From the Cover: Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein

The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.     

Structure and function of the complete internal fusion loop from Ebolavirus glycoprotein 2

Ebolavirus (Ebov), an enveloped virus of the family Filoviridae, causes hemorrhagic fever in humans and nonhuman primates. The viral glycoprotein (GP) is solely responsible for virus-host membrane fusion, but how it does so remains elusive. Fusion occurs after virions reach an endosomal compartment where GP is proteolytically primed by cathepsins. Fusion by primed GP is governed by an internal fusion loop found in GP2, the fusion subunit. This fusion loop contains a stretch of hydrophobic residues, some of which have been shown to be critical for GP-mediated infection. Here we present liposome fusion data and NMR structures for a complete (54-residue) disulfide-bonded internal fusion loop (Ebov FL) in a membrane mimetic. The Ebov FL induced rapid fusion of liposomes of varying compositions at pH values at or below 5.5. Consistently, circular dichroism experiments indicated that the α-helical content of the Ebov FL in the presence of either lipid-mimetic micelles or small liposomes increases in samples exposed to pH ≤5.5. NMR structures in dodecylphosphocholine micelles at pH 7.0 and 5.5 revealed a conformational change from a relatively flat extended loop structure at pH 7.0 to a structure with an ～90° bend at pH 5.5. Induction of the bend at low pH reorients and compacts the hydrophobic patch at the tip of the FL. We propose that these changes facilitate disruption of lipids at the site of virus-host cell membrane contact and, hence, initiate Ebov fusion.     

Structure of a core fragment of glycoprotein H from pseudorabies virus in complex with antibody

Compared with many well-studied enveloped viruses, herpesviruses use a more sophisticated molecular machinery to induce fusion of viral and cellular membranes during cell invasion. This essential function is carried out by glycoprotein B (gB), a class III viral fusion protein, together with the heterodimer of glycoproteins H and L (gH/gL). In pseudorabies virus (PrV), a porcine herpesvirus, it was shown that gH/gL can be substituted by a chimeric fusion protein gDgH, containing the receptor binding domain (RBD) of glycoprotein D fused to a truncated version of gH lacking its N-terminal domain. We report here the 2.1-Å resolution structure of the core fragment of gH present in this chimera, bound to the Fab fragment of a PrV gH-specific monoclonal antibody. The structure strongly complements the information derived from the recently reported structure of gH/gL from herpes simplex virus type 2 (HSV-2). Together with the structure of Epstein-Barr virus (EBV) gH/gL reported in parallel, it provides insight into potentially functional conserved structural features. One feature is the presence of a syntaxin motif, and the other is an extended “flap” masking a conserved hydrophobic patch in the C-terminal domain, which is closest to the viral membrane. The negative electrostatic surface potential of this domain suggests repulsive interactions with the lipid heads. The structure indicates the possible unmasking of an extended hydrophobic patch by movement of the flap during a receptor-triggered conformational change of gH, exposing a hydrophobic surface to interact with the viral membrane during the fusion process.     

猪源新城疫病毒F蛋白裂解位点对BHK-21细胞特异性膜融合的影响

目的研究猪源新城疫病毒F蛋白裂解位点区氨基酸序列对其特异性膜融合作用的影响。方法采用基因定点突变及基因重组的方法,测定突变猪源新城疫病毒JL01株F蛋白裂解位点区基因序列,并将阳性突变质粒与猪源新城疫病毒HN基因共转染BHK-21细胞,通过面积判断法和细胞计数法分析细胞融合效率。结果突变株RF4细胞融合效率为0.58±0.10,与野毒株的0.55±0.02接近。RF5融合效率较高,为0.69±0.05;RF3融合效率较低,为0.25±0.02。结论猪源新城疫病毒F蛋白裂解位点的改变对其特异性膜融合作用有一定影响。     

Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids

Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze the structural and mechanical properties of scaffold-containing (B), empty (A), and DNA-containing (C) nuclear capsids. Atomic force microscopy experiments revealed that A and C capsids were mechanically indistinguishable, indicating that the presence of DNA does not account for changes in mechanical properties during capsid maturation. Despite having the same rigidity, the scaffold-containing B capsids broke at significantly lower forces than A and C capsids. An extraction of pentons with guanidine hydrochloride (GuHCl) increased the flexibility of all capsids. Surprisingly, the breaking forces of the modified A and C capsids dropped to similar values as those of the GuHCl-treated B capsids, indicating that mechanical reinforcement occurs at the vertices. Nonetheless, it also showed that HSV1 capsids possess a remarkable structural integrity that was preserved after removal of pentons. We suggest that HSV1 capsids are stabilized after removal of the scaffold proteins, and that this stabilization is triggered by the packaging of DNA, but independent of the actual presence of DNA.     

Conformational thermostabilization of the beta1-adrenergic receptor in a detergent-resistant form

There are approximately 350 non-odorant G protein-coupled receptors (GPCRs) encoded by the human genome, many of which are predicted to be potential therapeutic targets, but there are only two structures available to represent the whole of the family. We hypothesized that improving the detergent stability of these receptors and simultaneously locking them into one preferred conformation will greatly improve the chances of crystallization. We developed a generic strategy for the isolation of detergent-solubilized thermostable mutants of a GPCR, the beta1-adrenergic receptor. The most stable mutant receptor, betaAR-m23, contained six point mutations that led to an apparent T(m) 21 degrees C higher than the native protein, and, in the presence of bound antagonist, betaAR-m23 was as stable as bovine rhodopsin. In addition, betaAR-m23 was significantly more stable in a wide range of detergents ideal for crystallization and was preferentially in an antagonist conformation in the absence of ligand.     

Myelin-associated glycoprotein mediates membrane fusion and entry of neurotropic herpesviruses

Varicella-zoster virus (VZV) and herpes simplex virus (HSV) are prevalent neurotropic herpesviruses that cause various nervous system diseases. Similar to other enveloped viruses, membrane fusion is an essential process for viral entry. Therefore, identification of host molecules that mediate membrane fusion is important to understand the mechanism of viral infection. Here, we demonstrate that myelin-associated glycoprotein (MAG), mainly distributed in neural tissues, associates with VZV glycoprotein B (gB) and promotes cell-cell fusion when coexpressed with VZV gB and gH/gL. VZV preferentially infected MAG-transfected oligodendroglial cells. MAG also associated with HSV-1 gB and enhanced HSV-1 infection of promyelocytes. These findings suggested that MAG is involved in VZV and HSV infection of neural tissues.     

丙型肝炎病毒F蛋白反式激活蛋白2基因在酵母细胞中的表达

目的为探讨丙型肝炎病毒(HCV)F蛋白反式激活蛋白2(HCV FTP2)的功能,在真核生物酵母细胞中表达HCV FTP2基因.方法以HepG2细胞来源的mRNA作为模板,经过逆转录聚合酶链反应(RT-PCR)扩增HCV FFP2基因,克隆到pGEM-T载体中,双酶切后回收连接到酵母表达质粒pGBKT7中表达.提取酵母蛋白质,进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blot免疫印迹分析.结果成功构建HCV FTP2基因酵母表达载体,Western blot免疫印迹显示HCV FTP2基因在酵母细胞中表达成功.表达产物相对分子质量27kD.结论HCV FTP2在酵母中表达成功.     

