Structure Analysis of Viral Assembly Mechanisms

描述（由申请人提供）：未成熟的逆转录病毒从感染细胞中释放出来后，经历一个成熟过程，在这个过程中，Gag被病毒蛋白酶切割成MA、CA和NC，引发大的形态变化并产生传染性病毒粒子。在成熟的病毒粒子内，MA仍然与病毒包膜相关，而CA则与约250个CA六聚体和12个CA五聚体组装成一个富勒烯锥体，包裹着与NC复合物的RNA基因组。在上一个资助周期中，我们通过二维晶体的电子晶体学确定了全长HIV-1 CA的9-E分辨率冷冻电镜密度图。N端结构域（NTD）和c端结构域（CTD）的高分辨率结构对接产生了一个分子模型，该模型指导了稳定NTD六聚体的二硫键的插入。进一步的诱变破坏了连接相邻六聚体的CTD二聚体的稳定性，从而实现了增溶和三维结晶。原子分辨率x射线结构表明，CA六聚体是由一个相对刚性的NTD亚基内环组成，周围是一个可移动的CTD亚基带。CTD带的迁移性可能是富勒烯锥体中产生连续弯曲衣壳晶格的潜在机制。同样的二硫策略被用来生成稳定的CA五聚体，我们正在完成第一个高分辨率x射线结构。对于下一个融资周期，我们将追求3个具体目标：(1)我们将投入40%的精力继续我们成熟衣壳晶格的结构研究。除了完成五聚体的x射线结构外，我们还将确定变直径CA管的亚纳米低温电镜重建。利用六聚体和五聚体的高分辨率结构，并以六聚体在CA管中的相互作用为指导，我们将使用计算方法建立锥形衣壳的原子模型。(2)我们将投入40%的精力进行未成熟Gag晶格的结构研究。我们已经产生了Gag突变体，显示螺旋衍射，并作为未成熟晶格的体外模拟物。与我们对成熟晶格的研究类似，低温电子显微镜和分子对接将产生一个模型，该模型将指导可溶Gag六聚体的工程，用于低温电子显微镜和x射线晶体学研究。(3)我们将投入20%的精力来探索组装过程中可能成熟的中间体的结构。我们已经生成了具有亚纳米衍射的gag样小鼠白血病病毒的多种二维晶体形式，我们的工作假设是，对这些不同晶体形式的分析可能揭示成熟过程中发生的结构重排。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见解，这将对设计新的治疗策略很重要。

DESCRIPTION (provided by applicant): 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-E 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 3 specific aims: (1) We will devote 40% 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 40% 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. (3) We will devote 20% effort to explore the structures of possible maturation intermediates during assembly. We have generated multiple 2D crystal forms of a Gag-like construct of murine leukemia virus that display subnanometer diffraction, and our working assumption is that analysis of these varying crystal forms may reveal structural rearrangements that occur during maturation. 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.