MECHANISM OF REGULATION OF HIV-1 INFECTION BY CHEMOKINES

这是PI第一次独立支持请求的第二次重新提交，我们在该请求中提议研究原代巨噬细胞（相对于淋巴细胞）中HIV-1感染的β-趋化因子调控所涉及的分子机制。 趋化因子受体家族的成员CXCR 4和CCR 5已被证明分别作为HIV-1 T嗜性（X4）和M嗜性（R5）毒株的融合辅因子。 在人类中发现的CCR 5纯合缺陷等位基因（CCR 5 delta 32）与HIV-1感染抗性的相关性证明了CCR 5在HIV-1传播中的重要性。 此外，这一观察结果表明，靶向HIV-1-CCR 5相互作用可能提供一种干扰HIV-1感染的方法。然而，虽然大量的工作已经表明，天然配体CCR 5（β-趋化因子; MIP-1 α，MIP-1 β，和RANTES）抑制HIV-1在淋巴细胞中的复制，我们的研究结果表明，增强而不是抑制作用的β-趋化因子对HIV-1在原代巨噬细胞中的复制。 我们已经开始研究这种现象背后的机制，我们的初步数据表明，在巨噬细胞中，β-趋化因子治疗仅诱导短暂抑制HIV-1进入，随后上调病毒复制（淋巴细胞中HIV-1复制的抑制结合了进入和进入后水平的持续抑制）。 由于巨噬细胞是HIV-1在病毒传播过程中的一个重要靶点，因此本提案的主要目标是确定促进β-趋化因子处理的巨噬细胞中HIV-1复制上调的机制。 我们建议表征β-趋化因子对CCR 5表达的调节，并确定巨噬细胞中CCR 5表达的改变（与淋巴细胞相比）是否有助于观察到的现象。此外，我们建议确定G蛋白和下游效应激活的β-趋化因子结合CCR 5受体在巨噬细胞中表达，并表征其对参与HIV-1在巨噬细胞中复制的信号通路的影响（淋巴细胞将平行测定比较）。 总之，这些拟议的研究应提供新的见解β-趋化因子调节HIV-1感染的机制，在初级巨噬细胞，一个重要的目标，病毒在传播HIV-1感染，从而有助于更好地了解艾滋病发病机制的独特方面。

This is the second re-submission of the PI's first independent support request in which we propose to study the molecular mechanisms involved in beta-chemokine regulation of HIV-1 infection in primary macrophages (versus lymphocytes). The members of the chemokine receptor family, CXCR4 and CCR5, have been shown to act as fusion cofactors for HIV-1 T-tropic (X4) and M-tropic (R5) strains, respectively. The discovery in humans of the association of a homozygous defective CCR5 allele (CCR5 delta32) with resistance to HIV-1 infection demonstrated the importance of CCR5 in HIV-1 transmission. Furthermore, this observation suggested that targeting the HIV-1-CCR5 interaction may provide a means of interfering with HIV-1 infection. However, while a large body of work has demonstrated that the natural ligands of CCR5 (beta-chemokines; MIP-1alpha, MIP-1beta, and RANTES) inhibit HIV-1 replication in lymphocytes, our results demonstrated an enhancing rather than an inhibitory effect of beta-chemokines on HIV-1 replication in primary macrophages. We have begun to address the mechanisms which underlie this phenomenon and our preliminary data suggest that in macrophages beta-chemokine-treatment induces only transient inhibition of HIV-1 entry followed by upregulation of virus replication (inhibition of HIV-1 replication in lymphocytes combines sustained inhibition at the entry and post-entry level). Since macrophages are an important target of HIV-1 during virus transmission, a major goal of this proposal is to define the mechanisms which facilitate upregulation of HIV-1 replication in beta-chemokine-treated macrophages. We propose to characterize the regulation of CCR5 expression by beta-chemokines, and to establish whether altered expression of CCR5 in macrophages (as compared to lymphocytes) contributes to the observed phenomenon. Further, we propose to identify G proteins and downstream effectors activated by beta-chemokine binding to CCR5 receptors expressed in macrophages, and to characterize their effects on the signaling pathways involved in HIV-1 replication in macrophages (lymphocytes will be assayed in parallel for comparison). Together, these proposed studies should provide new insights into the mechanisms by which beta-chemokines regulate HIV-1 infection in primary macrophages, an important target of the virus during transmission of HIV-1 infection, and thus contribute to a better understanding of the unique aspects of AIDS pathogenesis.