丙型肝炎病毒F蛋白抗原性及患者血清F抗体流行率的研究

目的探讨丙型肝炎病毒(HCV)F蛋白的抗原性及患者血清F抗体的流行率。方法利用11条引物延伸获处HCV f基因,以此为模板经聚合酶链反应扩增得到截短型f65基因片段,定向克隆至pET32a(+),重组子转化大肠杆菌Plyss菌株,异丙基β半乳糖苷诱导后用镍离子树脂纯化HCV F65蛋白。以F65蛋白为抗原,酶联免疫吸附法检测HCV感染者血清中F抗体。应用F65蛋白免疫新西兰大白兔制备多克降F抗体,再用葡萄球菌A蛋白树脂纯化兔血清中F抗体。结果成功构建了pET32a(+)f65重组子,表达并纯化了分子量为3．2×10~4的HCV F65蛋白。30份HCV患者血清F抗体的A_(450)为0．125+0．061．F抗体阳性率为63．3%。获得了兔源性F抗体,该抗体与HCV F65蛋白发生特异性结合反应,滴度为1：30000。结论HCV F65蛋白具有抗原性,可用来检测血清中F抗体。HCV患者血清中存在F抗体。兔源性F抗体可用来检测HCV F蛋白。     

抑制性消减杂交技术克隆和筛选丙型肝炎病毒F蛋白的反式调节基因

目的 构建丙型肝炎病毒（HCV）F蛋白反式激活相关基因差异表达的差异cDNA，克隆HCV-F蛋白反式激活相关基因。方法 以HCV-F表达质粒pcDNA3．1（-）-F转染HepG2细胞，以空载体pcDNA3．1（-）为对照；制备转染后的细胞裂解液，从中提取mRNA并合成cDNA，经RsaI酶切后将实验组cDNA分成两组，分别与两种不同的接头衔接，再与对照组cDNA进行两次消减杂交及两次抑制性聚合酶链反应，将产物与T／A载体连接，构建cDNA消减文库，并转染大肠杆菌进行文库扩增，随机挑选克隆聚合酶链反应后进行测序及同源性分析。结果 成功构建人HcVF蛋白反式激活相关基因差异表达的cDNA。扩增后得到56个200～1000bP插入片段的克隆，随机挑选其中28个插入片段测序，并通过生物信息学分析获得其全长基因序列，结果共获得19种编码基因，其中2个为未知功能的新基因。结论 筛选到的cDNA全长序列，包括一些与细胞生长调节、物质代谢和细胞凋亡密切相关的蛋白编码基因。     

Screening of hepatocyte proteins binding to F protein of hepatitis C virus by yeast two-hybrid system

AIM: To investigate the biological function of F protein by yeast two-hybrid system. METHODS: We constructed F protein bait plasmid by cloning the gene of F protein into pGBKT7, then recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH 109 was mated with yeast Y187 (a type) containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics. RESULTS: Thirty-six colonies were selected and sequenced. Among them, 11 colonies were zymogen granule protein, 5 colonies were zinc finger protein, 4 colonies were zinc-α-2-glycoprotein, 1 colony was sialyltransferase, 1 colony was complement control protein factor I, 1 colony was vitronectin, and 2 colonies were new genes with unknown function. CONCLUSION: The yeast two-hybrid system is an effective method for identifying hepatocyte proteins interacting with F protein of hepatitis C virus. F protein may bind to different proteins.     

