Interplay between reverse transcription and host restriction

在整个进化过程中，感染剂感染了原核生物和真核生物。事实上，存在着共同进化。 指生物体及其感染体，在宿主中产生保护性反应，并适应 传染病病原体的应对措施。传染性地方性逆转录病毒，如小鼠白血病病毒(MLV) 已经在老鼠体内存在了数百万年，并为我们提供了一个出色的模型系统来理解 哺乳动物宿主抑制病毒复制，反之，病毒如何抵消这种限制。一个系统 病毒的限制是由载脂蛋白B mRNA编辑酶，催化多肽3(A3)家族 在产生病毒的细胞中和在感染目标细胞后包装成逆转录病毒的蛋白质，或者 阻断单链DNA中反转录或脱氨基脱氧胞苷残基，导致尿嘧啶和 病毒基因组的G-to-A突变。逆转录病毒反转录产物(单链RNA、单链DNA和 DsDNA)也被宿主核酸传感器感知到。与病毒核酸结合的传感器导致产生 抗病毒的细胞因子和趋化因子，如I型干扰素，它“警告”周围的细胞武装 通过产生A3等蛋白质来抵抗感染。这些宿主的抗病毒事件被认为发生在 主要在细胞质中，A3蛋白和许多宿主感受器被认为在那里发挥作用。 逆转录病毒进入细胞时，病毒和宿主膜融合，衣壳沉积在 细胞质。反转录从衣壳内开始，衣壳解离，反转 转录是相互依赖的；因为DNA比RNA更坚硬，没有衣壳解离，相反 转录不能进行，相反，DNA的产生有助于衣壳的解离。相反的 转录复合体不仅由病毒RNA、DNA和病毒蛋白逆转录酶及 整合酶，但病毒衣壳和其他蛋白质，如MLV蛋白p12，是拴住 前病毒DNA到宿主染色质中实现整合。 最近，关于逆转录是仅发生在细胞质中还是发生在细胞质中，存在着很大的争论。 在核内或两者都有。我们的实验室率先使用活体小鼠模型来研究A3蛋白如何限制 并利用A3和核酸传感器基因敲除(KO)小鼠进行了基因工程 在这些研究中表达人类A3蛋白的动物。我们的数据，基于我们对A3KO小鼠和 细胞，这表明最初的步骤发生在细胞质中，但逆转录也可能发生在 细胞质。有了这些小鼠模型，我们有了进行体外、体外和体内研究的工具 确定A3介导的反向转录的限制和传感如何与反向整合 MLV及其自然宿主小鼠的转录和核进入。为了实现这一目标，我们提出了三个目标， 这将决定：I.APOBEC3蛋白在细胞中的什么位置阻断反转录或使病毒脱氨基 DNA；ii.反转录的哪个阶段以及宿主碱基切除修复酶在细胞中的什么位置， Ung，从APOBEC3G脱氨的病毒DNA中去除尿嘧啶及其对病毒的影响 病毒核酸的宿主感应是发生在细胞质、细胞核还是两者都发生 车厢。确定这些事件发生的时间和地点对于了解 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最好的 感染早期介入治疗的目标。

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 (A3) 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. Sensor binding 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 A3. These host anti-viral events are believed to occur largely in the cytoplasm, where A3 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 used A3 and nucleic acid sensor knockout (KO) mice and genetically engineered animals that express human A3 proteins in these studies. Our data, based on our analysis of A3 KO mice and cells, suggest that the initial step occurs in the cytoplasm but that reverse transcription may also occur 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, removes uracil from APOBEC3G-deaminated viral DNA and the consequences of this for viral escape; 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.