Association of HIV-1 gp120 with its coreceptors

在这一延续性建议中，我们寻求扩大和完善我们对相互作用的理解， HIV-1包膜糖蛋白与其辅助受体CCR 5之间的相互作用。在上一个融资周期，我们 证实了我们最初观察到的CCR 5的N-末端存在酪氨酸硫酸化， 在HIV-1进入中的关键作用。我们还证明了基于CCR 5 N-端序列的硫酸化肽， 末端可以阻断HIV-1进入人PBMC和巨噬细胞，并已开始详细绘制 HIV-1包膜糖蛋白中与CCR 5 N-末端相互作用的区域。最后，我们记录了 CXCR 4上类似硫酸酯部分的存在和作用。在这次更新中，将继续调查 HIV-1包膜糖蛋白和HIV-1辅助受体之间联系的分子细节。 为实现这一目标，提出了以下具体目标。 在具体目标1中，我们将进一步分析gp 120区域与个体的分子相互作用。 结构域的CCR 5，使用Ig-融合构建体和生物硫酸化的CCR 5肽，我们已经开发。 在具体目标2中，我们将通过利用_NR5（N-末端缺失）来检查HIV-1融合过程 CCR 5）和硫酸化CCR 5肽。我们在上一个资助期间已经证明，这一_NR5确实 本身不支持HIV-1进入，但当与N硫酸化CCR 5肽互补时， 这是一个很好的系统来剖析复杂的HIV-1融合过程。 在具体目标3中，我们试图记录CCR 5酪氨酸硫酸化的水平和调节， 原代HIV-1靶细胞。我们还将研究酪氨酸硫酸化对内化的贡献， CCR 5响应其趋化因子配体的再循环，这是趋化因子介导的抑制的关键过程 HIV-1进入

In this continuation proposal, we seek to expand and refine our understanding of the interaction between HIV-1 envelope glycoprotein and its coreceptor CCR5. During the last funding period, we confirmed our initial observation of the presence of tyrosine sulfation in the N-terminus of CCR5 and its critical role in HIV-1 entry. We also demonstrated that sulfated peptides based on the sequence of CCR5 N- terminus can block HIV-1 entry in human PBMC and macrophages and have begun to map in detail the regions in HIV-1 envelope glycoprotein that interact with CCR5 N-terminus. Finally, we have documented the presence and role of analogous sulfate moieties on CXCR4. In this renewal, will continue to investigate the molecular details of the association between HIV-1 envelope glycoprotein and the HIV-1 coreceptors. The following specific aims are offered to accomplish this goal. In Specific Aim 1, we will further analyze the molecular interactions of regions of gp120 with individual domains of CCR5, using the Ig-fusion constructs and biosulfated CCR5 peptides we have developed. In Specific Aim 2, we will examine the HIV-1 fusion process by utilizing _NR5 (N-terminal deleted CCR5) and sulfated CCR5 peptides. We have demonstrated during last funding period that this _NR5 does not support HIV-1 entry by itself, but does when complemented with N sulfated CCR5 peptides, offering an excellent system to dissect the complex HIV-1 fusion process. In Specific Aim 3, we seek to document the levels and regulation of CCR5's tyrosine sulfation in primary HIV-1 target cells. We will also study the contribution of tyrosine sulfation to the internalization and recycling of CCR5 in response to its chemokine ligands, a process criticalto chemokine mediated inhibition of HIV-1 entry.