Alkhatib G, Locati M, Kennedy P E, Murphy P M, Berger E A
The Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Virology. 1997 Aug 4;234(2):340-8. doi: 10.1006/viro.1997.8673.
HIV-1 infection requires the presence of specific chemokine receptors on CD4+ target cells to enable the fusion reactions involved in virus entry. CCR5 is a major fusion coreceptor for macrophage-tropic HIV-1 isolates. HIV-1 entry and fusion are mediated by the viral envelope glycoprotein (Env) and are inhibited by CCR5 ligands, but the mechanisms are unknown. Here, we test the role of G protein signaling and CCR5 surface downmodulation by two separate approaches: direct inactivation of CCR5 signaling by mutagenesis and inactivation of G(i)-type G proteins with pertussis toxin. A CCR5 mutant lacking the last 45 amino acids of the cytoplasmic C-terminus (CCR5306) was created that was expressed on transfected cells at levels comparable to cells expressing CCR5 and displayed normal chemokine binding affinity. CCR5 ligands induced calcium flux and receptor downmodulation in cells expressing CCR5, but not in cells expressing CCR5306. Nevertheless, CCR5 or CCR5306, when coexpressed with CD4, supported comparable HIV-1 Env-mediated cell fusion. Consistent with this, treatment of CCR5-expressing cells with pertussis toxin completely blocked ligand-induced transient calcium flux, but did not affect Env-mediated cell fusion or HIV-1 infection. Also, pertussis toxin did not block chemokine inhibition of Env-mediated cell fusion or HIV-1 infection. However, chemokines inhibited Env-mediated cell fusion less efficiently for CCR5306 than for CCR5. We conclude that the C-terminal domain of CCR5 is critical for G protein signaling and receptor downmodulation from the surface, but that neither function is required for CCR5 fusion coreceptor activity. The contrasting phenotypes of CCR5 and CCR5306 suggest that coreceptor downmodulation and direct blockage of Env interaction sites both contribute to chemokine inhibition of HIV-1 infection.
HIV-1感染需要CD4+靶细胞上存在特定的趋化因子受体，以促成病毒进入所涉及的融合反应。CCR5是嗜巨噬细胞HIV-1分离株的主要融合共受体。HIV-1的进入和融合由病毒包膜糖蛋白（Env）介导，并受CCR5配体抑制，但其机制尚不清楚。在此，我们通过两种不同方法测试G蛋白信号传导和CCR5表面下调的作用：通过诱变直接使CCR5信号失活，以及用百日咳毒素使G(i)型G蛋白失活。构建了一个缺失细胞质C末端最后45个氨基酸的CCR5突变体（CCR5306），其在转染细胞上的表达水平与表达CCR5的细胞相当，且显示出正常的趋化因子结合亲和力。CCR5配体在表达CCR5的细胞中诱导钙流和受体下调，但在表达CCR5306的细胞中则不然。然而，CCR5或CCR5306与CD4共表达时，支持相当的HIV-1 Env介导的细胞融合。与此一致的是，用百日咳毒素处理表达CCR5的细胞可完全阻断配体诱导的瞬时钙流，但不影响Env介导的细胞融合或HIV-1感染。此外，百日咳毒素不阻断趋化因子对Env介导的细胞融合或HIV-1感染的抑制作用。然而，趋化因子对CCR5306介导的Env细胞融合的抑制效率低于对CCR5的抑制。我们得出结论，CCR5的C末端结构域对于G蛋白信号传导和表面受体下调至关重要，但这两种功能对于CCR5融合共受体活性都不是必需的。CCR5和CCR5306的不同表型表明，共受体下调和Env相互作用位点的直接阻断都有助于趋化因子对HIV-1感染的抑制作用。
