Mechanism for Chemokine Receptor Fusogenic Activity

描述（由申请人提供）：艾滋病流行的规模推动了对HIV-1感染和发病机制的研究。趋化因子共受体在病毒进入中的作用和包膜糖蛋白（env） gp120亚基晶体结构的发现为研究HIV-1感染的起源开辟了重要途径。gp 120的V3环通过辅助受体的使用控制病毒的趋向性。CCR5被普通传播株的env使用，CXCR4被在感染后期进化的嗜t型株使用。在被CD4激活后，gp 120可以与一个辅助受体相互作用，从而触发膜融合和病毒进入。本研究的重点是阐明gp120与协同受体相互作用的机制，协同受体是阻断HIV-1感染的关键攻击点。尽管CCR5的n端和疏水核心带螺旋间环都足以赋予含有无活性趋化因子受体片段的杂交体共受体活性，但只有CXCR4的后一个区域似乎参与了与gpl20的相互作用。我们已经证明，具有V3冠变体的gp120亚基缺乏利用CCR5的疏水核心和螺旋间环进行env介导融合的能力，并且与野生型受体的结合受损。表明这两种结构相互作用。这项研究的核心假设是，受体的细胞外结构域短暂地结合V3环，诱导一种构象，这种构象允许gp120的新生辅受体结构和保守区域进一步相互作用。为了验证这一假设，我们提出了以下具体目标：1)表征CCR5 n端和第二胞外环域在共受体活性中的作用；2)深入了解CCR5和CXCR4疏水核心和螺旋间环与共受体活性拮抗剂（包括配体和拮抗剂）相互作用的构象；3)利用独特的CCR5和CXCR4信号变体，通过将这些辅助受体与酵母菌信息素途径功能偶联而产生的信号变体，表征辅助受体信号传导对HIV-1复制生物学的影响。拟议的实验将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，指导开发破坏HIV-1感染中前线共受体作用的策略。

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.