Role of Tetraspan Glycoproteins (CD63) in HIV Replication

描述(申请人提供)：HIV感染原代人类细胞通常需要Env gp120与主要受体CD4和趋化因子受体CCR5或CXCR4相互作用，然后由Env gp41诱导融合。其他因素和过程也可能参与其中，它们的重要性可能在主要靶细胞、巨噬细胞(M？)和T细胞。通过对髓系特异性单抗文库的筛选，发现抗CD63单抗可以阻止HIV进入原代M细胞，但不能阻止T细胞进入。CD63是Tetraspanin膜糖蛋白家族的成员，现在与HIV感染(MS#1)有关，其他Tetraspanins与HTLV-I(CD82)、FIV(CD9)和丙型肝炎病毒(CD81)的感染有关。最近的研究表明，包括CD63第二细胞外环(EC2)的GST融合蛋白对其具有抑制作用，这进一步支持了CD63在HIV感染中的特定作用。自上次提交以来的新数据也表明CD63在HIV内体处理中的作用，因此我们将研究早期事件以及HIV生命周期后期发生的事件，以更全面地描述CD63在HIV感染中的潜在作用。这些研究可能会导致新的抗逆转录病毒疗法的开发。我们将在五年内实现这些目标：目标1.为了验证CD63与早期HIV复制事件有关的假设，我们将研究M？和T细胞/细胞系。目的2.为了验证CD63通常是感染原发M细胞和T细胞所必需的假设，我们将评估病毒蛋白的运输和组装，特别是在有和没有CD63沉默和小抑制RNA(SiRNA)的情况下病毒蛋白的运输和组装。我们还将沉默各种Rab蛋白，这是一种细胞GTP酶，通过内体途径运输细胞(和病毒)蛋白，以确定受CD63影响的HIV运输的步骤。目的3.验证CD63在M？(和其他细胞)，我们将评估B亚型和非B亚型毒株对CD63和CD63-EC2的敏感性，包括那些仅使用CCR5的毒株(R5、CXCR4单独使用(X4)或双嗜性毒株(R5X4))。

DESCRIPTION (provided by applicant): HIV infection of primary human cells typically requires interaction of Env gp120 with both the primary receptor CD4 and a chemokine receptor, either CCR5 or CXCR4, followed by Env gp41-induced fusion. Involvement of other factors and processes are likely, and their importance may differ between the principle target cells, macrophages (M?) and T-cells. Upon screening of a myeloid-specific monoclonal antibody (mAb) library, anti-CD63 mAb was found to block HIV entry into primary M?, but not T-cells. CD63 is a member of the tetraspanin membrane glycoprotein family, now implicated in HIV infection (MS #1), and other tetraspanins have been implicated in infection with HTLV-I (CD82), FIV (CD9) and Hepatitis C virus (CD81). Recent studies have shown inhibition with a GST fusion protein that includes the CD63 second extracellular loop (EC2), which further supports a specific role for CD63 in HIV infection. New data since the last submission also suggests a role for CD63 in endosomal processing of HIV, and so we will look at both early events, as well as events that occur later in the HIV life cycle, to more fully delineate the potential role of CD63 in HIV infection. These studies may lead to development of novel antiretroviral therapies. We will pursue these aims over five years: Aim 1. To test the hypothesis that CD63 is involved with early HIV replication events, we will investigate virus uptake and initiation of reverse transcription in M? and T cells/cell lines. Aim 2. To test the hypothesis that CD63 is generally required for infection of primary M?, as well at T cells/cell lines, we will assess trafficking and assembly of viral proteins, particularly Gag (and also Env) with and without CD63 silencing with small inhibiting RNAs (siRNA). We will also silence varions Rab proteins, which are cellular GTPases that traffic cellular (and viral) proteins through endosomal pathways in order to identify steps of HIV trafficking affected by CD63. Aim 3. To test the hypothesis that CD63 plays a role in infection of M? (and other cells) by diverse primary HIV 1 strains, we will assess anti-CD63 and CD63-EC2 susceptibility of both subtype B and non-subtype B strains, including those that use CCR5 exclusively (R5, CXCR4 exclusively (X4) or dual-tropic strains (R5X4).