二十面体病毒衣壳自组装的热力学参数

球状病毒衣壳采用二十面体对称。本文就二十面体病毒蛋白质骨架的自组装提出一个统计热力学模型。统计结果表明病毒衣壳二十面体对称不是自由能最小化的结果，而是衣壳内部蛋白结构参数最优化的结果。     

A型流感病毒基质蛋白M1的二聚化机制

流感病毒基质蛋白1(matrix protein 1,M1)位于病毒包膜之下,形成一层壳状结构(衣壳),可与病毒的血凝素、神经氨酸酶、包膜和病毒遗传物质发生相互作用,在病毒的组装与复制过程中起着关键作用.不过,除N端有晶体结构外,全长M1蛋白的结构尚未解析.因此,人们对全长M1蛋白如何二聚化、然后多聚化形成病毒衣壳的过程知之甚少.为了解M1蛋白的二聚化机制,首先从M1的N端片段晶体结构出发,用蛋白质结构预测方法获得M1的全长结构模型;其次,对可能的M1二聚体进行分子动力学模拟,分析其二聚化界面的氨基酸,由此发现了一种通过M1蛋白C端片段结合而形成的二聚体;最后,为验证模拟结果,用电镜三维重构方法分析了全长M1二聚体的构象,并提出了M1多聚化的机理模型.     

虎纹蛙病毒的形态结构及其致病性研究

在透射电镜下观察了病毒感染细胞超薄切片中的病毒 ,发现细胞内病毒为二十面体对称结构 ,由衣壳、核心体和其间的非电子致密层构成 ,大小分别为 (12 5± 7)nm (N =2 5 ) (角对角 )、 (84± 7)nm (N =17)和13nm ,在感染细胞质中形成病毒发生基质 ,成熟病毒粒子在胞质中可积聚呈晶格状排列 ;病毒自细胞出芽释放时获得源于细胞的囊膜。回归感染证实虎纹蛙病毒的致病性 ,该病毒对虎纹蛙幼体和幼蛙敏感 ,浸泡和肌注感染死亡率为 10 0 % ;病毒可感染成蛙 ,但并不致死 ;肝脏和肾脏是病毒的感染器官 ;培养细胞内和释放进入培养液中的病毒粒子对虎纹蛙均具有感染性 ,说明虎纹蛙病毒的囊膜不是感染所必需的 ,缺乏囊膜的核衣壳也具有感染性。     

利用质谱技术鉴定寨卡病毒非结构蛋白NS1的细胞内相互作用蛋白

寨卡病毒(Zika Virus)属于黄病毒科中的黄病毒属,虽然很早就已经被人类所发现,但是一直到2015年在南美巴西的大规模爆发,才引起了广泛的关注.寨卡病毒对人类的感染往往引起包括小头畸形和格林-巴利综合征在内的多种症状.寨卡病毒的基因组为单链正链RNA,其基因组可以编码翻译并剪切加工出3个结构蛋白,分别为膜蛋白,囊膜蛋白和核衣壳蛋白,以及7个非结构蛋白(NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).相关研究已经证明,NS1蛋白与同属黄病毒属的登革病毒的发病有紧密的联系,而且根据其蛋白结构推测其可能与寨卡病毒穿越血脑屏障有关.因此鉴别NS1与细胞内的相互作用蛋白对于发现寨卡病毒在细胞内的转运,转录,以及装配都有重大的意义.在此,该课题构建并在HEK293细胞中表达包含Flag和Strep两种标签的NS1融合蛋白,通过免疫沉淀的方法将与NS1结合的蛋白利用标签蛋白进行分离,利用高分辨生物质谱技术,对蛋白进行分析鉴定.通过分别带有Flag与Strep标签的相互作用蛋白分析,发现了16个两种标签共同的结合蛋白,其进一步的通路分析证明这些蛋白于与病毒转录、病毒复制和免疫反应多个通路有关,相关的研究结果为今后进一步研究寨卡病毒的复制机制以及开发抗病毒药物提供了重要的参考价值.     

