Mechanism for Chemokine Receptor Fusogenic Activity

描述（由申请人提供）：艾滋病流行的严重性推动了对HIV-1感染和发病机制的研究。发现（趋化因子）辅助受体在病毒进入和包膜糖蛋白（env）的gp 120亚基上的晶体结构的作用，为研究HIV-1感染的开始开辟了重要途径。gp 120的V3环通过辅助受体的使用来控制病毒的嗜性，CCR 5被通常传播的毒株的env使用，CXCR 4被在感染后期进化的嗜T性毒株使用。在被CD 4激活后，gp 120可以与辅助受体相互作用，从而触发膜融合和病毒进入。该研究的重点是阐明gp 120和辅助受体之间的相互作用机制，这是阻断HIV-1感染的关键攻击点。而具有CCR 5螺旋间环的N-末端和疏水核心各自足以赋予含有非活性趋化因子受体片段的杂合体共受体活性，只有CXCR 4的后一个区域似乎参与了与gp 120的相互作用。我们已经证明，具有V3冠状变体的gp 120亚基缺乏利用CCR 5的疏水核心和螺旋间环进行env的能力。介导的融合，并且与野生型受体的结合受损，表明这两种结构相互作用。所提出的研究的核心假设是受体的胞外结构域瞬时结合V3环，以诱导允许涉及新生辅助受体结构和gp 120保守区域的进一步相互作用的构象。提出了以下具体目的来检验该假设：1）表征CCR 5 N-末端和第二胞外环结构域在共受体活性中的作用，2）改进对CCR 5和CXCR 4疏水核心和螺旋间环的构象的了解，所述螺旋间环参与与共受体活性的拮抗剂（包括配体和拮抗剂）的相互作用，3）使用独特的CCR 5和CXCR 4信号传导变体来表征辅助受体信号传导对HIV-1复制生物学的影响，所述独特的CCR 5和CXCR 4信号传导变体通过将这些辅助受体与酵母中的信息素途径功能性地偶联而产生。拟议中的实验将联合收割机结合酵母遗传学和分子动力学模拟的力量，建立一个结构基础，指导发展战略，破坏前线辅助受体在HIV-1感染中的作用。

DESCRIPTION (provided by applicant): The magnitude of the AIDS epidemic has fueled research on the mechanisms involved in HIV-1 infection and pathogenesis. Discovery of the role of (chemokine) coreceptors in viral entry and the crystal structure on the gp120 subunit of the envelope glycoprotein (env) have opened critical avenues for studying the inception of HIV-1 infection. The V3 loop of gp 120 governs viral tropism through coreceptor usage CCR5 is used by env of commonly transmitted strains and CXCR4 by T-tropic strains that evolve in late phases of infection. After activation by CD4, gp 120 can interact with a coreceptor, thereby triggering membrane fusion and viral entry. The focus of the proposed research is to elucidate the mechanism of the interaction between gp120 and coreceptor, a critical point of attack to block HIV-1 infection. Whereas the N-terminus and hydrophobic core with interhelical loops of CCR5 are each sufficient to confer coreceptor activity to hybrids containing segments of inactive chemokine receptors, only that latter region of CXCR4 appears to be involved in the interaction with gpl20 We have demonstrated that gp120 subunits with V3 crown variant lack the ability to utilize the hydrophobic core and interhelical loops of CCR5 for env-mediated fusion and have impaired binding to the wild type receptor, suggesting that these two structures interact. The central hypothesis underlying the proposed research is that extracellular domains of the receptors transiently bind the V3 loop to induce a conformation that is permissive for further interactions involving the nascent coreceptor structures and conserved regions of gp120. The following specific aims are proposed to test this hypothesis 1) To characterize the role of CCR5 N-terminus and second extracellular loop domains in coreceptor activity, 2) To refine insight into the conformation of the CCR5 and CXCR4 hydrophobic core and interhelical loops involved in the interaction with antagonists of coreceptor activity, including ligands and antagonists, 3) To characterize the consequences of coreceptor signaling on the biology of HIV-1 replication using unique CCR5 and CXCR4 signaling variants generated by functionally coupling these coreceptors to the phermone pathway in yeast. The proposed experiments will combine the power of yeast genetics and molecular dynamic simulations to establish a structural basis that will guide the development of strategies to disrupt the role of frontline coreceptors in HIV-1 infection.