STRUCTURAL BIOLOGY OF VIRUS ASSEMBLY

该项目旨在阐明控制细胞凋亡的分子机制。 病毒衣壳的组装具有双重目标， 抗病毒化合物的靶点，并深入了解 大分子复合物的组装。我们的主要成果 在过去一年中， (i)越来越多的证据表明，一些衣壳起着功能性作用， 特定基因组相关过程发生的隔室，即 它们不仅仅是病毒基因组所在的封闭容器 被限制住了对于B型肝炎病毒（HBV），RNA前基因组是 在衣壳内逆转录，因此， 大约20个A孔，允许核苷酸进入和核苷酸流出。 消化的RNA。L-A病毒衣壳也作为生物合成的 腔室：在这种情况下，转录本通过 洞大到足以容纳ssRNA，但小到足以阻止 dsRNA模板 (ii)我们完成并发表了HBV衣壳的结构， 前所未有的高分辨率（对于电子显微镜）为9 A， 开发计算方法，帮助研究其他病毒 和大分子复合物。此外，还进行了详细的分析。 完成牛乳头状病毒，可视化其螺旋 壳粒间接头。对于HBV，达到的分辨率足够 来观察构成150个残基的一半的a螺旋 程序集域，但不足以允许跟踪链。 实现后一个目标的进展是通过精确定位其 通过在位置150处附加半胱氨酸，结合11个原子的 金团簇，并通过冷冻电镜观察团簇。c-末端 聚集在所有5倍和6倍轴下。该观察结果 提供了一个“分子拥挤”的解释， 我们之前发现的形态发生开关 组装更多的大（T=4）或小（T=3）衣壳。的成功 这个实验表明，我们也许能够绘制出 同样的方式，最终定义蛋白质折叠。 (iii)包封的噬菌体T7 dsDNA被显示为包装成一个包封体。 紧密缠绕的同轴线轴， 计算建模，利用突变体与 提供两个定义明确，相互 垂直，视图时，观察薄的冰膜。这一结果 代表了第一个决定性的三维测定， 任何类型的浓缩（静止）染色体的结构。T7 DNA 包装非常紧密，相邻的B型之间有间距 只有25 A的双链体，中心到中心，与六边形匹配 相位晶体相反，L-A的dsRNA最有可能是A型， 根据我们用STEM显微镜测量的单位长度质量来判断， 它的封装要松散得多（大约40 A，中心到中心）。这种松散 基因组必须经历的搅动运动 在L-A衣壳内，当它被推动通过固定化的聚合酶时， 在复制和转录过程中。

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.