Mutagenesis of tyrosine and di-leucine motifs in the HIV-1 envelope cytoplasmic domain results in a loss of Env-mediated fusion and infectivity.
Mutagenesis of tyrosine and di-leucine motifs in the HIV-1 envelope cytoplasmic domain results in a loss of Env-mediated fusion and infectivity.
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DOI：
10.1186/1742-4690-8-37
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发表时间：
2011-05-14
期刊：
影响因子：
3.3
通讯作者：
Hunter E
中科院分区：
文献类型：
--
作者：
Bhakta SJ;Shang L;Prince JL;Claiborne DT;Hunter E
人类免疫缺陷病毒（HIV）包膜糖蛋白（Env）的gp 41组分含有具有多个高度保守的酪氨酸（Y）和双亮氨酸（LL）基序的长胞质结构域（CD）。研究表明，位于Env第712-715位残基的主要内吞作用基序（Y 712 HRL）远端的基序可能有助于Env在病毒生命周期中的功能。为了研究这些基序在Env的生物合成、运输和功能中的生物学贡献，我们构建了两组突变体，其中保守的Y-和LL-基序在存在或不存在Y 712的情况下依次被替代残基取代。然后构建靶向单个基序的另外的突变体。当在不存在其他病毒蛋白的情况下表达时，所有突变体Env通过多种抗体维持至少WT水平的Env表面染色。Y 712突变（Y 712 C）导致含有该变化的所有突变体的表面表达增加至少4倍。Y-和LL-基序的顺序诱变导致Env融合性的一般进行性降低。然而，添加突变的双亮氨酸和酪氨酸基序以外的酪氨酸在残基768导致最显着的影响Env纳入病毒粒子，病毒感染性，和病毒与靶细胞融合。从这里报道的研究中，我们表明，Y-和LL-基序的突变，有效地消除了两亲性的裂解肽2（LLP 2）结构域或破坏YW和LL基序在一个区域跨越残基795-803（YWWNLLQYW），只是C-末端的LLP 2，可以显着干扰HIV-1 Env的生物学功能和废除病毒复制。由于这些突变蛋白在细胞表面表达，我们得出结论，酪氨酸和双亮氨酸残基的gp 41的胞质结构域内发挥关键作用，在HIV-1复制，是不同的靶向质膜。
The gp41 component of the Human Immunodeficiency Virus (HIV) envelope glycoprotein (Env) contains a long cytoplasmic domain (CD) with multiple highly conserved tyrosine (Y) and dileucine (LL) motifs. Studies suggest that the motifs distal to major endocytosis motif (Y712HRL), located at residues 712-715 of Env, may contribute to Env functionality in the viral life cycle. In order to examine the biological contribution of these motifs in the biosynthesis, transport, and function of Env, we constructed two panels of mutants in which the conserved Y- and LL-motifs were sequentially substituted by alternative residues, either in the presence or absence of Y712. Additional mutants targeting individual motifs were then constructed. All mutant Envs, when expressed in the absence of other viral proteins, maintained at least WT levels of Env surface staining by multiple antibodies. The Y712 mutation (Y712C) contributed to at least a 4-fold increase in surface expression for all mutants containing this change. Sequential mutagenesis of the Y- and LL-motifs resulted in a generally progressive decrease in Env fusogenicity. However, additive mutation of dileucine and tyrosine motifs beyond the tyrosine at residue 768 resulted in the most dramatic effects on Env incorporation into virions, viral infectivity, and virus fusion with target cells. From the studies reported here, we show that mutations of the Y- and LL-motifs, which effectively eliminate the amphipathic nature of the lytic peptide 2 (LLP2) domain or disrupt YW and LL motifs in a region spanning residues 795-803 (YWWNLLQYW), just C-terminal of LLP2, can dramatically interfere with biological functions of HIV-1 Env and abrogate virus replication. Because these mutant proteins are expressed at the cell surface, we conclude that tyrosine and di-leucine residues within the cytoplasmic domain of gp41 play critical roles in HIV-1 replication that are distinct from that of targeting the plasma membrane.
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