人偏肺病毒F蛋白与细胞融合机制研究进展

人偏肺病毒(Human metapneumovirus,hMPV)是一种新发呼吸道病毒。hMPV感染可引起广泛的呼吸道疾病,目前已越来越受到人们的重视。多数副粘病毒与宿主细胞膜的融合过程依赖吸附蛋白和融合蛋白的共同参与。hMPV的特别之处在于其融合蛋白(F)既可以结合受体,又可以介导膜融合。本文从hMPV包膜表面具有双重功能的F蛋白入手,简要介绍F蛋白的结构和生理功能,重点阐述F蛋白介导的细胞融合机制和特性,对近几年来国内外研究进展进行了回顾与展望。     

偏肺病毒G蛋白:一个多变蛋白的多重角色

偏肺病毒包括人偏肺病毒和禽偏肺病毒,是感染人和禽的一种重要病原。G蛋白是偏肺病毒粒子表面的一种糖蛋白,属II型跨膜蛋白。不同种偏肺病毒G蛋白的大小和同源性差异巨大,发挥的生物学功能也显著不同,如在病毒的吸附过程、病毒介导的细胞融合、对病毒在体内外的复制能力影响以及免疫保护作用都有很大不同。同时,偏肺病毒G蛋白在免疫抑制和免疫逃避中也起着重要作用。目前国内开展的相关研究较少,本文对偏肺病毒G蛋白的最新研究成果和进展进行了综述和讨论,并对未来的相关研究进行了展望。     

副粘病毒附着蛋白在病毒融合过程中的作用*

副粘病毒附着蛋白 (AP)是病毒表面的一种主要糖蛋白 ,它能够诱导机体产生中和抗体。近年来的研究表明附着蛋白在病毒融合过程中的作用不仅限于其对受体的识别和结合 ,它还促进融合蛋白 (F)介导病毒与宿主细胞的融合。由此可见 ,副粘病毒具有其特有的融合机理 ,因此研究附着蛋白在病毒融合过程中的作用是揭示副粘病毒融合机理的前提 ,同时也会为新型抑制药物的研究提供思路。     

副粘病毒附着蛋白在病毒融合过程中的作用

副粘病毒附着蛋白（AP）是病毒表面的一种主要糖蛋白,它能够诱导机体产生中和抗体。近年来的研究表明附着蛋白在病毒融合过程中的作用不公限于其对受体的识别和结合,它还促进融合蛋白（F）介导病毒与宿主细胞的融合。由此可见,副粘病毒具有其特有的融合机理,因此研究附着蛋白在病毒融合过程中的作用是揭示副粘病毒融合机理的前提,同时也会为新型抑制药物的研究提供思路。     

禽副粘病毒-2 膜融合相关多肽基因的构建与表达

囊膜病毒与宿主细胞的膜融合是病毒入侵宿主细胞的第一步 . 禽副粘病毒 -2 (APMV-2) 囊膜表面糖蛋白有 2 种,与宿主受体结合的血凝素神经氨酸酶 (HN) 及介导膜融合的融合糖蛋白 (F). HN 蛋白的茎部区域 (stalk region) 与球状头部区域 (globular head region) ,以及 F 蛋白的 3 段七肽重复区域 (heptad repeat , HR) 都可能与膜融合直接相关,将 5 段多肽进行基因的构建与表达研究 . 根据已发表的禽副粘病毒 -1 (APMV-1) 氨基酸序列,应用 BLAST 程序进行同源性分析,以确定 APMV-2 相应区域,使用搭桥 PCR 或普通 PCR 方法构建基因,分别克隆入表达载体 pGEX-6p- Ⅰ获得重组质粒,阳性重组质粒转化入大肠杆菌 BL21 (DE₃) ,表达后获得可溶性融合蛋白, 3C 蛋白酶酶切后的蛋白质混合物经 Glutathione-Sepharose 4B 亲和层析纯化,最终获得可溶性、高纯度的 5 段多肽 . 应用 LearnCoil-VMF 软件与 ExPASy 系列软件对蛋白质结构与功能进行预测与分析 . 分子筛实验结果表明, F 蛋白的 HR1 与 HR2 可形成六聚体结构,圆二色谱实验结果则表明,六聚体蛋白富含 α 琢螺旋结构 .     

