Efficient Fusion at Neutral pH by Human Immunodeficiency Virus gp41 Trimers Containing the Fusion Peptide and Transmembrane Domains.
Efficient Fusion at Neutral pH by Human Immunodeficiency Virus gp41 Trimers Containing the Fusion Peptide and Transmembrane Domains.
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DOI：
10.1021/acs.biochem.7b00753
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发表时间：
2018-02-20
期刊：
影响因子：
2.9
通讯作者：
Weliky DP
中科院分区：
文献类型：
--
作者：
Liang S;Ratnayake PU;Keinath C;Jia L;Wolfe R;Ranaweera A;Weliky DP
人类免疫缺陷病毒（HIV）是膜包膜的，并且初始感染步骤是病毒和细胞膜的连接/融合。该步骤由gp 41催化，gp 41是一种单程整合病毒膜蛋白。该蛋白含有位于病毒外部的约170个残基的胞外域，其对于融合是重要的，并且包括融合肽（FP）、N-螺旋、环、C-螺旋和病毒膜近端外部区（MPER）。病毒粒子最初具有三个gp 41胞外域和三个gp 120蛋白之间的非共价复合物。gp 120含有约500个残基，其功能是通过与靶细胞膜的特异性蛋白受体结合来识别靶T细胞和巨噬细胞。gp 120从gp 41胞外域移开，并且胞外域被认为与靶细胞膜结合并介导膜融合。胞外域的二级和三级结构在具有gp 120的初始复合物和没有gp 120的最终状态中是不同的。在融合过程中还没有gp 41的成像，因此gp 41和膜结构之间的时间关系尚不清楚。本研究描述了大的gp 41结构，包括胞外域和跨膜结构域（TM）的生物物理和功能特性。在中性pH下观察到中性和阴离子囊泡的显著融合，这反映了HIV/细胞融合的预期条件。在HIV/细胞融合中可能接触宿主膜的FP以及分别界面接触和穿过HIV膜的MPER和TM增强融合。与囊泡的初始接触是由蛋白质三聚体进行的，所述蛋白质三聚体处于反映与gp 120的初始复合物的天然寡聚体状态，并且对于没有gp 120的胞外域也通常观察到。圆二色谱数据支持N-螺旋、C-螺旋和MPER的螺旋结构，以及FP和环的非螺旋结构。通过尺寸排阻色谱法（SEC）观察单体、三聚体和六聚体状态的分布，其依赖于增溶洗涤剂和构建体。这些SEC和其他数据被整合到一个完善的工作模型的HIV/细胞融合，包括解离的胞外域成gp 41单体，然后折叠成发夹并列的两个膜，和随后的融合催化的三聚体和六聚体的发夹。因此，单体和寡聚体gp 41状态可能满足HIV进入膜附着和融合的双重要求。本研究报告囊泡融合在生理pH值的超耐热HIV gp 41发夹三聚体，包括FP和TM段。这种最终的gp 41状态可能催化HIV/细胞融合步骤，随后的膜并置，其中后一步骤可能与发夹形成同时进行。此外，本研究和早期研究报告了发夹三聚体部分解离成单体。这些单体可能在进化上是有利的，因为它们有助于初始发夹形成，并且它们也可能是gp 41 N-和C-螺旋肽融合抑制剂的靶标。
Human immunodeficiency virus (HIV) is membrane-enveloped and an initial infection step is joining/fusion of viral and cell membranes. This step is catalyzed by gp41 which is a single-pass integral viral membrane protein. The protein contains a ~170-residue ectodomain located outside the virus that is important for fusion, and includes the fusion peptide (FP), N-helix, loop, C-helix, and viral membrane-proximal external region (MPER). The virion initially has non-covalent complexes between three gp41 ectodomains and three gp120 proteins. A gp120 contains ~500-residues and functions to identify target T-cells and macrophages via binding to specific protein receptors of the target cell membrane. Gp120 moves away from the gp41 ectodomain, and the ectodomain is thought to bind to the target cell membrane and mediate membrane fusion. The secondary and tertiary structures of the ectodomain are different in the initial complex with gp120 and the final state without gp120. There is not yet imaging of gp41 during fusion, so the temporal relationship between the gp41 and membrane structures is not known. The present study describes biophysical and functional characterization of large gp41 constructs that include the ectodomain and transmembrane domain (TM). Significant fusion is observed of both neutral and anionic vesicles at neutral pH which reflects the expected conditions of HIV/cell fusion. Fusion is enhanced by the FP, which in HIV/cell fusion likely contacts the host membrane, and the MPER and TM, which respectively interfacially contact and traverse the HIV membrane. Initial contact with vesicles is made by protein trimers which are in a native oligomeric state that reflects the initial complex with gp120, and also is commonly observed for the ectodomain without gp120. Circular dichroism data support helical structure for the N-helix, C-helix, and MPER, and non-helical structure for the FP and loop. Distributions of monomer, trimer, and hexamer states are observed by size-exclusion chromatography (SEC), with dependences on solubilizing detergent and construct. These SEC and other data are integrated into a refined working model of HIV/cell fusion that includes dissociation of the ectodomain into gp41 monomers followed by folding into hairpins that appose the two membranes, and subsequent fusion catalysis by trimers and hexamers of hairpins. The monomer and oligomer gp41 states may therefore satisfy dual requirements for HIV entry of membrane apposition and fusion. The present study reports vesicle fusion at physiologic pH by a hyperthermostable HIV gp41 hairpin trimer that includes the FP and TM segments. This final gp41 state may catalyze HIV/cell fusion steps that follow apposition of the membranes, where the latter step is likely concurrent with hairpin formation. In addition, the present and earlier studies report partial dissociation of the hairpin trimer into monomers. The monomers may be evolutionarily advantageous because they aid initial hairpin formation, and they may also be the target of gp41 N- and C-helix peptide fusion inhibitors.
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