STRUCTURAL STUDIES OF SSDNA VIRUSES: HIV

SSDNA 病毒的结构研究：HIV

• 批准号: 7955534
• 负责人: Mavis Agbandje-Mckenna
• 金额: $ 3.14万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955534
• 关键词: Affinity; Amino Acids; Animals; Binding; Biochemical; Biological; Caliber; Canis familiaris; Capsid; Carbohydrates; Cell Nucleus; Cell Surface Receptors; Cell division; Cells; Cellular Tropism; Characteristics; Clinical Treatment; Clinical Trials; Complex; Computer Retrieval of Information on Scientific Projects Database; Crystallization; Data; Data Collection; Dependovirus; Development; Disease; Endocytosis; Engineering; Family suidae; Felis catus; Funding; Gene Delivery; Gene Transduction Agent; Generations; Genes; Genome; Goals; Grant; HIV; Hamsters; Human; Immune; Immunosuppressive Agents; Infection; Institution; Laboratories; Life; Life Cycle Stages; Llama; Mediating; Mice Minute Virus; Mink; Modeling; Molecular; Monkeys; Mouse Strains; Mus; Nature; Outcome; Parvoviridae; Parvovirus; Pathogenicity; Pathway interactions; Phenotype; Preparation; Production; Property; Receptor Cell; Reporting; Research; Research Personnel; Resources; Roentgen Rays; Role; Seasons; Serotyping; Sialic Acids; Single Stranded DNA Virus; Site; Source; Structure; Surface; System; Testing; Time; United States National Institutes of Health; Vaccine Antigen; Variant; Viral; Viral Genes; Viral Vaccines; Virulence; Virulent; Virus; Work; X ray diffraction analysis; X-Ray Diffraction; base; cancer cell; cytokine; design; gene therapy; human disease; improved; in vivo; influenzavirus; member; mutant; pathogen; prototype; receptor; receptor binding; tissue tropism; trafficking; transduction efficiency; transgene expression; uptake; vector

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The aim of our projects on the ssDNA viruses is to identify structural determinants of receptor attachment, tissue tropism, in vivo pathogenicity, and transduction efficiencies between highly homologous Parvoviridae strains and serotypes. Our structural studies so far indicate that slight capsid surface alterations, resulting from amino acid differences, are associated with pronounced differences in biological properties during the viral life cycle of parvoviruses. The long-term goal of our studies are to utilize the structural information obtained for the design of viral vaccines, foreign antigenic delivery systems, viral gene therapy vectors for the treatment of animal and human diseases. This rapid access application is to obtain experimental beam time for X-ray diffraction data collection on the Adeno-associated virus serotypes 5, 8, and 9 (AAV5, AAV8 and AAV9) - being exploited for gene therapy applications, H-1 hamster parvovirus being exploited for cancer cell targeted gene delivery of cytokines, and the prototype strain of minute virus of mice (MVM) (a viral model for understanding virus host range adaptation) co-crystallized with carbohydrate components of its cell surface receptor. The AAV5 and MVMp data will be collected for capsids co-crystallized with sialic acid components of their cell surface receptors towards identifying the capsid site that facilitates infectious interaction. AAV5, AAV8, and MVM data