病毒跨膜蛋白的结构功能与抗病毒药物设计

根据病毒衣壳表面有无囊膜结构, 病毒可被分为无包膜病毒和有包膜病毒。包膜病毒的膜蛋白在病毒的吸附、侵入、脱壳、生物大分子合成、病毒粒子的装配与释放等生命周期中起重要作用。某些包膜病毒的膜蛋白对病毒侵入宿主细胞的膜融合是不可或缺的。结构分析显示, Ⅰ型和Ⅱ型病毒融合蛋白采用类似的膜融合方式。此外, 流行性感冒病毒的M2 蛋白、人类免疫缺陷病毒Ⅰ型( HIV-1) 的Vpu 蛋白、重症急性呼吸综合征冠状病毒( SARS-CoV) 3a蛋白等膜蛋白还具有离子通道的功能。针对这些病毒膜融合蛋白设计的抑制分子, 将为研发抗包膜病毒新型药物提供新思路和策略。本文以3 种病毒膜融合蛋白为例, 对其融合机制、跨膜蛋白离子通道功能及其在抗病毒药物设计中的应用作一简要综述。     

融合蛋白与病毒入膜机制研究进展

包膜病毒感染细胞的第一步即病毒与靶细胞膜的融合,它由病毒包膜上的融合蛋白诱发,融合蛋白与受体分子相互作用后暴露出融合肽,它伸向靶膜使两膜紧密接近后,多肽周围的脂质分子进一步重排,通过中间态最后发生融合,本文将介绍近年来病毒融合蛋白及入膜机制研究进展。     

副粘病毒包膜糖蛋白分子生物学研究进展

副粘病毒是一类重要的人和动物致病病毒。其中包括副流感病毒(Parainfluenza virus,PIV)、流行性腮腺炎病毒(Mumpsvirus,MuV)和新城疫病毒(Newcatle disease virus．NDV)。这些病毒在中性pH条件下,可以通过膜融合而感染宿主细胞。整个病毒由包膜、衣壳和核酸组成。其基因组为15kb左右的单股负链RNA,主要包括6组基因,用于编码6种病毒结构蛋白。     

非包膜病毒进入细胞基质的研究进展

病毒是最简单的生命形态,必须在宿主细胞中才能繁殖。为了增殖,病毒需要将它们的基因组运送到宿主细胞中。而要达到这一目的,病毒必须通过宿主细胞的主要屏障——细胞质膜,不同病毒会根据宿主细胞膜的特点运用不同的进入策略。根据病毒的外表是否具有脂双层膜可将病毒分成两类:包膜病毒和非包膜病毒。包膜病毒外表有一层来源于宿主细胞的脂双层膜,膜上整合了病毒编码的膜融合蛋白。包膜病毒主要通过膜融合蛋白的作用促使病毒的包膜与细胞膜融合,从而介导病毒核衣壳的侵人。对于非包膜病毒来说,由于缺乏包     

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。     

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。     

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.     

冠状病毒的基因组结构及特征

冠状病毒(Coronavirus)是具有包膜的正单链RNA病毒,基因组大小介于26 000与32 000 nt之间,编码刺突蛋白(S)、包膜蛋白(E)、膜蛋白(M)和核壳蛋白(N)等四种结构蛋白、复制酶(ORF1a/b)与若干辅助蛋白,部分病毒还具有血细胞凝集素酯酶(HE),这些蛋白除维持病毒结构,还有促进感染与抵抗宿主免疫反应等功能,其中刺突蛋白可与宿主细胞表面的受体结合,使病毒包膜和宿主细胞的膜融合以感染细胞.冠状病毒的感染会影响细胞的许多信号转导途径,引发免疫反应,是一类可感染哺乳动物与鸟类的病毒.     

