CRYSTALLOGRAPHIC STUDIES OF PLEOMORPHIC VIRUSES

许多重要的病毒和有包膜动物的核蛋白核心 病毒（如逆转录病毒）是不稳定的和多形性的， 管理其结构一般是未知的，由于缺乏 晶体学研究 晶体学最成功的是 由于易于制备均匀制剂， 并且这些可能形成良好有序的晶体。 颗粒稳定 主要通过蛋白质-RNA相互作用，当暴露于 甚至是温和的物理或化学变性剂。 原因很简单 这种类型的病毒通常最容易进行组装研究 因为当病毒体被破坏时， 拆卸。 豇豆褪绿斑驳病毒（CCMV）是这方面的典型 课 这是第一个在体外重组形成的二十面体病毒 传染性颗粒和关于CCMV和病毒组装的文献 有关苜蓿花叶病毒（ALMV）的研究论文超过100篇， 评论. 在美国国家科学基金会的资助下，人们发现CCMV 颗粒对超离心高度敏感，这导致 开发新的制备程序和生产 第一个高质量的CCMV晶体。 CCMV是一个超级 一组被称为三角病毒的病毒。 所有成员的衣壳 这一群体相对不稳定，但有一个明显的趋势， 从CCMV（T=3）到CCMV（T=3），蛋白质-蛋白质相互作用的重要性降低 黄瓜花叶病毒（CMV）转化为ALMV，ALMV形成杆状颗粒 在体内从亚基六聚体和五聚体到烟草条纹病毒 (TSV)，其在体内与亚基形成球状颗粒， 从RNA中释放出来时不会组装。 先前的研究表明， 这个超级群中所有亚基的结构都可能是β- 桶 该提案的长期目标是解决和解决4 不同的亚基结构，并确定氨基酸决定簇 衣壳稳定性和形态学。 为此，CCMV的结构 在3.2埃分辨率下解析。 180个标准病毒β桶 （190 AA）形成五聚体和真正的六聚体，通过 交织C末端多肽以形成新型T=3颗粒。 这种形态单位的组合与 DNA肿瘤病毒SV 40的四级结构。 CCMV的结构 将在3.2埃，黄瓜花叶的T=3结构细化 病毒将从目前的5.5埃分辨率扩展到 至少4.0埃分辨率; T=1重组的结构 ALMV亚基的产物将在4.0埃分辨率下测定， TSV二聚体亚基的结构将在2.6 埃分辨率。 衍射至所示分辨率的晶体 都在掌握之中。 异构装配产品和 将通过cryoEM分析这些病毒的分解中间体， 蛋白质建模 用于大肠杆菌表达和体外组装的系统 由合作者Mark Young开发的表达CCMV衣壳蛋白 将用于确认衣壳稳定性和形态的决定因素 用定点突变体（即反向遗传学）。

Many important viruses and nucleoprotein cores of enveloped animal viruses (e.g. retroviruses) are unstable and pleomorphic and principles governing their structure are generally unknown due to the lack of crystallographic studies. Crystallography has been most successful with robust particles because homogeneous preparations are readily produced and these are likely to form well ordered crystals. Particles stabilized predominantly by protein-RNA interactions are unstable when exposed to even mild physical or chemical denaturants. For this reason simple viruses of this type are often the most accessible for assembly studies because subunit tertiary structure is not disturbed when the virion is disassembled. Cowpea chlorotic mottle virus (CCMV) is typical of this class. It was the first icosahedral virus reassembled in vitro to form infectious particles and the literature on the assembly of CCMV and the related alfalfa mosaic virus (ALMV) exceeds 100 papers and numerous reviews. With support from an NSF grant it was discovered that CCMV particles are highly sensitive to ultracentrifugation and this lead to the development of a new preparation procedure and the production of the first good quality CCMV crystals. CCMV is the type member of a super group of viruses known as tricorna viruses. The capsids of all members of this group are relatively unstable but there is a clear trend of reduced importance of protein-protein interactions from CCMV (T=3) to cucumber mosaic virus (CMV) to ALMV, which forms bacilliform particles in vivo from subunit hexamers and pentamers, to tobacco streak virus (TSV), which forms spheroidal particles in vivo with subunits that will not assemble when released from RNA. Previous studies indicate that structures of all the subunits in this super group are likely to be beta- barrels. The long term goal of this proposal is to solve and superimpose the 4 different subunit structures and identify the amino acid determinants of capsid stability and morphology. Towards this end the structure of CCMV was solved at 3.2 angstrom resolution. 180 canonical virus beta-barrels (190AA) form pentamers and true hexamers that associate through interwoven C terminal polypeptides to form a new type of T=3 particle. This association of morphological units is strikingly similar to the quaternary structure of the DNA tumor virus SV40. The structure of CCMV will be refined at 3.2 angstrom, the T=3 structure of cucumber mosaic virus will be extended from its current 5.5 angstrom resolution to at least 4.0 angstrom resolution; the structure of the T=1 reassembly product of ALMV subunits will be determined at 4.0 angstrom resolution, and the structure of the TSV dimeric subunits will be determined at 2.6 angstrom resolution. Crystals diffracting to the resolutions indicated are in hand for all these viruses. Heterogeneous assembly products and disassembly intermediates of these viruses will be analyzed by cryoEM and protein modeling. Systems for E coli expression and in vitro assembly of expressed CCMV capsid protein developed by collaborator Mark Young will be used to confirm determinants of capsid stability and morphology with site directed mutants (i.e. reverse genetics).