will also be collected at pHs (6.0, 5.5 and 4.0) that mimic the endocytic pathway data in an effort to elucidate the nature of the capsid transistions associated with parvovirus trafficking to the nucleus. We are seasoned users of CHESS and have determined several structures from data collected at A1 and F1. The Parvoviridae are spherical, non-enveloped, T=1 icosahedral viruses, with a wide range of natural hosts which includes humans, monkeys, pigs, dogs, cats, mink and mice. They are the cause of a number of serious diseases, especially in the young of the species that they infect, due to a requirement for cellular factors, produced during active cell division. Parvoviral capsids are ~260 ¿ in diameter and encapsidate a ssDNA genome of ~5000 bases. Our correlation of available structural information on parvovirus capsids with molecular and biochemical data indicates that capsid surface variations modulate biological differences, such as receptor attachment, host range, pathogenicity and antigenicity, among members of the parvoviruses and between strains/serotypes of the same virus. Our studies are focused on two aspects of parvovirology: (I) Understanding viral adaptation to new hosts and mechanisms of host pathogenicity, exploiting Minute virus of mice (MVM) and H-1 hamster parvovirus (H-1); Highly homologous MVM strains have disparate tissue tropism and infection pathogenicity, with one or two amino acids conferring a lethal outcome. Our work MVM has focused determining the structures of the homologous MVMp (prototype strain) and MVMi (the immunosuppressive strain) as well as virulent mutants of MVMp, alone and in complex with the sialic acid component of their infectious cell surface receptor. We have shown that the regions controlling their tissue tropism and pathogenicity phenotypes are on the capsid surface and configure a receptor attachment site1,-3. This observation suggests that host adaptation likely utilizes alteration of host receptor recognition, reminiscent of the host adaptation/jumping that has been reported for the influenza virus. The H-1 project is new to the lab and is aimed at structural characterization towards improving its use for cancer cell targeted gene delivery. (II) Structural characterization of host cellular entry, trafficking and transduction efficiency towards improved viral-based gene delivery, exploiting the Adeno-associated virus (HIV AAV) serotypes that hold significant promise for the correction of human diseases. The AAVs can be used to transfer non-host genes efficiently into primary cells in vivo, and in most cases, expression of the transgene appears to be long lived. Several serotypes are now in clinical trials of the treatment of animal and human diseases. The cellular tropism of the different AAV serotypes is determined by the ability of the virus capsid to bind host cell receptors with different terminal carbohydrates that mediate cell entry followed by uptake via endocytosis. The receptor binding regions are thought to be on capsid surfaces regions that differ between the serotypes. Our work is focused on characterizing the structures of the representative members of the AAV clades (AAV1-AAV9), their