Elucidating the Regulation of HIV RNA Functions: Translation Genome Packaging

逆转录病毒全长RNA在病毒复制中起着两个重要作用：Gag/Gag- pol翻译的模板和病毒粒子中的基因组。我们之前的结果表明，大多数HIV-1颗粒具有两个病毒RNA基因组拷贝，这表明包装受到严格调控。我们进行的研究表明，HIV-1的包装不受RNA质量的调节，而是通过识别二聚体RNA来调节。我们现在已经确定了将RNA包装成HIV-1病毒粒子所必需和充分的序列。此外，我们已经确定了几个高度结构化的RNA元件在HIV-1复制中的作用。我们还研究了全长RNA的翻译，并描绘了影响HIV-1 RNA核输出的宿主因素以及HIV-1 RNA从细胞核向细胞质输出的动力学。这些实验旨在深入了解HIV-1 RNA如何发挥其功能。【背景】全长HIV-1 RNA（以下简称HIV-1 RNA）作为Gag/Gag- pol翻译的模板和病毒粒子基因组。HIV-1 RNA需要通过宿主复杂的细胞调控才能从细胞核输出到细胞质。一旦输出，HIV-1 RNA就可以被翻译和/或包装，并且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-1 RNA是如何发挥作用的。作为一种未剪接的RNA， HIV-1 RNA需要绕过细胞看门人从细胞核输出并到达细胞质。HIV-1 RNA包含一种RNA结构，即Rev应答元件（RRE）。病毒蛋白Rev与RRE结合，并与宿主蛋白CRM1相互作用，允许HIV-1 RNA输出。最近的研究表明，RNA输出的调控可能比以前想象的更复杂，可能涉及CRM1和RanGTP以外的多种宿主因子。我们正在研究输出途径如何影响细胞质HIV-1 RNA运输和与RNA相关的蛋白质。最近的研究表明，HIV-1使用异质转录起始位点，这些位点的使用可以影响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转录起始位点的使用对RNA结构和功能的影响。此外，我们正在与Brandon Keele博士合作研究转录起始位点控制RNA功能的保守性。为了更好地理解HIV-1基因表达，我们跟踪了活细胞中单个HIV-1 rna的翻译。我们发现，在给定的时间内，平均有一半的细胞质HIV-1 rna被积极翻译。此外，翻译和非翻译rna在细胞质中很好地混合；因此，Gag生物发生发生在整个细胞质中，而不局限于特定的亚细胞位置。由于Gag是一种RNA结合蛋白并包装HIV-1 RNA，关于HIV-1基因表达的一个长期存在的问题是Gag是否调节其自身的翻译。我们观察到，尽管Gag具有RNA结合能力，但其表达并不改变翻译HIV-1 RNA的比例。尽管翻译RNA和非翻译RNA都可以到达主要的HIV-1组装位点质膜，Gag选择性地将非翻译RNA包装到组装复合体中。这些研究表明，尽管HIV-1 RNA具有翻译模板和病毒基因组两种功能，但单个RNA分子一次只执行一种功能。____ HIV-1转录从多个相邻位点开始，产生的RNA物种在5‘端只有几个核苷酸的差异，包括那些具有一个（1G）或三个（3G） 5’鸟苷的RNA物种。引人注目的是，1G RNA比3G RNA更容易被打包成病毒粒子。我们使用生化和病毒学分析来研究HIV-1如何区分这两种几乎相同的HIV-1 rna。我们发现1G RNA，而不是3G RNA，主要折叠成暴露RNA:RNA和RNA:Gag相互作用重要元素的结构。此外，我们已经鉴定出突变体，其中1G和3G RNA折叠成相似的结构，导致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-1 RNA在病毒复制过程中起着两个重要作用：作为病毒粒子基因组和作为Gag/Gag- pol翻译的模板。先前对两个HIV-1分子克隆的研究已经得出结论，TSS的使用影响了未剪接的HIV-1 RNA的结构和功能。为了研究这种复制策略的进化起源，我们确定了15种灵长类慢病毒感染细胞和病毒粒子中HIV-1 RNA的转录起始位点（TSS）。所有检测到的HIV-1分离株，包括几种传播的创始人病毒，都使用了多种TSS，并选择了一种特定的RNA物种进行包装。此外，这些特征在与HIV-1祖病毒相关的siv中观察到，表明这些特征起源于祖先病毒。HIV-2、HIV-2相关siv和其他siv也表现出多重TSS和特异性未剪接RNA物种的优先包装。这些发现表明，在HIV-1出现之前，多次使用TSS和选择性包装特定的非剪接RNA物种。

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.