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，其中我们提出研究β趋化因子调节原代巨噬细胞（相对于淋巴细胞）HIV-1感染的分子机制。趋化因子受体家族的成员CXCR4和CCR5已被证明分别作为HIV-1嗜t型（X4）和嗜m型（R5）菌株的融合辅助因子。在人类中发现纯合子缺陷CCR5等位基因（CCR5 delta32）与HIV-1感染抗性相关，证明了CCR5在HIV-1传播中的重要性。此外，这一观察结果表明，靶向HIV-1- ccr5相互作用可能提供一种干扰HIV-1感染的手段。然而，尽管大量研究表明CCR5的天然配体（β趋化因子；mip -1 α， mip -1 β和RANTES）抑制淋巴细胞中HIV-1的复制，但我们的研究结果表明β趋化因子对原代巨噬细胞中HIV-1的复制具有增强而不是抑制作用。我们已经开始解决这一现象背后的机制，我们的初步数据表明，在巨噬细胞中，β趋化因子治疗只诱导HIV-1进入的短暂抑制，随后是病毒复制的上调（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）。由于巨噬细胞在病毒传播过程中是HIV-1的重要靶点，因此本研究的主要目标是确定β趋化因子处理的巨噬细胞中促进HIV-1复制上调的机制。我们拟通过β趋化因子表征CCR5表达的调控，并确定巨噬细胞中CCR5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，我们建议鉴定巨噬细胞中与CCR5受体结合的β趋化因子激活的G蛋白和下游效应物，并表征它们对巨噬细胞中参与HIV-1复制的信号通路的影响（淋巴细胞将被平行检测以进行比较）。总之，这些拟议的研究应该为β -趋化因子在原代巨噬细胞中调节HIV-1感染的机制提供新的见解，原代巨噬细胞是HIV-1感染传播过程中病毒的重要靶点，从而有助于更好地理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.