interactions with host receptor molecules, and entry. We are also working to identify the dominant antigenic regions of these capsids with the goal to engineering a second generation of gene delivery vectors with host immune evasion properties. This beam time application is for five projects; MVM, H-1, AAV5, AAV8, and AAV9. We have determined the structures of MVMi and MVMp1, plus those of virulent MVMp strains to 3.2 ¿ (in preparation) ¿¿" with data collected at CHESS, APS, BNL and at the Daresbury Laboratories (UK). Our current studies are aimed at continued characterization of the capsid regions used for receptor recognition and pH-mediated transitions associated with endosomal trafficking to the nucleus for replication. We have co-crystals of MVMp-sialic acids that specifically bind MVM capsids3 and crystals grown at pHs 6.0-4.0 that mimic the endocytic pathway. For H-1 we have crystals of empty capsids. For the AAVs, we have crystals of AAV5 and AAV8 at pHs that mimic the endocytic pathway and for AAV9, for which there is currently no structure information, we have small crystals for testing diffraction quality. We also have crystals of AAV5 co-crystallized with the sialic acid component of its receptor. We have already determined the crystal structures of wild type AAV5 and AAV8 at pH 7.54-6. F1 beam time allocation will enable us to collect X-ray diffraction data for our continued structure function studies on these viruses discussed above. The long-term goal of this research is to obtain a fundamental understanding of the structural determinants of the natural variations that exists in the Parvoviridae, along with information on their interactions with host cellular components and mutational data. Such information is important for understating the characteristics of emerging pathogens. We also aim to use this data towards the development of vaccines, antigen delivery systems, and gene therapy vectors for the treatment of animals and humans. References: 1. Kontou, M., L. Govindasamy, H.-J. Nam, N. Bryant, A. L. Llamas-Saiz, C. Foces-Foces, E. Hernando, M.-P Rubio, R. McKenna, J. M. Almendral., M. Agbandje-McKenna. 2005. Structural determinants of tissue tropism and in vivo pathogenicity for the parvovirus minute virus of mice. J. Virol., 79:10931-10943. 2. Alberto L¿¿pez-Bueno, A, M-P. Rubio, N. Bryant, R. McKenna, M. Agbandje-McKenna, J. M. Almendral. 2006. Host-selected amino acid changes at the sialic acid binding pocket of the parvovirus capsid modulate cell binding affinity and determine virulence. J. Virol., 80: 1563-1573. 3. Nam, H.-J., B. Gurda-Whitaker, W. Y. Gan, S. Ilaria, R. McKenna, P. Mehta, R. A. Alvarez, M. Agbandje-McKenna. 2006. Identification of the sialic acid structures recognized by minute virus of mice and the role of binding affinity in virulence adaptation. J. Bio. Chem., 281:25670-25677. 4. DiMattia, M., L. Govindasamy, H.C. Levy, B. Gurda-Whitaker, A. Kalina, E. Kohlbrenner, J. A. Chiorini, R. McKenna, N. Muzyczka, S. Zolotukhin, M. Agbandje-McKenna. 2005. Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5. Acta Cryst., F61, 917-921. 5. Lane, M. D., H.-J. Nam, E. Padron, B. Gurda-Whitaker, E. Kohlbrenner, G. Aslanidi, B. Byrne, R. McKenna, N. Muzyczka, S. Zolotukhin, M. Agbandje-McKenna. 2005. Production, Purification, Crystallization, and preliminary X-ray analysis of Adeno-Associated