囊膜病毒膜融合的分子机制

囊膜病毒可能采用相似的病毒-宿主细胞膜融合机制,即病毒表面糖蛋白结合到宿主细胞受体后,启动了病毒融合蛋白的一系列构象变化,根据囊膜蛋白构象变化特征,囊膜病毒可采用两种以上的方式发生膜融合,并据此分为两类：Ⅰ型病毒膜融合和Ⅱ型病毒膜融合.Ⅱ型病毒膜融合以黄病毒为代表,其分子机制与Ⅰ型病毒膜融合不同,但不很清楚.而Ⅰ型病毒膜融合中,如艾滋病毒,流感病毒等,在囊膜蛋白变构形成稳定折叠的发夹三聚体结构时,拉近了两膜之间的距离,此过程释放出来的能量进一步促使两膜融合.膜融合使病毒蛋白及病毒RNA基因组释放到宿主细胞内而感染宿主.以上述研究为基础设计的C肽/N肽小分子抑制子, 可以在病毒糖蛋白中间体构象形成的短时间内,高效、特异地竞争结合其配体,从而阻止糖蛋白的进一步折叠,达到抑制病毒入侵的目的,为病毒疾病的防治提供了新思路和策略.针对艾滋病毒设计的C肽,即T20或Enfuvirtide在临床应用效果很好.以艾滋病毒和流感病毒为例,主要对Ⅰ型病毒膜融合的研究进展进行了讨论.     

干扰素诱导的跨膜蛋白抗病毒作用的研究进展

干扰素诱导的跨膜蛋白(Interferon-induced Transmembrane Proteins,IFITMs)是20世纪80年代发现的一种宿主限制因子蛋白,1996年发现该蛋白具有抗病毒作用,目前该蛋白的抗病毒作用及其作用机制已成为研究热点。研究表明IFITM能抑制多种病毒的复制,包括甲型流感病毒、人类免疫缺陷病毒-1、丙型肝炎病毒、埃博拉病毒和西尼罗病毒等。IFITM蛋白主要在病毒生命周期的早期,即病毒进入细胞质之前,发挥抑制病毒复制的作用。近来的研究表明,IFITM蛋白通过影响病毒包膜与内涵体膜的融合抑制病毒复制,但具体机制尚不明确。本文对IFITM的发现、结构、抗病毒作用以及潜在的作用机制进行了综述。     

Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review)

Enveloped animal viruses infect host cells by fusion of viral and target membranes. This crucial fusion event occurs either with the plasma membrane of the host cells at the physiological pH or with the endosomal membranes at low pH and is triggered by specific glycoproteins in the virus envelope. Both lipids and proteins play critical and co-operative roles in the fusion process. Interactions of viral proteins with their receptors direct which membranes fuse and viral fusion proteins then drive the process. These fusion proteins operate on lipid assemblies, whose physical and mechanical properties are equally important to the proper functioning of the process. Lipids contribute to the viral fusion process by virtue of their distinct chemical structure, composition and/or their preferred partitioning into specific microdomains in the plasma membrane called 'rafts'. An involvement of lipid rafts in viral entry and membrane fusion has been examined recently. However, the mechanism(s) by which lipids as dynamic raft components control viral envelope-glycoprotein-triggered fusion is not clear. This paper will review literature findings on the contribution of the two raft-associated lipids, cholesterol and sphingolipids in viral entry.     

Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review)

Enveloped animal viruses infect host cells by fusion of viral and target membranes. This crucial fusion event occurs either with the plasma membrane of the host cells at the physiological pH or with the endosomal membranes at low pH and is triggered by specific glycoproteins in the virus envelope. Both lipids and proteins play critical and co-operative roles in the fusion process. Interactions of viral proteins with their receptors direct which membranes fuse and viral fusion proteins then drive the process. These fusion proteins operate on lipid assemblies, whose physical and mechanical properties are equally important to the proper functioning of the process. Lipids contribute to the viral fusion process by virtue of their distinct chemical structure, composition and/or their preferred partitioning into specific microdomains in the plasma membrane called 'rafts'. An involvement of lipid rafts in viral entry and membrane fusion has been examined recently. However, the mechanism(s) by which lipids as dynamic raft components control viral envelope-glycoprotein-triggered fusion is not clear. This paper will review literature findings on the contribution of the two raft-associated lipids, cholesterol and sphingolipids in viral entry.     

