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

总结 人类转录组的大部分由长链非编码RNA（lncRNA）组成，其调节细胞的转录。 蛋白质编码基因的表达和功能，免疫细胞的发育，分化和对 病原体感染诱导lncRNA表达的整体变化。人类免疫缺陷病毒感染者中lncRNA的表达 细胞以及宿主lncRNA如何影响HIV感染的复制和持续是未知的。自发 HIV-1的功能性治愈发生在所有HIV患者的0.3-0.5%中。这些患者被称为精英控制者（EC）， 在没有治疗的情况下维持不可检测的HIV水平，维持稳定的CD 4 + T细胞计数， 不太可能传播艾滋病毒。内皮细胞能够自发控制HIV，并识别防御机制， 在这些患者中，有希望找到一种有效的治疗艾滋病毒感染的方法。以前的研究集中在蛋白质- 编码基因然而，有效的HIV控制可能涉及复杂的基因网络，包括lncRNA。我们 发现EC与HAART治疗的慢性HIV患者和健康人相比， 未感染的对照组。EC（SIEC）中抑制的lncRNA对HIV结局的功能影响尚不清楚。 我们已经观察到HIV感染的CD 4 +T细胞中细胞lncRNA表达的显著整体变化， 与未感染的细胞进行了对比大多数艾滋病毒的细胞功能失调（HIDE） lncRNA尚未确定。我们采用CRISPR/RfxCas 13 d（CasRx）沉默筛选来确定 SIEC和HIDE lncRNA对HIV复制的功能影响。我们的初步数据显示沉默 几种SIEC和HIDE lncRNA显著调节HIV复制。根据这些初步数据，我们 假设宿主lncRNA在HIV感染细胞中表达协调天然抗性和疾病 艾滋病毒感染的结果。我们将确定EC中特异性lncRNA的抑制是如何保护的， 阐明其功能的分子机制（目的1）。我们还将鉴定HIV去调节（HIDE）lncRNA 调节病毒复制并研究lncRNA介导的调节的潜在机制（目的2）。 我们将使用分子和生物化学技术的创新组合来实现这些目标，例如 细胞系中的CRISPR/CasRx沉默、转录组学、RNA反义纯化和质谱分析 模型以及原代CD 4 + T细胞。这项研究意义重大，因为它将识别细胞 影响HIV和疾病结果的自发控制的lncRNA，提供了前所未有的见解， lncRNA介导的HIV复制调节，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.