Introducing restriction factors into the genome of an AIDS virus host species

描述(由申请者提供)：特定物种的限制因素吸引了艾滋病毒/艾滋病研究人员的特殊研究兴趣。这种先天免疫是由APOBEC3蛋白、TRIM5pha、TRIMCyp和BST2/Tetherin等蛋白质介导的。为了感染新物种，逆转录病毒必须进化出特定的对策。值得注意的是，现在人们认为灵长类慢病毒的辅助基因(vif、vpx、vPu、nef，可能还有vpr)主要用于对抗物种特异性限制因子的抗病毒作用。限制因子科学通过GNE疗法或小分子途径在治疗中的应用引起了人们的浓厚兴趣。关于这些因素在体内、在人体内的实际作用，仍有许多未知之处。这包括它们是否可以被利用来预防经粘膜或经皮感染。这个想法很有说服力，因为这些因素可以在信号级联和/或适应性免疫建立之前立即发挥作用，并以细胞自主的方式发挥作用。这一领域的实验在很大程度上是在不同物种的培养细胞中进行的，取得了巨大的成功。对猕猴和人类的等位基因影响的观察也很有限(此前，对小鼠逆转录病毒的Fv1系统也是如此)。然而，对于慢病毒来说，对易感物种进行受控的身体水平的遗传操作是不可能的。我们不知道一个单一的限制因素或它们的组合能在多大程度上保护宿主，以及在三个广泛被认为的水平中的哪一个：初始感染、全身性病毒血症的发展和疾病(艾滋病)。限制因子从来没有通过引入对慢病毒敏感的物种的种系来进行实验测试。在这里，我们将首次进行这样的实验。我们将在一种天然的慢病毒中完成这项工作 罹患艾滋病并感染大流行艾滋病病毒的宿主。我们实现了高效的猫转基因，使这成为可能。我们将这个试点项目的结果作为R01提案的初步数据。与啮齿动物不同，家猫自然容易感染艾滋病病毒。此外，FIV还受到灵长类限制因素的限制，包括东半球和新大陆的TIMCyps。这是这种艾滋病模型的理想用途。它将使无法在小鼠、大鼠、兔子、猕猴或人身上进行的实验成为可能。我们将在上述三个潜在水平上确定限制因子(S)阻断FIV的能力。我们将回答是否可以通过添加限制因子基因使一个物种对其艾滋病病毒具有遗传免疫力。我们还将确定病毒是否以及如何进化作为回应。我们将反过来确定，当病毒复制时，受保护的细胞谱系是否被选择并在体内得到丰富。因此，这个项目在基础科学水平和翻译水平上都是创新的。我们相信我们是做这项工作的理想实验室，因为我们有多年处理猫和分析其组织的经验，我们有FIV和HIV的具体经验，我们有宿主细胞因素和限制的背景和知识，我们实现了高效的猫科动物转基因。

DESCRIPTION (provided by applicant): Species-specific restriction factors have attracted exceptional research interest from HIV/AIDS researchers. This kind of innate immunity is mediated by proteins such as APOBEC3 proteins, TRIM5alpha, TRIMCyp, and BST2/Tetherin. To infect new species, retroviruses must evolve specific countermeasures. Remarkably, it is now believed that the accessory genes (vif, vpx, vpu, nef, and probably vpr) of primate lentiviruses are largely devoted to countering the antiviral effects of species-specific restrictio factors. There is intensive interest in therapeutic application of restriction factor science via gne therapy or small molecule approaches. Much remains unknown about how these factors actually function in vivo, in a body. This includes whether they can be exploited to protect against trans-mucosal or percutaneous infection. The idea is compelling because these factors can act immediately, before signaling cascades and/or adaptive immunity are mounted, and in a cell-autonomous way. The experiments in this field have largely been conducted in cultured cells of diverse species, with great success. There are also limited observations of allelic influences in macaques and humans (and, previously, the Fv1 system for mouse retroviruses). However, controlled body-level genetic manipulation of a susceptible species has not been possible for lentiviruses. We do not know how much a single restriction factor or a combination of them can defend a host and at which of three broadly considered levels: initial infection, development of systemic viremia, and disease (AIDS). Restriction factors have never been experimentally tested by introduction into the germline of a species susceptible to a lentivirus. Here we will carry out such experiments for the first time. We will do this in a natural lentiviral host that develops AIDS and is afflicted with a pandemic AIDS virus. We enabled efficient cat transgenesis to make this possible. We include the results of this pilot project as preliminary data for this R01 proposal. Unlike rodents, the domestic cat is naturally susceptible to an AIDS virus. Moreover, FIV is restricted by primate restriction factors, including both Old and New World TRIMCyps. This is an ideal use of this AIDS model. It will enable experiments that cannot be done in mice, rats, rabbits, macaques or people. We will determine the ability of restriction factor(s) to block FIV at the three potential levels described above. We will answer whether a species can be rendered genetically immune to its AIDS virus by addition of restriction factor genes. We will also determine whether and how the virus evolves in response. We will determine, in turn, if protected cell lineages are selected and enriched in vivo as the virus replicates. Thus, this project is innovative at the basic science level and the translational level We believe we are an ideal lab to do this because we have years of experience working with the cat and analyzing its tissues, we have specific experience with FIV and HIV, we have background and knowledge in host cell factors and restriction, and we enabled efficient feline transgenesis.