Recruitment of host noncoding RNAs by HIV-1

描述(申请人提供)：人类免疫缺陷病毒1型(HIV-1)是导致获得性免疫缺陷综合征(AIDS)的原因，是导致严重发病率和死亡率的原因。尽管针对HIV-1蛋白的疗法的使用大大降低了艾滋病的死亡率，但抗逆转录病毒药物必须持续数十年。鉴于逆转录病毒蛋白的内在变异性，针对被HIV-1劫持的新宿主途径或对病毒复制至关重要的宿主成分的治疗可能非常有用。这个探索性项目的目的是测试这样一个假设，即HIV-1非随机地将新合成的宿主非编码RNA(NcRNAs)招募到病毒粒子中的倾向可以被利用来获得对HIV-1组装的鲜为人知的早期步骤的洞察，并确定潜在的靶点 抗病毒药物。虽然宿主ncRNA早在40多年前就被描述为经历逆转录病毒包装，但HIV-1包裹的ncRNAs的谱、这些ncRNAs被招募的机制以及它们在HIV-1生命周期中的贡献在很大程度上都是未知的。在初步实验中，我们使用高通量测序对HIV-1包装的ncRNAs进行了公正和全面的评估。我们的分析表明，除了已知的包装RNA，如信号识别颗粒的7SL RNA成分外，还存在几个意想不到的ncRNA。我们还发现，ncRNA加工中间体，通常是罕见的，因为它们只短暂地存在于细胞内，高度富含病毒粒子。总而言之，这些结果表明，早期阶段 在HIV-1组装中，与宿主细胞ncRNA生物发生途径相交，从而打开了进入这两个过程的窗口。我们的目标是确定HIV-1对有效包装ncRNA的病毒和细胞需求，并确定ncRNA对HIV-1组装和复制的贡献程度。我们的第一个目标是基于我们的发现，通过执行额外的生物复制和确定病毒粒子中高度代表性的ncRNA相对于病毒基因组RNA的丰度，HIV-1包装了特定的ncRNAs和ncRNA前体。我们的第二个目标是确定HIV-1招募特定宿主ncRNA的机制(S)以及这些RNA与HIV-1生命周期的功能相关性。我们将基于我们的发现来检验这一假设，即HIV-1优先招募新生的ncRNA，即与病毒组件(如基因组RNA或核衣壳)的相互作用对包裹体的形成至关重要。由于HIV-1招募了一些只在细胞核中检测到的ncRNAs，我们将测试HIV-1是否与新的宿主细胞监视途径对接，在该途径中，未经处理和未组装的ncRNA被输出到细胞质中快速腐烂。由于阐明宿主ncRNAs对HIV-1生命周期的贡献可以产生抗病毒靶标，我们将干扰丰富表达的ncRNAs的包装，并检测对病毒组装和传染性的影响。通过阐明包装的ncRNA的集合、它们的招募机制以及它们对HIV-1复制的贡献程度，这些实验可以确定新的治疗靶点。

DESCRIPTION (provided by applicant): Human immunodeficiency virus type 1 (HIV-1), the cause of acquired immune deficiency syndrome (AIDS), is responsible for significant morbidity and mortality. Although the use of therapies that target HIV-1 proteins has greatly reduced AIDS mortality, anti-retrovirals must be administered for decades. Given the inherent mutability of retroviral proteins, therapies that target new host pathways hijacked by HIV-1 or host components important for viral replication could be extremely useful. The objective of this exploratory project is to test the hypothesis that the propensity of HIV-1 to non-randomly recruit newly synthesized host noncoding RNAs (ncRNAs) into virions can be exploited to obtain insights into poorly understood early steps in HIV-1 assembly and to identify potential targets for antivirals. Although host ncRNAs were first described to undergo retroviral packaging more than forty years ago, the spectrum of ncRNAs encapsidated by HIV-1, the mechanisms by which these ncRNAs are recruited, and the extent to which they contribute to the HIV-1 lifecycle are all largely unknown. In preliminary experiments, we used high-throughput sequencing to obtain an unbiased and comprehensive assessment of the ncRNAs packaged by HIV-1. Our analyses revealed that, in addition to known packaged RNAs such as the 7SL RNA component of the signal recognition particle, several unexpected ncRNAs were present. We also found that ncRNA processing intermediates, which are usually rare because they exist only transiently within cells, were highly enriched in virions. Together, these results indicate that an early stage in HIV-1 assembly intersects with host cell ncRNA biogenesis pathways, thus opening windows into both of these processes. Our goals are to identify the viral and cellular requirements for efficient ncRNA packaging by HIV-1 and to determine the extent to which the ncRNAs contribute to HIV-1 assembly and replication. Our first aim is to build on our findings that HIV-1 packages specific ncRNAs and ncRNA precursors by performing additional biological replicates and by determining the abundance of highly represented ncRNAs in virions relative to the viral genomic RNA. Our second aim is to determine the mechanism(s) by which HIV-1 recruits specific host ncRNAs and the functional relevance of these RNAs to the HIV-1 lifecycle. We will test the hypothesis, based on our findings that HIV-1 preferentially recruits nascent ncRNAs, that interactions with viral components, such as genomic RNA or nucleocapsid, are critical for encapsidation. Because HIV-1 recruits some ncRNAs that have only been detected in nuclei, we will test whether HIV-1 interfaces with a new host cell surveillance pathway in which unprocessed and unassembled ncRNAs are exported to the cytoplasm for rapid decay. Since elucidating the contributions that host ncRNAs make to the HIV-1 lifecycle could yield antiviral targets, we will interfere with packaging of abundantly represented ncRNAs and examine effects on virus assembly and infectivity. By elucidating the set of packaged ncRNAs, their mechanisms of recruitment and the extent to which they contribute to HIV-1 replication, these experiments could identify new therapeutic targets.