Association of HIV-1 gp120 with its coreceptors

描述（由申请人提供）：在本延续提案中，我们寻求扩大和完善我们对HIV-1包膜糖蛋白及其辅助受体CCR 5之间相互作用的理解。在上一个资助期间，我们证实了我们最初观察到的CCR 5 N端酪氨酸硫酸化的存在及其在HIV-1进入中的关键作用。我们还证明了基于CCR 5 N-末端序列的硫酸化肽可以阻断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，我们已经证明了CXCR 4上类似硫酸酯基团的存在和作用。在这次更新中，将继续研究HIV-1包膜糖蛋白和HIV-1辅助受体之间关系的分子细节。为实现这一目标，提出了以下具体目标。在具体目标1中，我们将使用我们开发的Ig融合构建体和生物硫酸化CCR 5肽，进一步分析gp 120区域与CCR 5单个结构域的分子相互作用。在具体目标2中，我们将通过利用delta NR 5（N-末端缺失的CCR 5）和硫酸化CCR 5肽来检查HIV-1融合过程。我们在上一个资助期间已经证明，这种delta NR 5本身并不支持HIV-1进入，但当与N硫酸化CCR 5肽互补时，可以提供一个极好的系统来剖析复杂的HIV-1融合过程。在具体目标3中，我们试图记录原代HIV-1靶细胞中CCR 5酪氨酸硫酸化的水平和调节。我们还将研究酪氨酸硫酸化对CCR 5响应其趋化因子配体的内化和再循环的贡献，这是趋化因子介导的HIV-1进入抑制的关键过程。

DESCRIPTION (provided by applicant): 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 delta NR5 (N-terminal deleted CCR5) and sulfated CCR5 peptides. We have demonstrated during last funding period that this delta 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 critical to chemokine mediated inhibition of HIV-1 entry.