Cryo-Electron Tomography Analysis of HIV-1 Maturation

描述（由申请人提供）：我们建议通过冷冻电子断层扫描确定在整个成熟过程中的选定阶段停止的HIV-1 VLP的结构。该方法将使我们能够确定（1）病毒在成熟过程中经历的形态学变化，（2）表达包膜蛋白（Env）的未成熟和成熟HIV-1 VLP的结构及其在基质（MA）层中的排列方式，以及（3）颗粒内其他病毒组分的位置。为了实现这些目标，我们与犹他州大学的研究人员合作，计划（1）纯化在几个“发育”阶段停止的HIV-1突变体，（2）通过冷冻电子断层扫描成像HIV-1突变体，并通过计算方法计算结构，（3）确定成熟过程中特定病毒蛋白质中存在的结构变异性。我们的研究将提供完整、完整的冷冻水合HIV-1 VLP的最高分辨率结构，其他研究人员可以从中确定其他蛋白质的位置和数据，从而设计新的治疗方法来对抗或根除HIV-1感染。

DESCRIPTION (provided by applicant): We propose to determine the structure of HIV-1 VLPs halted at selected stages throughout maturation by cryo-electron tomography. This method will enable us to determine (1) the morphological changes the virus undergoes during maturation, (2) the structure of immature and mature HIV-1 VLPs with the Envelope protein (Env) expressed and how it is arranged in the matrix (MA) layer, and (3) the positions of other viral components within the particles. To achieve these aims, in collaboration with researchers at the University of Utah, we plan to (1) purify HIV-1 mutants halted at several 'developmental' stages, (2) image the HIV-1 mutants by cryo-electron tomography and calculate the structures through computational methods, and (3) determine the structural variability present in the specific viral proteins during maturation. Our studies will provide the highest resolution structures of whole, intact frozen-hydrated HIV-1 VLPs from which other researchers may determine the placement of other proteins and data from which new therapies may be designed to combat or eradicate HIV-1 infection.