Salzwedel K, West J T, Hunter E
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Virol. 1999 Mar;73(3):2469-80. doi: 10.1128/JVI.73.3.2469-2480.1999.
Mutations were introduced into the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope glycoprotein, gp41, within a region immediately adjacent to the membrane-spanning domain. This region, which is predicted to form an alpha-helix, contains highly conserved hydrophobic residues and is unusually rich in tryptophan residues. In addition, this domain overlaps the epitope of a neutralizing monoclonal antibody, 2F5, as well as the sequence corresponding to a peptide, DP-178, shown to potently neutralize virus. Site-directed mutagenesis was used to create deletions, substitutions, and insertions centered around a stretch of 17 hydrophobic and uncharged amino acids (residues 666 to 682 of the HXB2 strain of HIV-1) in order to determine the role of this region in the maturation and function of the envelope glycoprotein. Deletion of the entire stretch of 17 amino acids abrogated the ability of the envelope glycoprotein to mediate both cell-cell fusion and virus entry without affecting the normal maturation, transport, or CD4-binding ability of the protein. This phenotype was also demonstrated by substituting alanine residues for three of the five tryptophan residues within this sequence. Smaller deletions, as well as multiple amino acid substitutions, were also found to inhibit but not block cell-cell fusion. These results demonstrate the crucial role of a tryptophan-rich motif in gp41 during a post-CD4-binding step of glycoprotein-mediated fusion. The basis for the invariant nature of the tryptophans, however, appears to be at the level of glycoprotein incorporation into virions. Even the substitution of phenylalanine for a single tryptophan residue was sufficient to reduce Env incorporation and drop the efficiency of virus entry approximately 10-fold, despite the fact that the same mutation had no significant effect on syncytium formation.
突变被引入到人类免疫缺陷病毒1型（HIV-1）跨膜包膜糖蛋白gp41的胞外结构域中，该区域紧邻跨膜结构域。这个区域预计会形成一个α螺旋，包含高度保守的疏水残基，并且色氨酸残基异常丰富。此外，该结构域与一种中和单克隆抗体2F5的表位重叠，以及与一种肽DP-178对应的序列重叠，DP-178已被证明能有效中和病毒。利用定点诱变技术，围绕一段17个疏水且不带电荷的氨基酸（HIV-1 HXB2株的666至682位残基）进行缺失、替换和插入操作，以确定该区域在包膜糖蛋白成熟和功能中的作用。删除整个17个氨基酸的片段消除了包膜糖蛋白介导细胞间融合和病毒进入的能力，而不影响该蛋白的正常成熟、运输或CD4结合能力。通过将该序列中的五个色氨酸残基中的三个替换为丙氨酸残基，也证明了这种表型。较小的缺失以及多个氨基酸替换也被发现能抑制但不阻断细胞间融合。这些结果证明了gp41中富含色氨酸的基序在糖蛋白介导的融合的CD4结合后步骤中的关键作用。然而，色氨酸不变性的基础似乎在于糖蛋白整合到病毒颗粒的水平。即使将单个色氨酸残基替换为苯丙氨酸也足以降低Env的整合，并使病毒进入效率下降约10倍，尽管相同的突变对合胞体形成没有显著影响。
