Quantitative High-Resolution Genetic Profiling of a Gammaherpesvirus

描述（由申请人提供）：疱疹病毒与许多人类疾病有关。伽玛疱疹病毒、eb病毒（EBV）和卡波西肉瘤相关疱疹病毒（KSHV）参与人类淋巴样癌和其他癌症的发生。伽玛疱疹病毒有溶解性和潜伏性两种感染形式。利用小鼠γ疱疹病毒-68 （MHV-68）作为模型系统，可以研究γ疱疹病毒的生命周期。由于病毒的编码能力有限，许多病毒蛋白进化为具有多个结构域的多功能蛋白。为了获得疱疹病毒多结构域蛋白的高分辨率功能定义，需要一种高通量遗传操作方法，该方法可以在不影响邻近结构域的情况下精确破坏特定的功能结构域。本提案的总体目标是建立一个定量的高分辨率的伽玛疱疹病毒基因组功能遗传谱。将mu转座子介导的15核苷酸随机插入诱变方法与定量、高通量毛细管基因分型方法相结合，建立了功能性基因分析平台。该方法的原理首先是通过使用包含15-nt随机插入的oriLyt突变文库确定MHV-68裂解复制起源（oriLyt）中oriLyt复制功能所需的顺式元件来建立的。其次，γ疱疹病毒编码的转录因子RTA在裂解性感染和潜伏期再激活期间启动裂解性基因表达程序是必需的。该方法在完整病毒基因组背景下的可行性已经通过MHV-68 RTA启动子顺式调控元件和RTA蛋白微域的功能谱得到了证明，这是病毒复制所必需的。该方法的发展将为MHV-68全基因组的高分辨率功能分析奠定基础。该建议将为细胞培养中MHV-68病毒复制建立全基因组和高分辨率的功能图谱，这将大大加快疱疹病毒的功能基因组研究。小鼠肺中MHV-68复制的功能谱也将被确定。通过比较体内和体外功能谱，我们将确定MHV-68序列在体内与病毒复制特异性相关。

DESCRIPTION (provided by applicant): Herpesviruses are associated with many human ailments. Gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are involved in the genesis of human lymphoid and other cancers. Gammaherpesviruses undergo both lytic and latent forms of infection. The gammaherpesviral life cycle can be studied by using a murine gammaherpesvirus-68 (MHV-68) as a model system. Since viruses have limited coding capacity, many of the viral proteins are evolved as multifunctional with several domains. To obtain high-resolution functional definition of herpesviral multidomain proteins, a high-throughput genetic manipulation method, which can allow precise disruption of a particular functional domain without affecting neighboring domains, is required. The overall goal of this proposal is to establish a quantitative high-resolution functional genetic profile of a gammaherpesvirus genome. A functional genetic profiling platform has been developed by combining a Mu-transposon mediated 15-nucleotide random insertional mutagenesis method and a quantitative, high-throughput capillary genotyping method. The principle of this method has first been established by determining the cis-elements in the MHV-68 origin of lytic replication (oriLyt) that are required for oriLyt replicatory function by using an oriLyt mutant library containing 15-nt random insertions. Second, the gammaherpesvirus encoded transcription factor RTA is required for initiating the lytic gene expression program during lytic infection and reactivation from latency. The feasibility of the method in the context of the intact viral genome has been documented with a functional profile of the MHV-68 RTA promoter cis-regulatory elements and RTA protein microdomains which are essential for virus replication. Developing this method will establish a foundation for high resolution functional profiling of the entire MHV-68 genome. This proposal will develop a genome-wide and high- resolution functional profile for MHV-68 viral replication in cell culture, which will greatly expedite functional genomic studies of herpesviruses. The functional profile of MHV-68 replication in the lung in mice will be determined as well. By comparing in vivo to in vitro functional profiles we will identify MHV-68 sequences specifically relevant to viral replication in vivo. PUBLIC HEALTH RELEVANCE: Herpesviruses are associated with many human ailments. Gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are involved in the genesis of human lymphoid and other cancers. Since viruses have limited coding capacity, many of the viral proteins are evolved as multifunctional with several domains. To obtain a high-resolution functional definition of herpesviral multidomain proteins, a high-throughput genetic manipulation method, which can allow precise disruption of a particular functional domain without affecting neighboring domains will be developed. The information will facilitate the identification new therapeutic targets against herpesvirus replication and associated diseases.