The in vivo function of A3A and A3G during retrovirus infection

描述(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病毒的威胁。因此，他们开发了多种机制，在前病毒整合之前限制逆转录病毒感染。宿主限制因素包括具有胞苷脱氨酶活性(CDA)的APOBEC3蛋白，这些蛋白在逆转录病毒复制过程中发挥作用，并抑制逆转录转座。人类A3(HA3)家族有7个成员(A3A-A3H)，而啮齿动物只有一个A3基因。所有的A3蛋白都有CDA结构域，可以在逆转录的单链DNA中将胞苷转化为尿苷，导致病毒DNA的突变。虽然人类A3蛋白在组织培养中得到了广泛的研究，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造的转基因小鼠并表达两种人类A3蛋白，A3A和A3G，目的是确定它们在体内限制逆转录病毒的能力。A3A在体外抑制病毒和逆转录元件逆转录转位方面具有强大的作用，而A3G则抑制逆转录病毒，如HIV-1。此外，有研究表明，A3蛋白在预防人畜共患病方面发挥了关键作用，事实上，在体外，已有几种人类A3蛋白被证明可以限制小鼠逆转录病毒。由于其强大的抗病毒活性，增加A3活性目前正在作为抗病毒治疗的靶点进行研究，但这是否有可能导致细胞DNA损伤或产生抗药性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，这两种逆转录病毒是小鼠的自然宿主，只被小鼠A3(MA3)部分限制。我们的实验室(苏珊·罗斯博士在宾夕法尼亚大学的实验室)率先使用转基因小鼠，包括MA3基因敲除小鼠，研究体内宿主和逆转录病毒的相互作用。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。这项研究将对A3蛋白在体内的功能提供深入的了解， 并有可能创造新的模型来测试治疗人类逆转录病毒感染的治疗策略。

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.