Substrate-induced changes in the structural properties of LacY

The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H⁺ across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugar- and H⁺-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H⁺- and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H⁺ symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154➝Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154➝Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.The lactose permease of Escherichia coli (LacY) of the major facilitator superfamily (MFS) (1, 2) catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H⁺ (sugar/H⁺ symport) (3–6). Uphill (i.e., active) symport of galactoside against a concentration gradient is achieved by transduction of free energy released from the downhill movement of H⁺ with the electrochemical H⁺ gradient (Δ$\tilde{\mu }$_H⁺; interior negative and/or alkaline). Conversely, because coupling between sugar and H⁺ is obligatory, downhill galactoside transport from a high to a low sugar concentration is coupled to uphill H⁺ transport with the generation of Δ$\tilde{\mu }$_H+, the polarity of which depends upon the direction of the sugar concentration gradient (7–10).LacY monomers reconstituted into proteoliposomes are functional (11, 12), and X-ray crystal structures reveal 12, mostly irregular, transmembrane α-helices organized into two pseudosymmetrical 6-helix bundles surrounding a large interior hydrophilic cavity open to the cytoplasm (13–16). At the apex of the hydrophilic cavity, which is at the approximate middle of the molecule, the galactoside- and H⁺-binding sites are located. Side chains important for sugar recognition are located in both the N- and the C-terminal 6-helix bundles, whereas those involved in H⁺ binding are largely in the C-terminal 6-helix bundle. Most X-ray structures obtained thus far exhibit a tightly sealed periplasmic side with the sugar-binding site at the apex of the cavity and inaccessible from the periplasm and an open cytoplasmic side (an inward-facing conformation). LacY is structurally highly dynamic, and binding of a galactoside closes the deep inward-facing cavity with opening of a complementary outward-facing cavity (reviewed in refs. 17, 18). Therefore, transport involves a large conformational change that allows alternating access of sugar- and H⁺-binding sites to either side of the cellular membrane, and a recent structure indicates that an occluded intermediate is involved (19). Although structural models of LacY provide insight into the conformational states involved in transport, a crystal structure represents a static structural snapshot, and therefore an understanding of how sugar binding triggers the cascade of events that results in dynamic alternating access remains unclear. Furthermore, because these interactions alter the physical properties of LacY (reviewed in ref. 9), the energetic, kinetic, and mechanical properties of LacY that fulfill different functional roles during transport remain to be characterized.Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been applied to localize and quantify interactions that stabilize structural elements of an increasing number of native membrane proteins (20–25). Because SMFS can be used with membrane proteins embedded in native or synthetic lipid membranes under physiological conditions, the method has been used to assess interactions that change upon substrate binding, insertion of mutations, and assembly or lipid composition of the membrane (26–35). Moreover, when operated in the dynamic mode, dynamic single-molecule force spectroscopy (DFS) localizes and quantifies the kinetic, energetic, and mechanical properties of the structural elements in a membrane protein in a physiologically relevant environment (20, 21).LacY binds galactopyranosides, and 4-nitrophenyl-α-d-galactopyranoside (αNPG) is among the lactose analogs with highest affinity (∼30 µM) (36). In the absence of substrate, LacY preferentially occupies an inward-facing open conformation, and substrate binding causes closing of the inward-facing cavity with opening of a reciprocal outward-facing cavity (reviewed in refs. 17, 18) with an occluded intermediate conformation (19). To understand the structural perturbations and properties associated with these conformations, we describe here the conformational, kinetic, energetic, and mechanical properties of LacY in the apo state and how these properties change upon substrate binding. SMFS and DFS are used to characterize the properties of individual structural segments of LacY and to describe how these regions change properties upon galactoside binding. To understand further how a single point mutation alters LacY, the conformationally restricted LacY mutant C154G (37), which crystallized originally (13), was also investigated. All measurements were conducted with wild-type (WT) or mutant C154G LacY embedded in a phospholipid membrane under physiological conditions. The findings quantify the structural properties of WT LacY, which change drastically upon sugar binding. In contrast, the structural properties of mutant C154G LacY remain largely unaffected by ligand binding.     

乙型脑炎病毒膜蛋白B区多肽抗原的表达、纯化及血清学评价

目的在原核表达系统中对乙型脑炎病毒包膜糖蛋白(E蛋白)的B区多肽抗原进行高效表达、纯化及血清学评价。方法利用PCR技术从乙脑减毒活疫苗中扩增编码B区的DNA片段,酶切消化后连接到表达载体pET22b上,用此连接产物转化大肠埃希菌BL21(DE3)并表达B区多肽抗原,表达产物经液相层析纯化;用乙脑病人血清对纯化后的B区多肽抗原进行评价。结果重组质粒pET22b-JEB经双酶切,其插入的外源基因片段为372 bp,与预期DNA片段大小一致。将纯化前与纯化后的蛋白作SDS-PAGE电泳,均可见一条约16kD的外源基因蛋白带,与计算的相对分子质量相符。经WB和ELISA血清学评价,表明重组B区多肽抗原具有较高的灵敏性及特异性。结论成功构建了表达载体pET22b-JEB,在原核系统中表达的B区多肽抗原具有良好的血清学检测价值。     

