The in vivo function of A3A and A3G during retrovirus infection

描述（由申请人提供）：逆转录病毒可感染多种物种，包括人类。逆转录病毒DNA整合到基因组中，导致持续感染。哺乳动物基因组也不断受到内源性逆转录病毒和逆转录因子的威胁。因此，他们开发了多种机制，以限制逆转录病毒感染前病毒整合。在宿主限制因子中，有具有胞苷脱氨酶活性（CDA）的APOBEC 3蛋白，其在逆转录病毒复制期间起作用并抑制逆转录转座。人类A3（hA 3）家族有7个成员（A3 A-A3 H），而啮齿类动物只有一个A3基因。所有A3蛋白都具有CDA结构域，并且可以将逆转录的单链DNA中的胞苷转化为尿苷，从而导致病毒DNA的突变。虽然人A3蛋白已在组织培养中被广泛研究，但对其在体内的作用知之甚少。在这里，我建议使用我最近创建的转基因小鼠，并表达2种人类A3蛋白，A3 A和A3 G，目的是确定它们在体内限制逆转录病毒的能力。A3 A在体外限制病毒和逆转录元件逆转录转座中具有有效作用，而A3 G抑制逆转录病毒如HIV-1。此外，已经表明A3蛋白在预防人畜共患病中起关键作用，并且实际上，几种人A3蛋白已经显示出在体外限制小鼠逆转录病毒。由于其有效的抗病毒活性，目前正在研究增加A3活性作为抗病毒治疗的靶点，但这是否有可能导致细胞DNA损伤或耐药逆转录病毒尚不清楚。因此，我们将使用这些转基因小鼠来测试它们对小鼠乳腺肿瘤病毒（MMTV）和小鼠白血病病毒（MLV）感染的影响，小鼠是逆转录病毒的天然宿主，并且仅部分受小鼠A3（mA 3）限制。我们的实验室（宾夕法尼亚大学Susan Ross博士的实验室）率先使用转基因小鼠，包括mA 3敲除小鼠，研究体内宿主-逆转录病毒相互作用。因此，这个项目将为我提供逆转录病毒和转基因小鼠模型生成方面的培训，同时借鉴我以前在小鼠病毒发病机制方面的培训。这项研究将提供深入了解A3蛋白在体内的功能， 并且还具有创建用于测试治疗人类逆转录病毒感染的治疗策略的新模型的潜力。

DESCRIPTION (provided by applicant): Retroviruses can infect a variety of species, including humans. Retroviral DNA integrates into the genome, resulting in persistent infections. Mammalian genomes are also under constant threat by endogenous retroviruses and retroelements. They thus have developed multiple mechanisms to restrict retroviral infections prior to proviral integration. Among the host restriction factors are APOBEC3 proteins with cytidine deaminase activity (CDA) that act during retroviral replication and that inhibit retrotransposition. There are 7 human A3 (hA3) family members (A3A-A3H), while rodents have a single A3 gene. All A3 proteins have CDA domains and can convert cytidines to uridines in reverse-transcribed single-stranded DNA, resulting in mutation of the viral DNA. While the human A3 proteins have been extensively studied in tissue culture, little is known about their action in vivo. Here, I propose to use transgenic mice that I recently created and express 2 human A3 proteins, A3A and A3G, with the goal of determining their ability to restrict retroviruses in vivo. A3A has a potent role in restricting viruses and retroelement retrotransposition in vitro, while A3G inhibits retroviruses such as HIV-1. Moreover, it has been suggested that A3 proteins play a critical role in preventing zoonoses and indeed, several human A3 proteins have been shown to restrict mouse retroviruses in vitro. Because of their potent antiviral activity, increasing A3 activity is currently being investigated as a target of ani-viral therapy but whether this has the potential to lead to cellular DNA damage or to drug-resistant retroviruses is not known. We will thus use these transgenic mice to test their effects on mouse mammary tumor virus (MMTV) and murine leukemia virus (MLV) infection, retroviruses for which the mouse is the natural host and which are only partially restricted by mouse A3 (mA3). Our laboratory (Dr. Susan Ross' laboratory at the University of Pennsylvania) pioneered the use of genetically modified mice, including mA3 knockout mice, to study host-retrovirus interactions in vivo. Thus, this project will provide me with training in retroviruses ad the generation of transgenic mouse models, while drawing on my previous training in viral pathogenesis in mice. This study will provide insight into the function of the A3 proteins in vivo, and also has the potential to create new models for testing therapeutic strategies for treating retroviral infections in humans.