HIV-1 TAR derived miRNA: Implications for Latency and Pathogenesis

描述(申请人提供)：人类免疫缺陷病毒I型(HIV-1)是获得性免疫缺陷综合症(AIDS)的病原体。艾滋病是一种复杂和多方面的疾病，它是由艾滋病毒感染免疫系统细胞而发展起来的。病毒有一个多步骤的生命周期，以病毒的转录控制为中心，受病毒Tat蛋白和称为反式激活反应(TAR)元件的RNA元件之间的相互作用调节。我们最近证明了TAR被参与RNA干扰的蛋白质(RNAi)利用，并被加工成病毒微RNA(MiRNA)，从而驱动病毒LTR的重塑，导致异染色质的形成。我们研究的长期目标是了解RNAi机制如何影响HIV-1病毒复制。RNA干扰(RNAi)是高等真核生物中一种保守的调控机制。RNAi途径分别从RNA的长双链延伸或RNA发夹产生小干扰RNA(SiRNA)或微型RNA(MiRNA)。然后，siRNA或miRNA将效应复合体引导到同源的mRNA序列，并通过几种机制中的一种来调节基因表达的抑制。HIV-1TAR元件是在所有HIV-1转录本的5‘端发现的茎和环结构。TAR的存在对于TAT蛋白激活病毒转录是重要的。然而，TAR的结构--一个57个核苷酸的发夹，由~24个碱基茎和一个6个碱基末端环组成--与细胞前微型RNA(Pre-miRNA)非常相似。这些细胞内的前miRNA被Dier酶作用，产生能够调节基因表达的miRNA。我们最近证明了TAR可以被细胞内的Disher裂解形成病毒miRNA。这种miRNA可以在HIV-1感染的细胞系和原代细胞中检测到。进一步的实验证据表明，这种病毒miRNA可能参与了转录潜伏期的诱导和细胞基因表达的改变。本项目的目的是确定TAR来源的miRNA在病毒复制中的机制和作用。我们的假设是，TAR来源的miRNA在病毒生命周期中发挥关键作用，既是参与建立和维持转录潜伏期的机制，也是改变细胞基因表达的机制。我们这项研究的基本原理是基于我们实验室的初步数据，以及在其他人类病毒中存在病毒miRNA，如人类巨细胞病毒、人类疱疹病毒8、爱泼斯坦-巴尔病毒和桃潜伏花叶类病毒。以下具体目标将解决我们的假设：(I)HIV-1 TAR miRNA对病毒基因表达的作用机制是什么？(Ii)病毒miRNA在改变细胞基因表达方面扮演什么角色？从这些研究中获得的数据将阐明RNAi在HIV-1感染中的作用，并为抑制病毒复制提供新的可能性。与公共卫生相关：目前可用于治疗艾滋病毒-1的药物并不代表最终的治愈方法。通过RNA干扰控制病毒的研究和对RNAi在病毒生命周期中的重要性的进一步了解，可能会导致发现艾滋病毒/艾滋病治疗的新选择。

DESCRIPTION (provided by applicant): Human Immunodeficiency Virus type I (HIV-1) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a complex and multifaceted disease that develops from the infection of cells of the immune system by the HIV virus. The virus has a multi-step life cycle that revolves around the transcriptional control of the virus as regulated by interaction between the viral Tat protein and an RNA element known as the transactivation response (TAR) element. We have recently demonstrated that TAR is utilized by proteins involved in RNA interference (RNAi) and is processed into a viral microRNA (miRNA) that drives remodeling of the viral LTR, leading to heterochromatin formation. The long-term goal of our research is to understand how the RNAi mechanism affects HIV-1 viral replication. RNA interference (RNAi) is a regulatory mechanism conserved in higher eukaryotes. The RNAi pathway generates small interfering RNA (siRNA) or micro RNA (miRNA) from either long double stranded stretches of RNA or RNA hairpins, respectively. The siRNA or miRNA then guides an effector complex to a homologous sequence of mRNA and regulates suppression of gene expression through one of several mechanisms. The HIV-1 TAR element is a stem and loop structure found at the 5' end of all HIV-1 transcripts. The presence of TAR is important for activation of viral transcription by the protein Tat. However, the structure of TAR - a 57 nucleotide hairpin consisting of ~24 basepair stem and a 6 base terminal loop - is very similar to cellular pre- micro RNA (pre-miRNA). These cellular pre-miRNA are acted upon by the enzyme Dicer to yield miRNA that is capable of regulating gene expression. We have recently demonstrated that TAR is cleaved by cellular Dicer to form a viral miRNA. This miRNA is detectable in HIV-1 infected cell lines and primary cells. Further experimental evidence suggests that this viral miRNA may be involved in the induction of transcriptional latency and in the alteration of cellular gene expression. The objective of this project is to determine the mechanism and role of TAR derived miRNA in viral replication. Our hypothesis is that TAR derived miRNA plays a critical role in the viral life cycle as both a mechanism involved in the establishment and maintenance of transcriptional latency and in the alteration of cellular gene expression. Our rationale for this study is based on preliminary data from our lab and the presence of viral miRNA in other human viruses such as human cytomegalovirus, human herpesevirus 8, Epstein Barr virus and peach latent mosaic viroid. The following specific aims will address our hypothesis: (I) What is the mechanism of action of HIV-1 TAR miRNA on viral gene expression? (II) What roles does the viral miRNA play in altering cellular gene expression? Data obtained from these studies will elucidate the role of RNAi in HIV-1 infection and provide new possibilities for inhibiting viral replication. PUBLIC HEALTH RELEVANCE: Currently available drugs for the treatment of HIV-1 do not represent a definitive cure. Research into control of the virus by RNA interference and an increased understanding of the importance of RNAi to the viral life cycle could potentially lead to the discovery of new options in the treatment of HIV/AIDS.