Role of cellular long non-coding RNAs in HIV replication and disease outcome

摘要 人类转录组的大部分由长的非编码RNA(LncRNAs)组成，它调节 蛋白质编码基因的表达和功能、免疫细胞的发育、分化和对 病原体。感染导致lncRNA表达的全球变化。人类免疫缺陷病毒感染组织中lncRNAs的表达 细胞以及宿主lncRNAs如何影响艾滋病毒感染的复制和持久性尚不清楚。一种自发的 HIV-1的功能性治愈在所有HIV患者中的比例为0.3-0.5%。这些患者被称为精英控制者(EC)， 在没有治疗的情况下保持无法检测到的艾滋病毒水平，保持稳定的CD4+T细胞计数，以及 不太可能传播艾滋病毒。ECS举例说明了对艾滋病毒的自发控制，以及识别防御机制 对这些患者来说，找到治疗艾滋病毒感染的功能性疗法是有希望的。此前的研究主要集中在蛋白质-- 编码基因。然而，有效的艾滋病毒控制可能涉及复杂的基因网络，包括lncRNAs。我们 发现EC与HAART治疗的慢性HIV患者和健康人相比，有几个lncRNA显著抑制 未感染的对照组。在EC(SIEC)中抑制的lncRNAs对HIV结局的功能影响尚不清楚。 我们观察到HIV感染的CD4+T细胞中细胞内lncRNA表达的显著变化 与我们初步研究中未感染的细胞相比。大多数HIV病毒解除管制后的细胞功能(HIDE) LncRNAs还有待确定。我们使用了CRISPR/RfxCas13d(CasRx)-沉默屏幕来确定 SIEC和HIDE lncRNA对HIV复制的功能影响。我们的初步数据显示，沉默 几个SIEC和HIDE LncRNAs显著调控HIV复制。根据这些初步数据，我们 假设宿主在HIV感染细胞中的lncRNA表达协调了自然抵抗力和疾病 艾滋病毒感染的结局。我们将确定在内皮细胞中抑制特定的lncRNAs是如何起保护作用的 阐明其作用的分子机制(目标1)。我们还将识别HIV解除管制(隐藏)的lncRNA 调控病毒复制和研究lncRNA介导的调控机制(S)(目标2)。 我们将利用分子和生化技术的创新组合来实现这些目标，例如 CRISPR/CasRx-沉默、转录、RNA反义纯化和质谱学 模型以及原代CD4+T细胞。这项拟议的研究意义重大，因为它将识别细胞 影响HIV自发控制和疾病结局的IncRNA，提供了前所未有的洞察力 LncRNA介导的HIV复制调控，为开发新的方法奠定了基础 干预。

Summary The majority of the human transcriptome consists of long non-coding RNAs (lncRNAs), which regulate the expression and function of protein-coding genes, immune cell development, differentiation, and response to pathogens. Infections induce global changes in lncRNA expression. The expression of lncRNAs in HIV-infected cells and how host lncRNAs impact the replication and persistence of HIV infection are unknown. A spontaneous functional cure of HIV-1 occurs in 0.3-0.5% of all HIV patients. These patients, termed elite controllers (EC), maintain undetectable levels of HIV in the absence of treatment, maintain stable CD4+ T cell counts, and are less likely to transmit HIV. The ECs exemplify spontaneous control of HIV, and identifying defense mechanisms in these patients holds promise for finding a functional cure for HIV infection. Previous studies focused on protein- coding genes. However, effective HIV control is likely to involve complex gene networks, including lncRNAs. We found several lncRNAs significantly suppressed in EC vs. HAART-treated chronic HIV-patients and healthy uninfected controls. The functional impact of lncRNAs suppressed in EC (SIEC) on HIV outcomes is unknown. We have observed significant global changes in cellular lncRNA expression in HIV-infected CD4+T cells compared to uninfected cells in our preliminary studies. Cellular functions of most of the HIV deregulated (HIDE) lncRNAs are yet to be determined. We employed a CRISPR/RfxCas13d (CasRx)-silencing screen to determine the functional impact of SIEC and HIDE lncRNAs on HIV replication. Our preliminary data showed that silencing of several SIEC and HIDE lncRNAs significantly regulated HIV replication. Based on these preliminary data, we hypothesize that host lncRNA expression in HIV-infected cells orchestrates natural resistance and disease outcome in HIV infection. We will determine how the suppression of specific lncRNAs in ECs is protective and elucidate molecular mechanisms of their function (Aim 1). We will also identify HIV deregulated (HIDE) lncRNAs modulating viral replication and investigate the mechanism(s) underlying lncRNA-mediated regulation (Aim 2). We will pursue these aims using innovative combinations of molecular and biochemical techniques, such as CRISPR/CasRx-silencing, transcriptomics, RNA antisense purification, and Mass-Spectrometry in cell line models as well as primary CD4+ T cells. The proposed research is significant because it will identify cellular lncRNAs that influence spontaneous control of HIV and disease outcomes, deliver unprecedented insight into lncRNA-mediated regulation of HIV replication, lay the groundwork for the development of new approaches to intervention.