KSHV-encoded Small Peptites

描述(申请人提供)：从KSHV基因组中ORF50 DNA模板的相反链转录出一个3.0kb(T3.0)的多聚腺苷化RNA，并被注释为非编码RNA。ORF50编码复制和转录激活因子(RTA)，控制病毒在潜伏和裂解生命周期之间的切换。我们发现T3.0编码几个小肽，其中一个(命名为病毒小肽-1或VSP-1)与RTA相互作用，并通过泛素-蛋白酶体途径阻止其降解。因此，VSP-1促进了KSHV基因的表达和裂解复制。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，很大一部分序列被转录和注释为非编码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肽，它们在生物过程的调节中起着关键作用。其次，我们的发现揭示了一种调节基因表达和病毒生命周期的新机制。我们认为，小肽代表着一个尚未开发的重要生物学来源。为了验证这一假说，并估计生物学中小肽世界的大小，我们建议探索小肽对哺乳动物细胞和病毒生物学的意义，具体目的如下。(1)利用蛋白质组学方法从KSHV基因组中鉴定小肽。将从KSHV感染细胞和纯化的病毒粒子中制备浓缩的小肽库，并进行质谱分析。将建立一个特殊的潜在KSHV小肽数据库，并用于分析MS原始光谱，以鉴定KSHV小肽。(2)我们将选择Aim One中鉴定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)，用遗传学的方法来评估病毒小肽在KSHV生活史中的功能。通过这一探索性研究，我们希望解决由先前注释的非编码RNA编码的小肽是否是哺乳动物细胞和病毒的共同特征这一问题，并论证这些小肽在生物学上的生物学意义。这一研究可能为小肽的生物调控开辟新的领域。

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.