犬呼肠病毒3型感染BHK-21细胞的超微结构观察

本实验采用免疫电镜负染、细胞超薄切片透射电镜观察 ,证实所分离的病毒为犬呼肠病毒。病毒粒子由致密的核心和包围着核心的双层衣壳组成 ,有的为空心衣壳 ,多呈晶格状排列 ;病毒在胞浆增殖 ,装配 ,通过细胞崩解释放 ;细胞浆内可见大量的微丝与微管结构 ,认为与病毒复制有关。胞浆与胞核中均可见到内容物不同 ,形态各异的包涵体     

猪圆环病毒2型衣壳蛋白的可溶性表达

利用原核表达系统构建猪圆环病毒2型病毒(PCV2)衣壳蛋白.首先构建具有谷胱甘肽转移酶标签的重组质粒p GEX4T1-ORF2,并将其转化至大肠杆菌BL21(DE3)中,表达并纯化得到可溶性的融合蛋白.进一步通过纳米粒度仪及免疫印迹等技术,分别研究了PCV2蛋白的粒径分布以及免疫原性.实验结果表明,BL21-p GEX4T1-ORF2菌株构建成功,能够可溶性地表达猪圆环病毒2型病毒衣壳蛋白,且蛋白具有免疫原性.     

SNF11蛋白的表达纯化

SWI/SNF复合体是由多个亚基组成的复合物在真核细胞中具有较高的保守性,能通过其自身ATP酶的活性调节染色质的结构,从而影响大量基因的表达,在人类神经系统的发育形成和功能发挥中起重要作用.SNF11是酵母SWI/SNF复合体中的一个亚基组分,能与核心亚基SWI2/SNF2相互作用.在本研究中,构建了p ET.32M.3C-SNF11的表达质粒,大量表达出SNF11的重组蛋白,通过Ni柱亲和层析和分子筛层析技术得到较纯的SNF11蛋白,并用分析型分子筛层析技术分析了蛋白的构象和聚集状况,为进一步研究SNF11蛋白与SWI/SNF复合体中其他亚基组分的相互作用提供了一定的基础.     

雄性特异性噬菌体

雄性特异性噬菌体可分为DNA型和RNA型两大类。DNA型噬菌体粒体为细长丝状,衣壳由管状疏水蛋白质和导向蛋白质构成。疏水蛋白属于α—螺旋结构。病毒衣壳包被着一个共价闭合的单股环状DNA分子,分子量约为1.5——2×10~6道尔顿。所有RNA噬菌体都是雄性特异性的,病毒粒体为正二十面体。RNA噬菌体只含三个基因,只能编码三个可识别的蛋白质。它们的基因组及其三种蛋白质的一级结构都已测定清楚。RNA基因组作为模板,既能控制复制,又可起mRNA的作用,以指导翻译,在发育过程中呈现出特有的独特性。     

人源CLC-7/Ostm1复合物结构揭示一种新构象（英文）

CLC-7是位于溶酶体膜上的Cl^-/H⁺交换转运蛋白。CLC-7及其β亚基Ostm1的缺陷均会导致骨硬化症和神经退行性疾病。本文解析了一个具有3.6?分辨率的人源CLC-7/Ostm1复合物的冷冻电镜结构。该结构揭示了CLC-7/Ostm1异源四聚体的一种新构象，其中CLC-7的胞质结构域可能由于高度柔性而缺失。柔性的细胞质结构域无法限制CLC-7的跨膜结构域，从而允许两个亚基相对远离，使得CLC-7跨膜结构域的二聚界面与已知具有稳定胞质结构域的结构相比减小了约一半。这种二聚界面的变化影响了多个骨硬化症相关残基的相互作用。     

含果蝇肌动蛋白基因的重组棉铃虫核型多角体病毒的构建

本实验利用昆虫杆状病毒表达载体，将果蝇肌动蛋白基因5Cactin克隆入杆状病毒表达载体，以棉铃虫单核衣壳核多角体病毒为亲本病毒，在脂质体介导下共转染棉铃虫细胞，空斑纯化得到重组病毒，以深入研究棉铃虫病毒在入侵、复制及装配过程中，宿主肌动蛋白的动态变化对病毒复制的作用与影响。     

