STRUCTURAL BIOLOGY OF VIRUS ASSEMBLY

病毒组装的结构生物学

• 批准号: 6160807
• 负责人: A C STEVEN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6160807
• 关键词: bacteriophage T7; bovine papillomavirus; capsid; coliphages; conformation; crosslink; cryoscience; double stranded RNA; hepatitis B virus group; herpes simplex virus 1; image processing; protein biosynthesis; protein folding; protein structure function; scanning transmission electron microscopy; structural biology; virus DNA; virus RNA; virus assembly; virus protein

This project aims to elucidate the molecular mechanisms that control the assembly of viral capsids with the twin goals of defining prospective targets for antiviral compounds and gaining insights into regulation of the assembly of macromolecular complexes in general. Our major results for the past year are: (i) Evidence is accruing that some capsids serve as functional compartments in which specific genome-related processes take place, i.e. they are not simply closed containers to which viral genomes are confined. For hepatitis B virus (HBV), the RNA pregenome is retrotranscribed inside the capsid, which accordingly, is fenetrated with about 20 A holes that allow the entry of nucleotides and egress of digested RNA. The L-A virus capsid serves also operates as a biosynthetic chamber: in this case, transcripts are fed out into the cytoplasm through holes large enough to admit ssRNA but small enough to prevent egress of the dsRNA template. (ii) We completed and published the structure of the HBV capsid at the unprecedentedly high resolution (for electron microscopy) of 9 A, developing computational methods that should aid work on other viruses and macromolecular complexes. A comparably detailed analysis was also accomplished for bovine papillovavirus, visualizing its helical inter-capsomer linkers. For HBV, the resolution achieved was sufficient to visualize the a-helices that make up about half of the 150-residue assembly domain, but not high enough to allow tracing of the chain. Progress towards the latter goal was made by precisely localizing its C-terminus by appending a cysteine at position 150, binding an 11-atom gold cluster to it, and visualizing the cluster by cryo-EM. The C-termini congregate together under all 5-fold and 6-fold axes. This observation provides a "molecular crowding" explanation for how this peptide operates as the morphogenetic switch that we previously found to specify whether more large (T=4) or small (T=3) capsids are assembled. The success of this experiment suggests that we may be able to map other residues in the same way, ultimately defining the protein fold. (iii) Encapsidated bacteriophage T7 dsDNA was shown to be packed as a tightly wound coaxial spool by a combination of cryo-EM with computational modeling, taking advantage of a mutant with the serendipitous property of providing two well defined, mutually perpendicular, views when observed in thin ice films. This result represents the first conclusive determination of the three-dimensional structure of a condensed (quiescent) chromosome of any kind. T7 DNA is packed remarkably tightly, with a spacing between neighboring B-form duplexes of only 25 A, center-to-center, matching that in a hexagonal phase crystal. In contrast, the dsRNA of L-A is most likely A-form, to judge by our measurements of mass-per-unit-length by STEM microscopy and it is packed much more loosely (about 40 A, center-to-center). This loose packing allows for the churning motion that the genome must undergo within the L-A capsid as it is propelled past the immobilized polymerase during replication and transcription.

该项目旨在阐明控制细胞死亡的分子机制。 病毒衣壳的组装具有定义预期的双重目标 抗病毒化合物的靶点和对调节的洞察力 一般的大分子络合物的组装。我们的主要成果 过去一年的情况如下： (I)越来越多的证据表明，某些衣壳具有功能性 发生特定基因组相关过程的隔室，即 它们不仅仅是病毒基因组的封闭容器 被限制了。对于乙肝病毒(乙肝)，RNA前基因组是 在衣壳内逆转录，相应地，衣壳被 大约20安培的洞，允许核苷酸进入和离开 消化的核糖核酸。L-A病毒衣壳也是一种生物合成 小室：在这种情况下，转录通过以下途径送入细胞质 孔大到足以容纳单链RNA，但小到足以防止 DsRNA模板。 (Ii)我们已在 前所未有的高分辨率(对于电子显微镜)9A， 开发计算方法，以帮助研究其他病毒 和大分子复合体。还进行了比较详细的分析。 完成了对牛乳头状病毒的研究，可视化了其螺旋结构 胶囊间连接物。对于乙肝病毒，所实现的分辨率是足够的。 以可视化约占150个残基的一半的a-螺旋 程序集域，但不足以允许跟踪链。 通过精确地将其本地化，朝着后一目标取得了进展 C末端通过在150位附加半胱氨酸，与11个原子结合 黄金星团到它，并通过冷冻-EM可视化该星团。C-终端 聚集在所有5折和6折的轴线下。这一观察结果 为这种多肽的工作原理提供了“分子拥挤”的解释。 作为我们之前发现的形态发生开关来指定是否 更多的大(T=4)或小(T=3)衣壳被组装。的成功之处 这项实验表明，我们或许能够绘制出其他残基在 用同样的方法，最终定义蛋白质折叠。 (Iii)被包裹的噬菌体T7 dsDNA被包装为 用低温电磁场结合紧密缠绕同轴线轴 计算建模，利用变种的 提供两个定义良好的、相互的 垂直，在薄冰膜中观察时的景象。这个结果 代表了对三维空间的第一个决定性的确定 任何类型的浓缩(静止)染色体的结构。T7 DNA是 排列得非常紧密，相邻的B型之间有间距 只有25A的双工，中心到中心，与六角形的相匹配 相态晶体。相比之下，L-A的dsRNA最有可能是A型， 根据我们用扫描电子显微镜和扫描电子显微镜测量的单位长度质量 它的包装要松散得多(大约40A，从中心到中心)。这是松动的 包装允许基因组必须经历的搅动运动 在L-A衣壳内，因为它被推进通过固定的聚合酶 在复制和转录过程中。