜近端外部区域（MPER）是gp41的功能结构域之一，由于它是中和抗体的靶点，因此一直是大量研究的焦点。在本研究中，我们通过丙氨酸扫描诱变，在利用CXCR4共受体的毒株（HXB2）和利用CCR5的毒株（JRFL）中检测了MPER（660-683）中的23个氨基酸残基。尽管gp41序列具有高度保守性，但MPER中丙氨酸突变在两种毒株中的影响有所不同。在细胞-细胞融合的情况下，HXB2中的大多数突变具有融合活性，且蛋白表达水平不低于野生型的50%。然而，HXB2中约30%的突变体在病毒进入时表现出严重的融合活性缺陷。JRFL毒株MPER中的突变在细胞-细胞融合和病毒进入方面比HXB2中的突变具有更显著的影响。两种毒株中突变影响存在巨大差异这一事实表明，MPER与融合肽和/或其他NHR结构域等非保守序列相互作用的可能性，以及对Env复合物在膜融合过程中发生的构象变化可能产生的远距离结构影响。
