Elucidating the Regulation of HIV RNA Functions: Translation Genome Packaging

逆转录病毒全长RNA在病毒复制中有两个重要作用：Gag/Gag-Pol翻译模板和病毒粒子中的基因组。我们之前的结果显示，大多数HIV-1颗粒都有两个病毒RNA基因组副本，表明包装受到严格调控。我们已经进行了研究，并表明HIV-1的包装不是由RNA质量调节的，而是通过识别二聚体RNA来调节的。我们现在已经定义了将RNA包装成HIV-1病毒粒子所必需的和充分的序列。此外，我们还确定了几个高结构的RNA元件在HIV-1复制中的作用。我们还在研究全长RNA的翻译，并描绘了影响HIV-1RNA核输出的宿主因素以及HIV-1RNA从核到细胞质的输出动力学。这些实验试图深入了解HIV-1RNA是如何发挥作用的。_背景：HIV-1全长RNA(以下简称HIV-1RNA)作为Gag/Gag-Pol翻译模板和病毒基因组。HIV-1RNA需要通过复杂的细胞调节从细胞核输出到细胞质。一旦出口，HIV-1RNA可以被翻译和/或包装，需要在这两种功能之间取得平衡。在这个项目中，我们试图更好地了解HIV-1RNA是如何发挥作用的。_作为一种非剪接的RNA，HIV-1RNA需要绕过细胞守门人从细胞核输出到细胞质。HIV-1RNA包含一个RNA结构，即REV反应元件(RRE)。病毒蛋白REV与RRE结合，并与宿主蛋白CRM1相互作用，从而允许HIV-1RNA的出口。最近的研究表明，RNA输出的调控可能比之前想象的更复杂，可能涉及除CRM1和RanGTP之外的多种宿主因素。我们正在研究出口途径如何影响HIV-1RNA细胞质运输和与RNA相关的蛋白质。最近有研究表明，HIV-1使用异源转录起始位点，这些位点的使用会影响RNA的功能。我们已经与Karin Musier-Forsyth博士建立了合作关系，研究HIV-1转录起始点的使用对RNA结构和功能的影响。此外，我们正在与Brandon Keele博士合作研究使用转录起始点来控制RNA功能的保守性。_成就：为了更好地了解HIV-1基因的表达，我们跟踪了活细胞中单个HIV-1 RNA的翻译。我们发现，平均而言，在给定的时间内，一半的细胞质HIV-1 RNA正在被主动翻译。此外，翻译和非翻译的RNA在细胞质中很好地混合在一起；因此，Gag的生物发生发生在整个细胞质中，而不限于特定的亚细胞位置。因为Gag是一种RNA结合蛋白，包装着HIV-1RNA，所以关于HIV-1基因表达的一个长期存在的问题是，Gag是否调节自己的翻译。我们观察到，尽管Gag具有与RNA结合的能力，但它的表达并不改变翻译HIV-1RNA的比例。虽然翻译和非翻译的RNA都可以进入质膜，这是HIV-1的主要组装部位，但GAG选择性地将非翻译的RNA包装到组装复合体中。这些研究表明，尽管HIV-1RNA具有翻译模板和病毒基因组两种功能，但单个RNA分子一次只能执行一种功能。_HIV-1转录从多个相邻的位点开始，产生的RNA物种只在5‘端有几个核苷酸差异，包括那些含有一个(1G)或三个(3G)5’鸟苷的RNA物种。引人注目的是，1G RNA优先包装成病毒粒子，而不是3G RNA。我们使用生化和病毒学分析来研究HIV-1如何区分这两个几乎相同的HIV-1 RNA。我们发现，1G RNA，而不是3G RNA，主要折叠成结构，暴露对RNA：RNA和RNA：GAG相互作用重要的元件。此外，我们还鉴定了1G和3G RNA折叠成相似结构的突变体，导致了3G RNA的有效包装。因此，HIV-1根据其采用促进RNA二聚化和Gag结合的结构的能力来选择其病毒基因组。未剪接的HIV-1RNA在病毒复制过程中起着两个重要的作用：作为病毒粒子基因组和作为Gag/Gag-Pol翻译的模板。以前对两个HIV-1分子克隆的研究得出结论，TSS的使用会影响未剪接的HIV-1RNA的结构和功能。为了研究这一复制策略的进化起源，我们确定了HIV-1RNA在感染细胞中的转录起点(TSS)和15种灵长类慢病毒的病毒粒子。所有被检查的HIV-1分离株，包括几种传播的方正病毒，都使用了多个TS，并选择了一种特定的RNA物种进行包装。此外，在与HIV-1前体相关的SIV中观察到了这些特征，表明这些特征来自于祖先病毒。HIV-2、与HIV-2相关的SIV和其他SIV也表现出多个TS和特定非剪接RNA物种的优先包装。这些发现表明，在HIV-1出现之前，多个TS的使用和对特定非剪接RNA物种的选择性包装。

Retroviral full-length RNA serves two important roles in viral replication: the template for Gag/Gag-Pol translation and the genome in the virion. Our previous results showed that most HIV-1 particles have two copies of viral RNA genome, indicating that the packaging is tightly regulated. We have performed studies and showed that HIV-1 packaging is not regulated by RNA mass but by recognizing a dimeric RNA. We have now defined the sequences necessary and sufficient for packaging RNA into HIV-1 virions. Additionally, we have determined the role of several highly structured RNA elements in HIV-1 replication. We are also examining the translation of the full-length RNA, and delineating the host factors that affect HIV-1 RNA nuclear export and the dynamics of HIV-1 RNA export from the nucleus to the cytoplasm. These experiments seek to gain insights into how HIV-1 RNA serves its functions. ___BACKGROUND: The full-length HIV-1 RNA (hereafter referred to as HIV-1 RNA) serves as a template for Gag/Gag-Pol translation and as the virion genome. HIV-1 RNA needs to negotiate through the complex cellular regulation of the host to be exported from the nucleus to the cytoplasm. Once exported, HIV-1 RNA can be translated and/or packaged and needs to strike a balance between these two functions. In this project, we seek to gain a better understanding of how HIV-1 RNA serves its roles. ___As an unspliced RNA, HIV-1 RNA needs to bypass the cellular gatekeepers to be exported from the nucleus and reach the cytoplasm. HIV-1 RNA contains an RNA structure, the Rev responsive element (RRE). The viral protein Rev binds to the RRE and interacts with the host protein CRM1 to allow for the export of HIV-1 RNA. Recent studies revealed that the