Murine Model System for HIV Pathogenesis

描述(申请人提供)：尽管在揭开艾滋病毒在人类细胞中复制周期的机制方面取得了进展，但我们对病毒如何在体内传播、它如何导致免疫缺陷以及为什么免疫反应通常不能有效地中和它的了解有限。这些问题很难在人类身上研究，甚至在非人类灵长类动物中也很难研究。我们的广泛目标是建立一个小鼠模型系统，它将忠实地再现HIV-1与其人类宿主的相互作用。这将允许对病毒传播、复制和致病机制的各个方面进行相对廉价的检查，并使用许多变量，还将允许研究艾滋病毒疾病的候选宿主遗传因素。开发这样的模型将需要更好地了解病毒是如何传播的，以及限制艾滋病毒在小鼠细胞中复制的特定物种的限制。体外研究表明，树突状细胞(DC)在HIV的传播和复制中具有重要作用。树突状细胞吞噬HIV颗粒，长时间携带HIV，增强反式T细胞感染。DC-SIGN是HIV内吞作用的关键DC受体，这是在DC：T细胞共培养中有效复制HIV所必需的过程。HIV与DC相互作用的重要性尚未在体内得到验证。具体目标1将是使用转基因小鼠来确定HIV在暴露于粘膜内的DC后是否可以传播到引流淋巴组织中的细胞。我们将结合SCID-Hu小鼠和表达人CD4、CCR5和Cyclin T1的小鼠来检测DC-SIGN转基因小鼠。尽管来自多重转基因动物的T细胞和巨噬细胞可以感染艾滋病毒，但它们无法释放感染颗粒。具体目标2将是使用基因方法来识别人类基因，这些基因可以增强小鼠细胞中艾滋病毒的组装和/或释放，并允许在小鼠模型中更好地复制艾滋病毒。具体目标3将是确定APOBEC3G，一种针对HIV Vif蛋白降解的抗病毒蛋白，是否限制了原代小鼠细胞的复制。如果是这样的话，我们将开发方法来克服它的活动。VIF可诱导人APOBEC3G的降解，但不能诱导小鼠APOBEC3G的降解。RNA干扰和基因打靶将被用来减少小鼠酶的表达，我们将用能结合并诱导小鼠APOBEC3G降解的Vif来改造HIV。

DESCRIPTION (provided by applicant): Despite progress in unraveling the mechanism of the HIV replication cycle in human cells, we have limited understanding of how virus is transmitted in vivo, how it causes immunodeficiency, and why immune responses are generally ineffective in neutralizing it. These issues are difficult to study in humans and even in non-human primates. Our broad aim is to generate a murine model system that will faithfully reproduce the interactions of HIV- 1 with its human host. This would allow for relatively inexpensive examination of various aspects of viral transmission, replication, and pathogenesis with numerous variables, and it would permit studies of candidate host genetic factors in HIV disease. Development of such a model will require a better understanding of how virus is transmitted and of species-specific restrictions that limit HIV replication in murine cells. In vitro studies suggest that dendritic cells (DC) have important roles in HIV transmission and replication. DC endocytose HIV particles, harbor HIV for long periods of time, and enhance infection of T cells in trans. DC-SIGN is a key DC receptor for HIV endocytosis, a process required for efficient HIV replication in DC:T cell co-cultures. The importance of HIV interaction with DC has not yet been tested in vivo. Specific Aim 1 will be to use genetically modified mice to determine if HIV can be transmitted to cells in draining lymphatic tissues after exposure to DC within mucosa. We will examine mice transgenic for DC-SIGN in combination with SCID-Hu mice and mice expressing human CD4, CCR5, and cyclin T1. Although T cells and macrophages from the multiply transgenic animals can be infected with HIV, they fail to release infectious particles. Specific Aim 2 will be to employ genetic approaches to identify human genes that enhance HIV assembly and/or release from murine cells and that will permit better replication of HIV in the murine model. Specific Aim 3 will be to determine if APOBEC3G, an antiviral protein targeted for degradation by the HIV Vif protein, restricts replication in primary mouse cells. If so, we will develop means to overcome its activity. Vif induces degradation of human, but not mouse, APOBEC3G. RNA interference and gene targeting will be used to reduce expression of the mouse enzyme, and we will engineer HIV with Vif that can bind to and induce degradation of mouse apobec3g.