Structure Analysis of Viral Assembly Mechanisms

未成熟的逆转录病毒从受感染的细胞中释放出来后，在 哪种Gag被病毒蛋白酶切割成MA、CA和NC，引发大的 形态变化和产生感染性病毒粒子。在马萨诸塞州成熟的病毒粒子中 仍然与病毒包膜相关，而CA组装成富勒烯锥体，与 ~250个CA六聚体和12个CA五聚体，包裹着复杂的RNA基因组 与NC。在上一个资助周期中，我们确定了9？分辨率的低温电磁场密度图 全长HIV-1 CA的2D晶体电子结晶学研究。对接高- N-末端结构域(NTD)和C-末端结构域(CTD)的分辨结构 产生了一个分子模型，该模型指导了二硫键的插入，从而稳定了 NTD六角机。进一步的突变破坏了连接相邻的CTD二聚体的稳定性 六聚体，从而实现增溶和3D结晶。由此产生的原子- 分辨X射线结构表明，CA六聚体是由一个相对较小的 NTD亚基的刚性内环，被CTD亚基的移动带包围。移动性 CTD带很可能是产生连续 富勒烯圆锥体中的弯曲衣壳晶格。同样的二硫键策略随后被用于 产生稳定的CA五聚体，我们正在完成第一次高分辨率X射线 结构。对于下一个资金周期，我们将追求两个具体目标：(1)我们将致力于 50%的努力来继续我们对成熟衣壳晶格的结构研究。除了……之外 完成五聚体的X射线结构，我们将确定亚纳米低温电子显微镜 变径CA管的重建具有高分辨率结构的 六聚体和五聚体，在CA管中六聚体相互作用的指导下，我们 将使用计算方法为圆锥形外壳建立原子模型。(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突变体显示出螺旋衍射并用作体外模拟的 不成熟的晶格。通过类似于我们对成熟晶格的研究，CryoEM和 分子对接将产生一种指导可溶性GAG工程的模型 用于低温电子显微镜和X射线结晶学研究的六角体。我们希望我们的 结构研究将继续提供对逆转录病毒组装原理的洞察 这对于设计新的治疗策略将是重要的。

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.