CRYSTALLOGRAPHIC STUDIES OF PLEOMORPHIC VIRUSES

许多重要的包膜动物病毒和核蛋白核心 病毒(如逆转录病毒)是不稳定的、多形性的和原理的 管理它们的结构通常是未知的，因为缺乏 结晶学研究结晶学的最成功之处在于 坚固的颗粒，因为均匀的制剂很容易产生 这些物质很可能形成有序的晶体。颗粒稳定 主要通过蛋白质-RNA相互作用在接触时不稳定 即使是温和的物理或化学变性剂。因此，原因很简单 这种类型的病毒通常是组装研究中最容易获得的 因为亚基的三级结构在病毒粒子 拆解了。豌豆褪绿斑驳病毒(CCMV)就是其中的典型代表 班级。这是第一个在体外重组形成的二十面体病毒 感染性颗粒和CCMV与病毒组装的文献 相关的紫花苜蓿花叶病毒(ALMV)发表论文100余篇，数量众多 评论。在NSF拨款的支持下，发现CCMV 颗粒对超速离心高度敏感，这会导致 一种新的制备工艺的开发和生产 第一个高质量的CCMV晶体。CCMV是超级成员的类型成员 被称为三角病毒的一组病毒。所有成员的隐蔽性 相对不稳定，但有一个明显的趋势 蛋白质-蛋白质相互作用的重要性从CCMV(T=3)降低到 黄瓜花叶病毒(CMV)到ALMV，形成杆状颗粒 体内从亚单位六聚体和五聚体到烟草条纹病毒 (TSV)，它在体内形成球形粒子，其亚基将 当从RNA中释放时不能组装。此前的研究表明， 这个超群中所有亚基的结构很可能是β- 桶。 这项建议的长期目标是解决和叠加4个 不同的亚基结构并鉴定其氨基酸决定因素 衣壳的稳定性和形态。为此，CCMV的结构 在3.2埃分辨率下求解。180个典型病毒贝塔桶 (190AA)形成五聚体和真六聚体，它们通过 C端多肽相互交织，形成一种新型的T=3粒子。 这种形态单位的关联惊人地类似于 DNA肿瘤病毒SV40的四级结构。CCMV的结构 会在3.2埃处细化，黄瓜花叶的T=3结构 病毒将从目前的5.5埃分辨率扩展到 最小4.0埃分辨率；T=1重组的结构 ALMV亚基的乘积将以4.0埃分辨率确定， 并且TSV二聚体亚基的结构将在2.6确定 Angstrom分辨率。晶体的衍射率达到指示的分辨率 所有这些病毒都在掌握之中。异类装配产品和 这些病毒的拆解中间产物将通过CryoEM和 蛋白质建模。大肠杆菌的表达和体外组装系统 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 与定点突变(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).