X-ray structure of the arenavirus glycoprotein GP2 in its postfusion hairpin conformation

Arenaviruses are important agents of zoonotic disease worldwide. The virions expose a tripartite envelope glycoprotein complex at their surface, formed by the glycoprotein subunits GP1, GP2 and the stable signal peptide. This complex is responsible for binding to target cells and for the subsequent fusion of viral and host-cell membranes for entry. During this process, the acidic environment of the endosome triggers a fusogenic conformational change in the transmembrane GP2 subunit of the complex. We report here the crystal structure of the recombinant GP2 ectodomain of the lymphocytic choriomeningitis virus, the arenavirus type species, at 1.8-Å resolution. The structure shows the characteristic trimeric coiled coil present in class I viral fusion proteins, with a central stutter that allows a close structural alignment with most of the available structures of class I and III viral fusion proteins. The structure further shows a number of intrachain salt bridges stabilizing the postfusion hairpin conformation, one of which involves an aspartic acid that appears released from a critical interaction with the stable signal peptide upon low pH activation.     

Hepatitis C virus F protein inhibits cell apoptosis by activation of intracellular NF-κB pathway

Aim:  To observe the influence of HCV F protein on apoptosis of HepG2 cells, and explore the association between F protein and NF-κB signal pathway.
Methods:  HCV 1b F gene containing HepG2-F cells and HCV 1b C gene containing HepG2-C cells were treated with 100 IU/mL TNF-α, and analyzed by flow cytometry, Western blotting, and dual luciferase reporter assay. Empty plasmid pcDNA3.1⁺ containing HepG2-3.1 cells were used as control.
Results:  (i) With the treatment of TNF-α for 18 h, the apoptosis rates (AR) of HepG2-F and HepG2-3.1 cells were 0.41% (± 0.11%) and 37.43% (± 2.03%) respectively, while that of HepG2-C was 4.07% (± 0.18%). At 36 h after TNF-α treatment, the AR of HepG2-F and HepG2-3.1 cells were 10.03% (± 0.41%) and 44.63% (± 3.37%), and that of HepG2-C was 14.95% (± 0.85%). (ii) After the treatment of TNF-α for 0.5–18 h, the p65 contents in the whole cells of HepG2-F and HepG2-3.1 showed no significant difference ( P  = 0.34, t  = 1.08), while the p65 contents in the nucleus of HepG2-F and HepG2-3.1 cells were 3.8–1.9 times and 1.8–1.0 times higher than that in the non-treated cells ( P  = 0.013, t  = 4.25). (iii) The relative luciferase unit (RLU) of the HepG2 cells, co-transfected with pcDNA3.1-F and pNF-κB-luc, and then treated with TNF-α (100 IU/mL) for 18 h, showed a pcDNA3.1-F dose-dependent increase.
Conclusion:  HCV F protein can over-activate NF-κB signal pathway, which makes HepG2-F cells able to resist TNF-α induced apoptosis.     

Influenza virus-mediated membrane fusion: determinants of hemagglutinin fusogenic activity and experimental approaches for assessing virus fusion

Hemagglutinin (HA) is the viral protein that facilitates the entry of influenza viruses into host cells. This protein controls two critical aspects of entry: virus binding and membrane fusion. In order for HA to carry out these functions, it must first undergo a priming step, proteolytic cleavage, which renders it fusion competent. Membrane fusion commences from inside the endosome after a drop in lumenal pH and an ensuing conformational change in HA that leads to the hemifusion of the outer membrane leaflets of the virus and endosome, the formation of a stalk between them, followed by pore formation. Thus, the fusion machinery is an excellent target for antiviral compounds, especially those that target the conserved stem region of the protein. However, traditional ensemble fusion assays provide a somewhat limited ability to directly quantify fusion partly due to the inherent averaging of individual fusion events resulting from experimental constraints. Inspired by the gains achieved by single molecule experiments and analysis of stochastic events, recently-developed individual virion imaging techniques and analysis of single fusion events has provided critical information about individual virion behavior, discriminated intermediate fusion steps within a single virion, and allowed the study of the overall population dynamics without the loss of discrete, individual information. In this article, we first start by reviewing the determinants of HA fusogenic activity and the viral entry process, highlight some open questions, and then describe the experimental approaches for assaying fusion that will be useful in developing the most effective therapies in the future.     

Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus

The influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs amantadine and rimantadine, whose use has been discontinued due to widespread drug resistance. Among the handful of drug-resistant mutants, S31N is found in more than 95% of the currently circulating viruses and shows greatly decreased inhibition by amantadine. The discovery of inhibitors of S31N has been hampered by the limited size, polarity, and dynamic nature of its amantadine-binding site. Nevertheless, we have discovered small-molecule drugs that inhibit S31N with potencies greater than amantadine’s potency against WT M2. Drug binding locks the protein into a well-defined conformation, and the NMR structure of the complex shows the drug bound in the homotetrameric channel, threaded between the side chains of Asn31. Unrestrained molecular dynamics simulations predicted the same binding site. This S31N inhibitor, like other potent M2 inhibitors, contains a charged ammonium group. The ammonium binds as a hydrate to one of three sites aligned along the central cavity that appear to be hotspots for inhibition. These sites might stabilize hydronium-like species formed as protons diffuse through the outer channel to the proton-shuttling residue His37 near the cytoplasmic end of the channel.     

戊型肝炎病毒衣壳蛋白及其中和表位的结构研究进展

戊型肝炎是由戊型肝炎病毒(Hepatitis E virus,HEV)引起的病毒性肝炎,被证实为人兽共患病,该病症对免疫力低下人群和已有基础性肝病的患者的危害更为显著。戊肝病毒主要通过粪口途径传播,猪是其主要的天然宿主。近年来,关于戊肝病毒的衣壳蛋白及其表位结构的研究取得了重要的进展,对于戊肝病毒的病毒学研究和戊肝疫苗的机制探讨起着重要的指导意义。本文将综述HEV病毒衣壳蛋白的结构研究和中和表位的结构基础,着重说明戊肝病毒中和表位存在型特异性和型交叉两种特征,提示型交叉表位与人兽共患现象的关系。     

Structure of the archaeal head-tailed virus HSTV-1 completes the HK97 fold story

It has been proposed that viruses can be divided into a small number of structure-based viral lineages. One of these lineages is exemplified by bacterial virus Hong Kong 97 (HK97), which represents the head-tailed dsDNA bacteriophages. Seemingly similar viruses also infect archaea. Here we demonstrate using genomic analysis, electron cryomicroscopy, and image reconstruction that the major coat protein fold of newly isolated archaeal Haloarcula sinaiiensis tailed virus 1 has the canonical coat protein fold of HK97. Although it has been anticipated previously, this is physical evidence that bacterial and archaeal head-tailed viruses share a common architectural principle. The HK97-like fold has previously been recognized also in herpesviruses, and this study expands the HK97-like lineage to viruses from all three domains of life. This is only the second established lineage to include archaeal, bacterial, and eukaryotic viruses. Thus, our findings support the hypothesis that the last common universal ancestor of cellular organisms was infected by a number of different viruses.     

Structure of the transmembrane regions of a bacterial cyclic nucleotide-regulated channel

The six-transmembrane helix (6 TM) tetrameric cation channels form the largest ion channel family, some members of which are voltage-gated and others are not. There are no reported channel structures to match the wealth of functional data on the non-voltage-gated members. We determined the structure of the transmembrane regions of the bacterial cyclic nucleotide-regulated channel MlotiK1, a non-voltage-gated 6 TM channel. The structure showed how the S1-S4 domain and its associated linker can serve as a clamp to constrain the gate of the pore and possibly function in concert with ligand-binding domains to regulate the opening of the pore. The structure also led us to hypothesize a new mechanism by which motions of the S6 inner helices can gate the ion conduction pathway at a position along the pore closer to the selectivity filter than the canonical helix bundle crossing.