KSHV-encoded Small Peptites

描述（由申请人提供）：从KSHV基因组中ORF 50 DNA模板的相对链转录3.0 kb（T3.0）的多聚腺苷酸化RNA，并已注释为非编码RNA。ORF 50编码复制和转录激活因子（RTA），其控制病毒在潜伏和裂解生命周期之间的转换。我们发现T3.0编码几种小肽，其中一种（命名为病毒小肽-1或vSP-1）与RTA相互作用，并通过泛素-蛋白酶体途径阻止RTA被降解。因此，vSP-1促进KSHV基因表达和裂解复制。这些发现的意义是多方面的。首先，在哺乳动物和病毒基因组中，大部分序列被转录并注释为非编码RNA。我们的研究表明，这些非编码RNA中的一些可能通过编码小肽发挥功能。哺乳动物细胞中可能存在大量的小分子肽，它们在生物学过程的调控中起着关键作用。其次，我们的发现揭示了一种新的基因表达调控机制以及病毒的生命周期。我们相信，小肽代表了一个重要的生物学未开发的来源。为了证明这一假设，并估计小肽世界在生物学中的重要性，我们提出了探索小肽对哺乳动物细胞和病毒生物学的意义，具体目标如下。(1)我们将使用蛋白质组学方法鉴定KSHV基因组中的小肽。将从KSHV感染的细胞以及纯化的病毒体制备富集的小肽池，并进行质谱（MS）分析。将开发一个特殊的潜在KSHV小肽数据库，并用于分析MS原始光谱，以鉴别KSHV小肽。(2)我们将选择在目的一中鉴定出的几个具有代表性的病毒小肽，以及两个T3.0编码的小肽（vSP-1和vSP- 2），利用遗传学方法评估病毒小肽在KSHV生活周期中的功能作用。通过这种探索性的研究，我们希望解决的问题，如果小肽编码的先前注释的非编码RNA是在哺乳动物细胞和病毒的共同特征，并证明这些小肽在生物学中的生物学意义。本研究为小分子肽的生物调控开辟了新的研究领域。

DESCRIPTION (provided by applicant): A polyadenylated RNA of 3.0 kb (T3.0) is transcribed from the opposite strand of ORF50 DNA template in the KSHV genome and has been annotated as a non-coding RNA. ORF50 encodes the replication and transcription activator (RTA) that controls switch of the virus between latent and lytic life cycle. We found that T3.0 encodes several small peptides and one of them (designated viral Small Peptide-1 or vSP-1) interacts with RTA and prevents it from being degraded through the ubiquitin-proteasome pathway. As a consequence, vSP-1 facilitates KSHV gene expression and lytic replication. The significance of these findings is multi-fold. First, in both mammalian and viral genomes, a large proportion of sequences are transcribed and annotated as noncoding RNAs. Our study suggests that some of these noncoding RNAs may function through encoding small peptides. There might be large numbers of small peptides in mammalian cells that play critical roles in regulation of biological processes. Second, our finding revealed a novel mechanism of regulation of gene expression as well as viral life cycle. We believe that small peptides represent an untapped source of important biology. To prove this hypothesis and to estimate the magnitude of small peptide world in biology, we proposed to explore the significance of small peptides to biology of mammalian cells and viruses with the specific aims as follows. (1) We would use proteomics approach to identify small peptides from the KSHV genome. Enriched small peptide pools will be prepared from KSHV-infected cells as well as purified virions and subjected to mass spectrometric (MS) analysis. A special potential KSHV small peptide database will be developed and used to analyze the MS raw spectra for identification of KSHV small peptides. (2) We will choose a few representative viral small peptides identified in aim one, together with two T3.0-encoded small peptides (vSP-1 and vSP- 2), to assess the functional roles of viral small peptides in KSHV life cycle using genetic approaches. Through this exploratory investigation, we hope to address the question if small peptides encoded by previously annotated noncoding RNAs are common features in mammalian cells and viruses and to demonstrate the biological significance of these small peptides in biology. This study may lead to a new field on biological regulation by small peptides.