副流感病毒5包膜糖蛋白的结构与功能

副流感病毒5(Parainfluenza virus 5,PIV5)属于单股负链不分节段的RNA病毒,迄今尚未发现PIV5与人类已知的疾病有关,主要被用作疫苗载体。其包膜上存在三种糖蛋白:融合(Fusion,F)蛋白、血凝素-神经氨酸酶(Hemaggulatinin-neuraminidase,HN)蛋白、小疏水性(Small hydrophobic,SH)蛋白。F蛋白能在同源性HN蛋白的协助下介导膜融合,HN蛋白具有受体识别、神经氨酸酶活性和促细胞融合活性,SH蛋白则在病毒致病机制中起作用。本文主要阐述了三种包膜糖蛋白的结构和功能,旨在为PIV5的研究提供一些参考。     

小鼠肝炎病毒S蛋白及其受体的研究现状

MHV表面S蛋白介导多种重要的生物学功能,包括对易感细胞受体的吸附、侵入阶段病毒与细胞膜的融合、病毒传播过程中细胞与细胞的融合,以及免疫激活、组织嗜性、病毒致病性的变异。S蛋白对受体mCEACAM的识别是MHV感染种属特异性和组织趋向性的最初决定因素,不同MHV毒株S1亚基的长度及核苷酸序列都呈现高度多态性,这些突变导致抗体表位和T细胞表位缺失,为病毒逃避免疫监视提供一条途径。     

副粘病毒血凝素-神经氨酸酶蛋白结构与功能的研究进展

副粘病毒的血凝素-神经氨酸酶和融合蛋白具有重要的生物学活性,其中前者具有受体识别活性、神经氨酸酶活性和促进融合蛋白的细胞融合作用.本文对近年来血凝素-神经氨酸酶结构和功能方面的研究进展进行了综述.     

组氨酸质子化触发病毒膜融合及其分子机制

膜融合是有包膜病毒入侵靶细胞的关键步骤,低pH、受体结合、二者兼具或其他尚未界定的机制均可触发病毒融合蛋白的构象重排,介导病毒包膜与靶细胞膜或内体膜间的融合。组氨酸(histidine,His)残基是唯一一个质子化状态变化(pKa~6~7)接近于病毒融合阈值(~pH6)的氨基酸,参与多种低pH依赖的病毒融合蛋白构象转变及膜融合,对其可能作用机制的阐述将有助于抗病毒药物的研制与发展。     

人呼吸道合胞病毒小疏水蛋白研究进展

人呼吸道合胞病毒是人呼吸道感染病原中的重要病原。SH蛋白是人呼吸道合胞病毒粒子表面的一种小分子糖蛋白,可形成五聚体的离子通道结构。SH蛋白是人呼吸道合胞病毒复制的非必需蛋白,但可以影响病毒融合蛋白介导的细胞融合,近年研究发现SH蛋白胞外区与血蓝蛋白形成的复合物可作为人呼吸道合胞病毒疫苗的抗原,产生的抗体在体内能清除病毒感染的细胞。本综述旨在对人呼吸道合胞病毒SH蛋白最新研究成果进行总结和讨论,并对未来针对SH蛋白的研究方向加以展望。     

Membrane Fusion

The fusion of biological membranes results in two bilayer-based membranes merging into a single membrane. In this process the lipids have to undergo considerable rearrangement. The nature of the intermediates that are formed during this rearrangement has been investigated. Certain fusion proteins facilitate this process. In many cases short segments of these fusion proteins have a particularly important role in accelerating the fusion process. Studies of the interaction of model peptides with membranes have allowed for increased understanding at the molecular level of the mechanism of the promotion of membrane fusion by fusion proteins. There is an increased appreciation of the roles of several independent segments of fusion proteins in promoting the fusion process.Many of the studies of the fusion of biological membranes have been done with the fusion of enveloped viruses with other membranes. One reason for this is that the number of proteins involved in viral fusion is relatively simple, often requiring only a single protein. For many enveloped viruses, the structure of their fusion proteins has certain common elements, suggesting that they all promote fusion by an analogous mechanism. Some aspects of this mechanism also appears to be common to intracellular fusion, although several proteins are involved in that process which is more complex and regulated than is fusion.     

A cell-free analysis of the endocytic pathway

The molecular control of the endocytic pathway is poorly understood. To obtain this information requires the use of cell-free systems which faithfully recreate the various endocytic events as they occur in the intact cell. Here I describe our approach to elucidating the mechanism which controls the fusion between different vesicles on the pathway.     

Dual‐specificity tyrosine phosphorylation‐regulated kinase 2 regulates osteoclast fusion in a cell heterotypic manner

Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign‐body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion‐founder cells that fuse to fusion‐follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells.     

