Interactions Between HIV Gag and Exosomal Proteins

HIV相关性痴呆（HAD）是HIV感染的一种重要共病。其不太严重的形式似乎对高效抗逆转录病毒疗法（HAART）具有抗性，并且HIV感染的巨噬细胞（MDM）和小胶质细胞是其发展的关键。由于它们在HIV感染的这个和其他方面的重要性，MDM中的周期已经被广泛研究，并且大多数研究人员认为巨噬细胞感染可以是长期的并且几乎不产生细胞病理学，使得这些细胞成为治疗个体中病毒的理想潜在储存库，并且虽然感染过程中的大多数步骤与淋巴细胞中的那些步骤平行，但是病毒感染过程中的大多数步骤与淋巴细胞中的那些步骤平行。 组装和排出是不同的，并且涉及胞质内组装和路由到多泡体（MVB）中，而不是在质膜上出芽。随后的病毒释放被认为是通过涉及血浆和MVB限制膜的直接融合的胞吐途径发生的。使用模型小胶质细胞/巨噬细胞感染，已被修改，使有低产量的病毒，我们已经确定了gag和膜联蛋白II（Anx 2），一个多效性蛋白参与外泌体的形成和融合之间的结合。这种蛋白质在巨噬细胞和小胶质细胞中高水平表达，特别是在受感染的大脑中。在本提案中，我们将在三个具体目标中发展这些初步结果。上 目的：进一步研究Anx 2在MDM中的表达减少对HIV感染的影响，并分析活化和非活化MDM中Anx 2蛋白表达的差异。在第二个具体目标中，我们将绘制p55/p24和Anx 2之间的结合位点，并将突变引入HIV前病毒，以确定对病毒感染的影响。在第三个具体目标中，我们将研究其表面表达和研究Anx 2在HIV和SIV感染的大脑中的分布，并使用荧光标记的分子进行搭配实验。这些结果将扩大我们对巨噬细胞慢病毒感染的理解，并可能为HIV和HAD的治疗干预提供额外的靶点。

HIV associated Dementia (HAD) is an important co-morbidity of HIV infection. Its less severe form appears to be resistant to highly active anti-retroviral therapy (HAART) and HIV infected macrophages (MDM) and microglia are key to its development. Because of their importance in this and other facets of HIV infection, the cycle in MDM has been studied extensively, and most investigators believe that macrophage infection can be long-lived and produce little cytopathology, making these cells ideal potential reservoirs for the virus in a treated individual, and that while most steps in the infectious process parallel those in lymphocytes, virus assembly and egress are different, and involve intracytoplasmic assembly and routing into multivesicular bodies (MVBs) in preference to budding at the plasma membrane. Subsequent release of virus is believed to take place via an exocytotic pathway involving direct fusion of plasma and MVB limiting membranes. Using a model microglia/macrophage infection that has been modified so that there is low production of virus, we have identified binding between gag and an annexin II (Anx2), a pleiotropic protein involved in exosomal formation and fusion. This protein is expressed to high levels in macrophages and microglia, particularly in the infected brain. In this proposal we will develop these preliminary results in three specific aims. In the first aim, we will further determine the effects of reduction of Anx2 in MDM on HIV infection, and analyze the differences between the protein expressed in activated vs. non-activated MDM. In the second specific aim, we will map the binding sites between p55/p24 and Anx2 and introduce mutations into HIV proviruses, to determine the effects on viral infection. In the third specific aim we will study its surface expression and study Anx2 distribution in the HIV and SIV infected brain, and perform collocation experiments using fluorescence tagged molecules. These results will expand our understanding of lentiviral infection of macrophages, and potentially provide an additional target for therapeutic intervention in HIV and HAD.