regulation of RNA export may be more complex than previously envisioned and may involve multiple host factors other than CRM1 and RanGTP. We are studying how export pathways affect cytoplasmic HIV-1 RNA transport and proteins associated with the RNA. ___It was recently shown that HIV-1 uses heterogenous transcriptional start sites and the usage of the sites can affect RNA function. We have established a collaboration with Dr. Karin Musier-Forsyth to examine the impact of HIV-1 transcription start site usage on RNA structures and functions. Additionally, we are collaborating with Dr. Brandon Keele to study the conservation of the usage of transcription start sites to control RNA functions. ___ACCOMPLISHMENTS: To gain a better understanding of HIV-1 gene expression, we tracked translation of individual HIV-1 RNAs in living cells. We found that, on average, half of the cytoplasmic HIV-1 RNAs are being actively translated at a given time. Furthermore, translating and nontranslating RNAs are well mixed in the cytoplasm; thus, Gag biogenesis occurs throughout the cytoplasm without being constrained to particular subcellular locations. Because Gag is an RNA-binding protein and packages HIV-1 RNA, a long-standing question regarding HIV-1 gene expression is whether Gag modulates its own translation. We observed that despite its RNA-binding ability, Gag expression does not alter the proportion of translating HIV-1 RNA. Although both translating and nontranslating RNAs can travel to the plasma membrane, the major HIV-1 assembly site, Gag selectively packages nontranslating RNA into the assembly complex. These studies illustrate that although HIV-1 RNA serves two functions, as a translation template and as a viral genome, individual RNA molecules carry out only one function at a time. ____ HIV-1 transcription initiates from multiple neighboring sites, generating RNA species that only differ by a few nucleotides at the 5' end, including those with one (1G) or three (3G) 5' guanosines. Strikingly, 1G RNA is preferentially packaged into virions over 3G RNA. We used biochemical and virological assays to investigate how HIV-1 distinguishes between these two nearly identical HIV-1 RNAs. We found that 1G RNA, but not 3G RNA, mainly folds into structures that expose elements important for RNA:RNA and RNA:Gag interactions. Additionally, we have identified mutants in which 1G and 3G RNAs fold into similar structures, resulting in efficient packaging of 3G RNA. Thus, HIV-1 selects its viral genome based on its capacity to adopt structures that facilitate RNA dimerization and Gag binding. Unspliced HIV-1 RNA serves two important roles during viral replication: as the virion genome and as the template for translation of Gag/Gag-Pol. Previous studies of two HIV-1 molecular clones have concluded that the TSS usage affects unspliced HIV-1 RNA structures and functions. To investigate the evolutionary origin of this replication strategy, we determined transcription start sites (TSS) of HIV-1 RNA in infected cells and virions for 15 primate lentiviruses. All the HIV-1 isolates examined, including several transmitted founder viruses, utilized multiple TSS and selected a particular RNA species for packaging. Furthermore, these features were observed in SIVs related to the progenitors of HIV-1, suggesting that these characteristics originated from the ancestral viruses. HIV-2, SIVs related to HIV-2, and other SIVs also exhibited multiple TSS and preferential packaging of specific unspliced RNA species. These findings indicate that multiple TSS usage and selective packaging of a particular unspliced RNA species predate the emergence of HIV-1.