Comparative analysis of the three-dimensional structure of Periplaneta fuliginosa densovirus

The three-dimensional structure of Periplaneta fuliginosa densovirus(pfDNV)is determined at 2.3nm resolution using the techniques of cryo-electron microscopy and image reconstruction.The pfDNV contains five structural proteins and 60 protein subunits arranged on a T=1 icosahedral shell with a relatively smooth surface.Its reconstruction reveals its distinct capsid structure from those observed in CPV and GmDNV.As in GmDNV,spike-like protrusions are not present in pfDNV at the threefold axes; while two small thorn-like protrusions are identified there However,different from CPV and GmDNV,cylindrical channels along the fivefold axes are closed in pfDNV;while a small thorn-like protrusions,which have not been reported in other parvovirus,are observed there in pfDNV although their function is yet to be investigated.The pfDNV has dimple-like depressions at the icosahedral twofold axes;but has no canyon-like regions encircling the fivefold axes.The icosahedrally well-ordered nucleic acid has also been observed in pfDNV,suggesting that the protein and nucleic acid probably form closed interaction.     

Three-dimensional structure of the wild-type RHDV

The three-dimensional (3D) structure of the wild-type rabbit hemorrhagic disease virus (RHDV) has been determined to a resolution of 3.2 nm by electron cryomicroscopy and computer image reconstruction techniques. The 3D density map exhibits characteristic structural features of a calicivirus: a T=3 icosahedral capsid with 90 arch-like capsomeres at the icosahedral and local 2-fold axes and 32 large surface hollows at the icosahedral 5- and 3-fold axes. This result confirms that the RHDV isolated in China is a member of the Caliciviridae family. A rather continuous capsid shell was found without channels. However, our RHDV structure also reveals some distinct structural characteristics not observed in other caliciviruses, including interconnected capsomeres and the lack of protuberance on the base of each of the surface hollows. Two types of particles were identified with similar outer capsid structure but different density distributions inside the capsid shells, which could not be distinguished by conventional negative staining electron microscopy. As the genomic and subgenomic RNAs are both packaged into particles for RHDV, we suggest that the two types of particles identified correspond to those containing either the genomic or subgenomic RNAs, respectively.     

Site-directed mutation affecting polyomavirus capsid self-assembly in vitro

Nonequivalent bonding of identical protein subunits occurs in the polyomavirus capsid were identical pentameric capsomeres occupy both hexavalent and pentavalent positions in the icosahedral surface lattice. The polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, has been isolated as capsomeres that self-assemble into capsid-like structures in vitro. The ability to switch bonding specificity in different symmetry environments therefore must be intrinsic to the VP1 molecule. In vitro self-assembly provides an assay for VP1 mutations affecting capsomere and capsid formation. We report here that a directed mutation in the VP1 expression vector, leading to a protein truncated at the carboxy terminus, results in a mutant VP1 that forms capsomeres, but not capsids, in the in vitro assembly assay. The carboxy terminus of VP1 therefore appears to be involved in the specific bonding responsible for the non-equivalent association of capsomeres.     

Crystal structure of the tricorn protease reveals a protein disassembly line.

The degradation of cytosolic proteins is carried out predominantly by the proteasome, which generates peptides of 7-9 amino acids long. These products need further processing. Recently, a proteolytic system was identified in the model organism Thermoplasma acidophilum that performs this processing. The hexameric core protein of this modular system, referred to as tricorn protease, is a 720K protease that is able to assemble further into a giant icosahedral capsid, as determined by electron microscopy. Here, we present the crystal structure of the tricorn protease at 2.0 A resolution. The structure reveals a complex mosaic protein whereby five domains combine to form one of six subunits, which further assemble to form the 3-2-symmetric core protein. The structure shows how the individual domains coordinate the specific steps of substrate processing, including channelling of the substrate to, and the product from, the catalytic site. Moreover, the structure shows how accessory protein components might contribute to an even more complex protein machinery that efficiently collects the tricorn-released products.     