Genetic variation of vertebral fusion patterns in coho salmon

The distribution of vertebral fusions along the spinal column differed significantly among crosses from two hatchery stocks of coho salmon, indicating a genetic basis for this character.     

Molecular mechanisms of membrane fusion: Steps during phospholipid and exocytotic membrane fusion

Exocytosis is considered as four separate steps: adhesion, fusion/pore formation, pore widening, and content discharge. Experiments on both synthetic and natural membranes are presented to show each of these steps. Major differences are seen in the two fusing systems. These differences are discussed in terms of molecular mechanisms of fusion.     

Structures and Mechanisms of Viral Membrane Fusion Proteins: Multiple Variations on a Common Theme

Recent work has identified three distinct classes of viral membrane fusion proteins based on structural criteria. In addition, there are at least four distinct mechanisms by which viral fusion proteins can be triggered to undergo fusion-inducing conformational changes. Viral fusion proteins also contain different types of fusion peptides and vary in their reliance on accessory proteins. These differing features combine to yield a rich diversity of fusion proteins. Yet despite this staggering diversity, all characterized viral fusion proteins convert from a fusion-competent state (dimers or trimers, depending on the class) to a membrane-embedded homotrimeric prehairpin, and then to a trimer-of-hairpins that brings the fusion peptide, attached to the target membrane, and the transmembrane domain, attached to the viral membrane, into close proximity thereby facilitating the union of viral and target membranes. During these conformational conversions, the fusion proteins induce membranes to progress through stages of close apposition, hemifusion, and then the formation of small, and finally large, fusion pores. Clearly, highly divergent proteins have converged on the same overall strategy to mediate fusion, an essential step in the life cycle of every enveloped virus.     

病毒融合蛋白的结构和功能

病毒融合蛋白可以分为三种类型,不同类型的病毒融合蛋白的结构差异很大,但是会采用相似的"发卡"构象实现融合.在一定条件下,病毒融合蛋白的疏水结构域,融合环或融合肽插入靶膜中,通过其自身折叠形成发卡使病毒和宿主的膜靠近.与此同时,融合蛋白构象变化会释放出足够的能量将双方膜打破并完成融合.本文中,我们总结了三种类型病毒融合蛋白的特征,并对其中央发卡三聚体结构域、跨膜结构域以及近膜结构域在融合过程中的作用进行了论述.     

Real-time Exchange of the Lipid-bound Intermediate and Post-fusion States of the HIV-1 gp41 Ectodomain

The envelope glycoprotein gp41 of the HIV-1 virus mediates its entry into the host cell. During this process, gp41 undergoes large conformational changes and the energy released in the remodeling events is utilized to overcome the barrier associated with fusing the viral and host membranes. Although the structural intermediates of this fusion process are attractive targets for drug development, no detailed high-resolution structural information or quantitative thermodynamic characterization are available. By measuring the dynamic equilibrium between the lipid-bound intermediate and the post-fusion six-helical bundle (6HB) states of the gp41 ectodomain in the presence of bilayer membrane mimetics, we derived both the reaction kinetics and energies associated with these two states by solution NMR spectroscopy. At equilibrium, an exchange time constant of about 12 seconds at 38 °C is observed, and the post-fusion conformation is energetically more stable than the lipid-bound state by 3.4 kcal mol^?1. The temperature dependence of the kinetics indicates that the folding occurs through a high-energy transition state which may resemble a 5HB structure. The energetics and kinetics of gp41 folding in the context of membrane bilayers provide a molecular basis for an improved understanding of viral membrane fusion.     

膜融合机制研究进展

膜的融合是一个基本的生命过程,在生物的生长发育中有着重要作用。通过融合,两套独立的双层脂分子合二为一,完成一定的生物功能。膜融合分子机制的关键在于其主要成分：融合蛋白。Ⅰ、Ⅱ类病毒融合蛋白形成“发夹”,胞内囊泡与目标膜各提供的融合蛋白形成“类亮氨酸拉链”,这些结构将独立的膜拉近,继而促使膜合为一体。细胞与细胞间融合蛋白的作用机制目前还未明确,在各种膜融合中,脂双层的变化可能是类似的,但介导融合的分子机制应该是不同的。目前,对于膜融合很多方面的理解还停留在假说阶段。理解了膜融合的过程和分子机制不仅将极大地促进生物学的发展,更重要是将为相关的疾病治疗打下坚实的基础。