Structure Analysis of Viral Assembly Mechanisms

当从感染细胞释放时，未成熟的逆转录病毒经历一个成熟过程， 其中Gag被病毒蛋白酶切割成MA、CA和NC，触发大的 形态变化并产生感染性病毒体。在成熟病毒体内，MA 仍然与病毒包膜相关，而CA组装为富勒烯锥， ~250个CA六聚体和12个CA五聚体，其包围复合的RNA基因组。 在NC。在上一个资助周期中，我们确定了一个9英寸分辨率的cryoEM密度图， 通过二维晶体的电子晶体学分析全长HIV-1 CA。对接高- N-末端结构域（NTD）和C-末端结构域（CTD）的解析结构 产生了一个分子模型，指导插入二硫键，稳定了 NTD六聚体。进一步的诱变使连接邻近的CTD二聚体不稳定。 六聚体，从而能够增溶和3D结晶。最终的原子- 分辨率X射线结构显示，CA六聚体是由一个相对 NTD子单元的刚性内环，由CTD子单元的移动的带围绕。流动性 CTD带很可能是一种潜在的机制， 富勒烯锥中的弯曲衣壳晶格。然后使用相同的二硫键策略， 产生稳定的CA五聚体，我们正在完成第一个高分辨率的X射线 结构在下一个资助周期，我们将追求两个具体目标：（1）我们将致力于 50%的努力，继续我们的成熟衣壳晶格的结构研究。除了 完成五聚体的X射线结构，我们将确定亚纳米cryoEM 具有可变直径的CA管的重建。具有高分辨率的结构， 六聚体和五聚体，并在CA管中的六聚体相互作用的指导下，我们 将使用计算方法来建立锥形衣壳的原子模型。(2)我们 将投入50%的精力来研究未成熟的Gag晶格的结构。我们有 产生的Gag突变体显示螺旋衍射，并作为体外模拟 未成熟的晶格通过与我们对成熟晶格的研究类比，cryoEM和 分子对接将产生一个模型，将指导工程可溶性Gag 用于cryoEM和X射线晶体学研究的六聚体。我们希望， 结构研究将继续提供对逆转录病毒装配原理的深入了解 这对设计新的治疗策略很重要。

Upon release from infected cells, immature retroviruses undergo a maturation process in which Gag is cleaved by the viral protease into MA, CA, and NC, triggering large morphological changes and producing infectious virions. Within the mature virion, MA remains associated with the viral envelope, while CA assembles as a fullerene cone with ~250 CA hexamers and 12 CA pentamers, which encloses the RNA genome complexed with NC. In the last funding cycle we determined a 9-¿ resolution cryoEM density map of full-length HIV-1 CA by electron crystallography of 2D crystals. Docking of high- resolution structures of the N-terminal domain (NTD) and C-terminal domain (CTD) yielded a molecular model that guided the insertion of disulfide bonds that stabilized the NTD hexamer. Further mutagenesis destabilized the CTD dimers that link adjacent hexamers, thereby enabling solubilization and 3D crystallization. The resulting atomic- resolution X-ray structures revealed that the CA hexamer is composed of a relatively rigid inner ring of NTD subunits, surrounded by a mobile belt of CTD subunits. Mobility of the CTD belt is likely to be an underlying mechanism for generating the continuously curved capsid lattice in the fullerene cone. The same disulfide strategy was then used to generate stable CA pentamers, and we are completing the first high-resolution X-ray structure. For the next funding cycle we will pursue 2 specific aims: (1) We will devote 50% effort to continue our structural studies of the mature capsid lattice. In addition to completing X-ray structures of the pentamer, we will determine subnanometer cryoEM reconstructions of CA tubes with variable diameters. With high-resolution structures of the hexamer and pentamer, and guided by the hexamer interactions in the CA tubes, we will use computational methods to build an atomic model for the conical capsid. (2) We will devote 50% effort to structural studies of the immature Gag lattice. We have generated Gag mutants that display helical diffraction and serve as an in vitro mimic of the immature lattice. By analogy with our studies of the mature lattice, cryoEM and molecular docking will yield a model that will guide the engineering of soluble Gag hexamers for cryoEM and X-ray crystallographic studies. We are hopeful that our structural studies will continue to provide insight into principles of retrovirus assembly that will be important for the design of new therapeutic strategies.