Insights into assembly from structural analysis of bacteriophage PRD1

The structure of the membrane-containing bacteriophage PRD1 has been determined by X-ray crystallography at about 4 A resolution. Here we describe the structure and location of proteins P3, P16, P30 and P31. Different structural proteins seem to have specialist roles in controlling virus assembly. The linearly extended P30 appears to nucleate the formation of the icosahedral facets (composed of trimers of the major capsid protein, P3) and acts as a molecular tape-measure, defining the size of the virus and cementing the facets together. Pentamers of P31 form the vertex base, interlocking with subunits of P3 and interacting with the membrane protein P16. The architectural similarities with adenovirus and one of the largest known virus particles PBCV-1 support the notion that the mechanism of assembly of PRD1 is scaleable and applies across the major viral lineage formed by these viruses.     

Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy

A half-century after the determination of the first three-dimensional crystal structure of a protein, more than 40,000 structures ranging from single polypeptides to large assemblies have been reported. The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-A resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (epsilon15) particle, by single-particle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity. However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond epsilon15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.     

人类乙型肝炎样病毒核心蛋白中第7位疏水性氨基酸重复肚段区域的突变可以抑制病毒核衣壳的组装

人类乙型肝炎病毒的核衣壳由核心蛋白的二聚体所组成．但是,核心蛋白亚单位与亚单位之间相互作用的机制至今尚不清楚．研究发现,在人类乙型肝炎样病毒──土拨鼠肝炎病毒（WHV）核心蛋白的氨基端,存在着4个保守的疏水氨基酸残基（氨基酸位置101～102）．它们分别是亮氨酸101,亮氨酸108,缬氨酸115和苯丙氨酸122．这4个疏水氨基酸残基以每隔6个氨基酸残基而重复出现1次．它们被称为“第7位疏水性氨基酸重复肽段（hhr）”．由于蛋白质中的疏水键往往在蛋白质的相互作用中起重要作用,因此就在培养细胞系统中研究WHV核心蛋白的hhr区域在…     

Atomic structure of single-stranded DNA bacteriophage phi X174 and its functional implications.

The mechanism of DNA ejection, viral assembly and evolution are related to the structure of bacteriophage phi X174. The F protein forms a T = 1 capsid whose major folding motif is the eight-stranded antiparallel beta barrel found in many other icosahedral viruses. Groups of 5 G proteins form 12 dominating spikes that enclose a hydrophilic channel containing some diffuse electron density. Each G protein is a tight beta barrel with its strands running radially outwards and with a topology similar to that of the F protein. The 12 'pilot' H proteins per virion may be partially located in the putative ion channel. The small, basic J protein is associated with the DNA and is situated in an interior cleft of the F protein. Tentatively, there are three regions of partially ordered DNA structure,     

Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus

The critical viral components for packaging DNA, recognizing and binding to host cells, and injecting the condensed DNA into the host are organized at a single vertex of many icosahedral viruses. These component structures do not share icosahedral symmetry and cannot be resolved using a conventional icosahedral averaging method. Here we report the structure of the entire infectious Salmonella bacteriophage epsilon15 (ref. 1) determined from single-particle cryo-electron microscopy, without icosahedral averaging. This structure displays not only the icosahedral shell of 60 hexamers and 11 pentamers, but also the non-icosahedral components at one pentameric vertex. The densities at this vertex can be identified as the 12-subunit portal complex sandwiched between an internal cylindrical core and an external tail hub connecting to six projecting trimeric tailspikes. The viral genome is packed as coaxial coils in at least three outer layers with approximately 90 terminal nucleotides extending through the protein core and the portal complex and poised for injection. The shell protein from icosahedral reconstruction at higher resolution exhibits a similar fold to that of other double-stranded DNA viruses including herpesvirus, suggesting a common ancestor among these diverse viruses. The image reconstruction approach should be applicable to studying other biological nanomachines with components of mixed symmetries.