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们关于单链DNA病毒的项目的目的是确定受体附着、组织向性、体内致病性以及高度同源的细小病毒科菌株和血清型之间的转导效率的结构决定因素。迄今为止，我们的结构研究表明，由于氨基酸差异而导致的轻微衣壳表面变化与细小病毒生命周期中生物学特性的显着差异有关。 我们研究的长期目标是利用获得的结构信息设计病毒疫苗、外源抗原递送系统、病毒基因治疗载体来治疗动物和人类疾病。 该快速访问应用程序旨在获得腺相关病毒血清型 5、8 和 9（AAV5、AAV8 和 AAV9）的 X 射线衍射数据收集的实验光束时间 - 用于基因治疗应用，H-1 仓鼠细小病毒用于细胞因子的癌细胞靶向基因传递，以及小鼠微小病毒 (MVM) 的原型毒株（一种病毒模型） 了解病毒宿主范围适应）与其细胞表面受体的碳水化合物成分共结晶。 将收集与其细胞表面受体的唾液酸成分共结晶的衣壳的 AAV5 和 MVMp 数据，以识别促进感染相互作用的衣壳位点。 还将在模拟内吞途径数据的 pH（6.0、5.5 和 4.0）下收集 AAV5、AAV8 和 MVM 数据，以努力阐明与细小病毒运输到细胞核相关的衣壳转变的性质。 我们是 CHESS 的经验丰富的用户，并根据 A1 和 F1 收集的数据确定了几种结构。 细小病毒科是球形、无包膜、T=1二十面体病毒，具有广泛的天然宿主，包括人、猴、猪、狗、猫、水貂和小鼠。 由于需要活跃细胞分裂过程中产生的细胞因子，它们是许多严重疾病的原因，特别是在它们感染的幼体中。 细小病毒衣壳直径约为 260 ¿，包裹着约 5000 个碱基的 ssDNA 基因组。 我们将细小病毒衣壳的现有结构信息与分子和生化数据进行关联，表明衣壳表面变化调节细小病毒成员之间以及同一病毒的毒株/血清型之间的生物学差异，例如受体附着、宿主范围、致病性和抗原性。 我们的研究集中在细小病毒学的两个方面： (I) 利用小鼠微小病毒（MVM）和H-1仓鼠细小病毒（H-1）了解病毒对新宿主的适应和宿主致病机制； 高度同源的 MVM 菌株具有不同的组织趋向性和感染致病性，其中一两个氨基酸会导致致命结果。 我们的 MVM 工作重点是确定同源 MVMp（原型菌株）和 MVMi（免疫抑制菌株）以及 MVMp 的有毒突变体的结构，单独或与感染性细胞表面受体的唾液酸成分复合。我们已经证明，控制其组织向性和致病性表型的区域位于衣壳表面并配置受体附着位点1,-3。 这一观察结果表明，宿主适应可能利用宿主受体识别的改变，让人想起已报道的流感病毒的宿主适应/跳跃。 H-1 项目是该实验室的新项目，旨在进行结构表征，以改善其在癌细胞靶向基因传递中的应用。 (II) 宿主细胞进入、运输和转导效率的结构表征，以改善基于病毒的基因传递，利用腺相关病毒 (HIV AAV) 血清型，为纠正人类疾病带来重大希望。 AAV 可用于将非宿主基因有效地转移到体内原代细胞中，并且在大多数情况下，转基因的表达似乎是长期存在的。 几种血清型目前正在进行治疗动物和人类疾病的临床试验。不同 AAV 血清型的细胞向性取决于病毒衣壳与具有不同末端碳水化合物的宿主细胞受体结合的能力，这些末端碳水化合物介导细胞进入，然后通过内吞作用进行摄取。 受体结合区域被认为位于不同血清型的衣壳表面区域上。 我们的工作重点是表征 AAV 进化枝 (AAV1-AAV9) 代表性成员的结构、它们与宿主受体分子的相互作用以及进入。 我们还致力于鉴定这些衣壳的显性抗原区域，目标是设计具有宿主免疫逃避特性的第二代基因递送载体。 此 Beam Time 应用程序适用于五个项目； MVM、H-1、AAV5、AAV8 和 AAV9。我们已经确定了 MVMi 和 MVMp1 的结构，以及毒性 MVMp 菌株的结构，其强度为 3.2 ¿（准备中） ¿”，并利用 CHESS、APS、BNL 和 Daresbury 实验室（英国）收集的数据。我们当前的研究旨在继续表征用于受体识别的衣壳区域以及与内体运输到细胞核相关的 pH 介导的转变。 复制。我们拥有特异性结合 MVM 衣壳3 的 MVMp-唾液酸共晶，以及在 pH 6.0-4.0 下生长的模拟内吞途径的晶体。 对于 H-1，我们有空衣壳晶体。 对于 AAV，我们有 AAV5 和 AAV8 的晶体，其 pH 值模拟内吞途径；对于 AAV9，我们目前还没有结构 信息，我们有用于测试衍射质量的小晶体。 我们还有 AAV5 与其受体唾液酸成分共结晶的晶体。我们已经确定了野生型AAV5和AAV8在pH 7.54-6下的晶体结构。 F1 光束时间分配将使我们能够收集 X 射线衍射数据，以便我们对上述这些病毒进行持续的结构功能研究。 这项研究的长期目标是获得基本的了解 细小病毒科中存在的自然变异的结构决定因素，以及它们与宿主细胞成分和突变数据相互作用的信息。 这些信息对于了解新出现的病原体的特征非常重要。我们还旨在利用这些数据来开发用于治疗动物和人类的疫苗、抗原递送系统和基因治疗载体。 参考： 1. Kontou，M.，L. Govindasamy，H.-J。 Nam，N. Bryant，A. L. Llamas-Saiz，C. Foces-Foces，E. Hernando，M.-P Rubio，R. McKenna，J. M. Almendral.，M. Agbandje-McKenna。 2005.小鼠细小病毒的组织向性和体内致病性的结构决定因素。 J.病毒学，79：10931-10943。 2. 阿尔贝托·洛佩斯-布埃诺 (Alberto L¿ pez-Bueno)，A、M-P。卢比奥、N. 布莱恩特、R. 麦肯纳、M. 阿格班杰-麦肯纳、J. M. 阿尔门德拉尔。 2006.细小病毒衣壳唾液酸结合袋上宿主选择的氨基酸变化调节细胞结合亲和力并确定毒力。 J.病毒学，80：1563-1573。 3. Nam, H.-J.、B. Gurda-Whitaker、W. Y. Gan、S. Ilaria、R. McKenna、P. Mehta、R. A. Alvarez、M. Agbandje-McKenna。 2006.小鼠微小病毒识别的唾液酸结构的鉴定以及结合亲和力在毒力适应中的作用。 J.比奥.化学，281：25670-25677。 4. DiMattia, M., L. Govindasamy, H.C. Levy、B. Gurda-Whitaker、A. Kalina、E. Kohlbrenner、J. A. Chiorini、R. McKenna、N. Muzyczka、S. Zolotukhin、M. Agbandje-McKenna。 2005. 腺相关病毒血清型5的生产、纯化、结晶和初步X射线结构研究。Acta Cryst., F61, 917-921。 5. Lane，M.D.，H.-J。 Nam、E. Padron、B. Gurda-Whitaker、E. Kohlbrenner、G. Aslanidi、B. Byrne、R. McKenna、N. Muzyczka、S. Zolotukhin、M. Agbandje-McKenna。 2005. 腺相关蛋白的生产、纯化、结晶和初步X射线分析