Role of APOBEC3 in in vivo Restriction of Retrovirus Infection

在整个进化过程中，感染因子已经感染了原核生物和真核生物。事实上， 生物体及其传染媒介的感染，随着宿主保护性反应的发展和适应性 传染源的对策。传染性地方性逆转录病毒，如鼠白血病病毒（MLV） 已经在小鼠体内存在了数百万年，为我们提供了一个出色的模型系统， 哺乳动物宿主抑制病毒复制，相反，病毒如何抵消这种限制。一个系统 载脂蛋白B mRNA编辑酶催化肽3（APOBEC 3） 蛋白质家族，其在病毒体产生细胞中包装成逆转录病毒，并在感染靶细胞后， 阻断逆转录或使单链DNA中的脱氧胞苷残基脱氨基， 尿嘧啶和病毒基因组中的G到A突变。逆转录病毒逆转录产物（ssRNA、ssDNA 和dsDNA）也被宿主核酸传感器检测。这些传感器与病毒核酸的结合导致 生产抗病毒细胞因子和趋化因子，如I型干扰素，它“警告”周围 细胞通过产生蛋白质如APOBEC 3来武装自己抵抗感染。这些宿主的抗病毒事件 被认为主要发生在细胞质中，其中APOBEC 3蛋白和许多宿主传感器被认为 来运作。 逆转录病毒进入细胞时，病毒和宿主膜融合，衣壳沉积在细胞膜上。 细胞质逆转录从衣壳内开始，衣壳解离并逆转录。 转录是相互依赖的;因为DNA比RNA更刚性，没有衣壳解离，逆转录 转录不能进行，相反，DNA的产生促进衣壳解离。反向 转录复合物不仅由病毒RNA、DNA和病毒蛋白质逆转录酶组成， 整合酶，但病毒衣壳和其他蛋白质，如MLV蛋白p12，这是必要的拴系 前病毒DNA与宿主染色质结合以实现整合。 最近，关于逆转录是否只发生在细胞质中， 或者两者都有。我们的实验室率先使用体内小鼠模型来研究A3蛋白如何限制 逆转录病毒感染，并已开发出A3基因敲除小鼠和基因工程动物，表达 人A3蛋白。基于我们对A3基因敲除小鼠和细胞的分析，我们的数据强烈表明， 逆转录的步骤发生在细胞质中。有了这些老鼠模型，我们就有了工具， 体外、离体和体内研究，以确定A3介导的反向转录物的限制和感应 整合了逆转录和核进入MLV及其天然宿主，小鼠。到 为了实现这一目标，我们提出了三个目标，这将决定：一。在细胞中，APOBEC 3蛋白阻止 逆转录或脱氨基病毒DNA; II.逆转录的哪个阶段， 宿主碱基切除修复酶UNG修复APOBEC 3G-脱氨基的病毒DNA; III.是否 病毒核酸的宿主感应发生在细胞质、细胞核或两个区室中。 确定这些事件发生的时间和地点对于理解逆转录病毒，包括艾滋病毒， 逃避宿主免疫，并确定哪些步骤可能是干预的最佳靶点。 在感染的早期阶段进行治疗。

Infectious agents have infected prokaryotes and eukaryotes throughout evolution. Indeed, there is co-evolution of organisms and their infectious agents, with development of protective responses in the hosts and adaptive countermeasures by the infectious agents. Infectious endemic retroviruses like murine leukemia virus (MLV) have existed in mice for millions of years and provide us with an outstanding model system to understand how mammalian hosts suppress virus replication and conversely, how viruses counteract this restriction. One system of viral restriction is conferred by the apolipoprotein B mRNA editing enzyme, catalytic peptide 3 (APOBEC3) family of proteins, which are packaged into retroviruses in virion-producing cells and after infection of target cells, either block reverse transcription or deaminate deoxycytidine residues in single-stranded DNA, resulting in uracils and G-to-A mutations in the viral genome. The products of retrovirus reverse transcription (ssRNA, ssDNA and dsDNA) are also sensed by host nucleic acid sensors. Binding of these sensors to viral nucleic acid leads to the production of anti-viral cytokines and chemokines, such as type I interferons, which “warns” surrounding cells to arm themselves against infection by producing proteins such as APOBEC3. These host anti-viral events are believed to occur largely in the cytoplasm, where APOBEC3 proteins and many host sensors are believed to function. Retroviruses enter cells when the viral and host membranes fuse and capsids are deposited in the cytoplasm. Reverse transcription initiates from within the capsid and capsid dissociation and reverse transcription are mutually dependent; because DNA is more rigid than RNA, without capsid dissociation, reverse transcription cannot proceed and conversely, the generation of DNA facilitates capsid dissociation. The reverse transcription complex not only consists of viral RNA, DNA and the viral proteins reverse transcriptase and integrase, but viral capsid and other proteins such as the MLV protein p12, which is needed for tethering of the proviral DNA to host chromatin to achieve integration. Recently, there has been much debate as to whether reverse transcription occurs solely in the cytoplasm or in the nucleus or both. Our lab pioneered the use of in vivo mouse models to study how A3 proteins restrict retrovirus infection and has developed A3 knockout mice and genetically engineered animals that express human A3 proteins. Our data, based on our analysis of A3 KO mice and cells, strongly suggest that the initial steps of reverse transcription occurs in the cytoplasm. With these mouse models, we have the tools to carry out in vitro, ex vivo and in vivo studies to determine how A3-mediated restriction and sensing of reverse transcripts are integrated with reverse transcription and nuclear entry for MLV and its natural host, the mouse. To accomplish this, we propose 3 aims, that will determine: I. Where in the cells APOBEC3 proteins block reverse transcription or deaminate viral DNA; II. What stage in reverse transcription and where in the cell the host base excision repair enzyme, UNG, repairs APOBEC3G-deaminated viral DNA; III. Whether host sensing of viral nucleic acid takes place in the cytoplasm, nucleus or both compartments. Determination of when and where these events occur is critical to understanding how retroviruses, including HIV, evade host immunity and the identification of which steps are likely to be the best targets for interventional therapies at the early stages of infection.