Disruption of Cellular RNA Processing by Kaposi's Sarcoma-Associated Herpesvirus

 描述(申请人提供)：卡波西肉瘤相关疱疹病毒(KSHV)是大多数艾滋病相关癌症的病原体。它在非洲许多地区流行，由于那里艾滋病毒负担特别高，卡波西肉瘤正在成为非洲大陆最常见的癌症之一。在艾滋病诱导的免疫抑制期间，KSHV复制不再得到有效控制，并与大量潜伏感染的细胞一起促进疾病进展。KSHV的扩增依赖于其对感染细胞的基因表达环境施加强大控制的能力。病毒用来调节基因表达的一个主要机制是诱导细胞质中信使RNA(信使RNA)的广泛降解。这种表型在病毒生命周期和免疫逃避中发挥着多种作用，并由一种名为SOX的病毒编码的核酸酶驱动。虽然SOX切割了大量的转录本，但它对RNA聚合酶II(Pol II)转录的RNA表现出明显的特异性，并且不具有显著的RNA结合活性。如何认识到这一点并使其达到目标，仍然是实地尚未回答的核心问题，也是这项提案的第一个目标的重点。我们将应用双分子 基于互补的蛋白质相互作用筛选，无偏向核糖核蛋白复合体的纯化，以及定向的功能研究，以准确地确定SOX是如何与其靶mRNAs结合的。识别和表征介导SOX靶向的因素是理解KSHV如何维持对基因表达的控制的核心，并同样可能为控制RNA命运的细胞策略提供新的见解。SOX对胞质mRNA的耗尽可能是由细胞感受到的，因此提供了一个独特的机会来揭示细胞对基因表达谱中广泛变化的反应，例如病原体诱导的变化。在这方面，最近在酵母中的结果表明，存在反馈机制来检测和响应基因表达不同阶段的变化。利用伽玛疱疹病毒作为工具，我们现在已经在哺乳动物细胞中发现了一条类似的途径，将mRNA降解与转录联系起来。我们称之为“转录启动”的这一途径的激活，会影响新生信使核糖核酸的合成速度。虽然这种宿主途径的正常作用可能是调节细胞基因的表达以应对压力，但我们假设在KSHV感染期间，其被SOX激活反而有助于促进病毒基因的POL II转录。定义这一途径是如何控制的，以及它如何影响病毒的生命周期 这些研究的结果应该提供新的见解，以了解看似遥远的基因表达阶段是如何整合的，以及像KSHV这样的病毒病原体如何利用这些细胞控制来提高它们的复制成功。

 DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of the majority of AIDS- associated cancers. It is endemic in many areas of Africa and, due to the extraordinarily high HIV burden there, Kaposi's sarcoma is emerging as one of the most common cancers on that continent. During AIDS-induced immunosuppression, KSHV replication is no longer effectively controlled, and, together with a large latently infected population of cells, contributes to disease progression. Amplification of KSHV is dependent on its ability to exert strong control over the gene expression environment of the infected cell. A primary mechanism the virus uses to regulate gene expression is to induce widespread degradation of messenger RNA (mRNA) in the cytoplasm. This phenotype plays numerous roles in the viral lifecycle and immune evasion, and is driven by a virally encoded nuclease termed SOX. Although SOX cleaves an extremely large number of transcripts, it exhibits clear specificity for RNA Polymerase II (Pol II) transcribed RNAs and does not have significant RNA binding activity. How it recognizes and is brought to its targets remains a central unanswered question in the field and is the focus of the first Aim of this proposal. We will apply bimolecular complementation-based protein interaction screening, unbiased ribonucleoprotein complex purification, and directed functional studies to pinpoint how SOX is directed to its target mRNAs. Identification and characterization of factors that mediate SOX targeting is central to understanding how KSHV maintains control of gene expression, and may similarly provide new insight into cellular strategies to govern RNA fate. The depletion of cytosolic mRNA by SOX is presumably sensed by the cell, and thus offers a unique opportunity to reveal cellular responses to broad changes in the gene expression landscape, such as those induced by pathogens. In this regard, recent results in yeast indicate the existence of feedback mechanisms to detect and respond to altered activity of different stages of gene expression. Using gamma-herpesviruses as tools, we have now discovered an analogous pathway in mammalian cells that links mRNA degradation with transcription. Activation of this pathway, which we term `transcriptional priming', impacts the rate of nascent mRNA synthesis. Though it is likely that the normal role of this host pathway is to regulate cellular gene expression in response to stress, we hypothesize that during KSHV infection its activation by SOX instead serves to enhance Pol II transcription of viral genes. Defining how this pathway is controlled and how it impacts the viral life cycle are the goals of Aim 2. Results from these studies should provide new insight into how seemingly distant stages of gene expression are integrated, and how viral pathogens like KSHV take advantage of these cellular controls to enhance their replicative success.