Lipids as modulators of membrane fusion mediated by viral fusion proteins

Enveloped viruses infect host cells by fusion of viral and target membranes. This fusion event is triggered by specific glycoproteins in the viral envelope. Fusion glycoproteins belong to either class I, class II or the newly described third class, depending upon their arrangement at the surface of the virion, their tri-dimensional structure and the location within the protein of a short stretch of hydrophobic amino acids called the fusion peptide, which is able to induce the initial lipid destabilization at the onset of fusion. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or with the endosomal membranes for pH-dependent viruses. Although, viral fusion proteins are parted in three classes and the subcellular localization of fusion might vary, these proteins have to act, in common, on lipid assemblies. Lipids contribute to fusion through their physical, mechanical and/or chemical properties. Lipids can thus play a role as chemically defined entities, or through their preferential partitioning into membrane microdomains called "rafts", or by modulating the curvature of the membranes involved in the fusion process. The purpose of this review is to make a state of the art on recent findings on the contribution of cholesterol, sphingolipids and glycolipids in cell entry and membrane fusion of a number of viral families, whose members bear either class I or class II fusion proteins, or fusion proteins of the recently discovered third class.     

Membrane-containing viruses with icosahedrally symmetric capsids

Viruses with an icosahedrally symmetric protein capsid and a membrane infect hosts from all three domains of life. Similar architectural principles are shared by different viral families, as exemplified by double-stranded DNA viruses such as PRD1 and STIV. During virus assembly, the membrane lipids are selectively acquired from the host cell. The X-ray structure of bacteriophage PRD1 revealed that the lipids are asymmetrically distributed between the two leaflets and facet length is controlled by a tape-measure protein. In most membrane-containing viruses, viral and host membranes fuse during viral entry. In the best-understood systems of the alphaviruses, flaviviruses and herpes viruses, fusion is mediated by viral glycoproteins. Recent structural advances reveal how very different protein architectures can be used to form trimeric extensions that extend into the target cell membrane and then fold back to mediate fusion of the target and viral membranes.     

Stochastic entry of enveloped viruses: fusion versus endocytosis

Infection by membrane-enveloped viruses requires the binding of receptors on the target cell membrane to glycoproteins, or "spikes," on the viral membrane. The initial entry mechanism is usually classified as fusogenic or endocytotic. However, binding of viral spikes to cell surface receptors not only initiates the viral adhesion and the wrapping process necessary for internalization, but can simultaneously initiate direct fusion with the cell membrane. Both fusion and internalization have been observed to be viable pathways for many viruses. We develop a stochastic model for viral entry that incorporates a competition between receptor-mediated fusion and endocytosis. The relative probabilities of fusion and endocytosis of a virus particle initially nonspecifically adsorbed on the host cell membrane are computed as functions of receptor concentration, binding strength, and number of spikes. We find different parameter regimes where the entry pathway probabilities can be analytically expressed. Experimental tests of our mechanistic hypotheses are proposed and discussed.     

Membrane fusion of enveloped viruses: Especially a matter of proteins

To infect mammalian cells, enveloped viruses have to deposit their nucleocapsids into the cytoplasm of a host cell. Membrane fusion represents a key element in this entry mechanism. The fusion activity resides in specific, virally encoded membrane glycoproteins. Some molecular properties of these fusion proteins will be briefly described. These properties will then be correlated to the ability of a virus to fuse with target membranes, and to induce cell-cell fusion. Some molecular and physical parameters affecting virus fusion—at the level of either viral or target membrane or both—and the significance of modelling virus fusion by using synthetic peptides resembling viral fusion peptides, will also be discussed.     

Rabies virus-induced membrane fusion.

Rabies virus is a member of the rhabdovirus family. It enters cells by a process of receptor mediated endocytosis. Following this step, the viral envelope fuses with the endosomal membrane to allow release of the viral nucleocapsid into the cytoplasm. Fusion is induced by the low pH of the endosomal compartment and is mediated by the single viral glycoprotein G, a homotrimeric integral membrane protein. Rabies virus fusion properties are related to different conformational states of G. By different biochemical and biophysical approaches, it has been demonstrated that G can assume at least three different states: the native (N) state detected at the viral surface above pH 7, the activated (A) hydrophobic state which interacts with the target membrane as a first step of the fusion process, and the fusion inactive (I) conformation. Differently from other fusogenic viruses for which low pH-induced conformational changes are irreversible, there is a pH dependent equilibrium between these states, the equilibrium being shifted toward the I-state at low pH. The objective of this review is to detail recent findings on rhabdovirus-induced membrane fusion and to underline the differences that exist between this viral family and influenza virus which is the best known fusogenic virus. These differences have to be taken into consideration if one wants to have a global understanding of virus-induced membrane fusion.     

Mechanisms for generating cell membrane antigens that are recognized by cytotoxic T lymphocytes

Studies with many viruses have revealed that viral specific protein synthesis is an obligatory step in generating antigens on target cells for antiviral cytotoxic T lymphocytes. This has been most clearly demonstrated with DI particles, virions that are structurally complete but lack infectious RNA. Adsorption of such particles onto target cell membranes does not render these cells susceptible to lytic attack by antiviral effector cells, unless some viral protein synthesis transpires. However, some viruses, such as Sendai virus, circumvent the requirement for viral protein synthesis via fusion of the viral envelope with the target cell membrane, a process mediated by a specialized fusion protein. Once inserted into the lipid bilayer, it is likely that viral components and self H-2 noncovalently associate so that the complex can be recognized by antiviral cytotoxic T cells. This idea is supported by the demonstration that viral proteins and H-2 containing membrane proteins, incorporated into reconstituted membrane vesicles or liposomes are recognized by cytotoxic T cells. These data further show that native rather than altered viral and H-2 molecules are the moieties recognized. Associations between antigen and H-2 have been detected by a variety of techniques and in some cases are not random but selective; that is, viral antigens perferentially associate with some H-2 alleles and not others. In summary, these findings indicate that although viral antigens are present in the mature virions, these components are not recognized by antiviral killer cells until integrated into the plasma membrane. This may be achieved either through direct fusion of the viral envelope with the target cell or following viral protein synthesis and insertion of viral antigens into the plasma membrane.     

Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane.

Enveloped animal viruses, such as Semliki Forest virus (SFV), utilize a membrane fusion strategy to deposit their genome into the cytosol of the host cell. SFV enters cells through receptor-mediated endocytosis, fusion of the viral envelope occurring subsequently from within acidic endosomes. Fusion of SFV has been demonstrated before to be strictly dependent on the presence of cholesterol in the target membrane. Here, utilizing a variety of membrane fusion assays, including an on-line fluorescence assay involving pyrene-labeled virus, we demonstrate that low-pH-induced fusion of SFV with cholesterol-containing liposomal model membranes requires the presence of sphingomyelin or other sphingolipids in the target membrane. The minimal molecular characteristics essential for supporting SFV fusion are encompassed by a ceramide. The action of the sphingolipids is confined to the actual fusion event, cholesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. Complex formation of the sphingolipids with cholesterol is unlikely to be important for the induction of SFV--liposome fusion, as sphingolipids that do not interact appreciably with cholesterol, such as galactosylceramide, effectively support the process. The remarkably low levels of sphingomyelin required for half-maximal fusion (1-2 mole%) suggest that sphingolipids do not play a structural role in the SFV fusion process, but rather act as a cofactor, possibly activating the viral fusion protein in a specific manner.     

The Role of histidine residues in low-pH-mediated viral membrane fusion

A central event in the invasion of a host cell by an enveloped virus is the fusion of viral and cell membranes. For many viruses, membrane fusion is driven by specific viral surface proteins that undergo large-scale conformational rearrangements, triggered by exposure to low pH in the endosome upon internalization. Here, we present evidence suggesting that in both class I (helical hairpin proteins) and class II (beta-structure-rich proteins) pH-dependent fusion proteins the protonation of specific histidine residues triggers fusion via an analogous molecular mechanism. These histidines are located in the vicinity of positively charged residues in the prefusion conformation, and they subsequently form salt bridges with negatively charged residues in the postfusion conformation. The molecular surfaces involved in the corresponding structural rearrangements leading to fusion are highly conserved and thus might provide a suitable common target for the design of antivirals, which could be active against a diverse range of pathogenic viruses.