High-Resolution Functional Profiling of a Gammaherpesvirus RTA Locus in the Context of the Viral Genome
High-Resolution Functional Profiling of a Gammaherpesvirus RTA Locus in the Context of the Viral Genome
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DOI：
10.1128/jvi.02302-08
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发表时间：
2009-02-15
影响因子：
5.4
通讯作者：
Sun, Ren
中科院分区：
文献类型：
--
作者：
Arumugaswami, Vaithilingaraja;Sitapara, Ronika;Sun, Ren
伽玛疱疹病毒、卡波西氏肉瘤相关疱疹病毒和爱泼斯坦-巴尔病毒与多种人类癌症有关。我们的目标是开发一种定量的、高通量的功能图谱系统，以在疱疹病毒基因组的背景下识别对病毒复制至关重要的病毒顺式元件和蛋白质亚域。在伽马-2疱疹病毒中，反式激活因子RTA是启动裂解基因表达和病毒重新激活所必需的。我们以RTA基因座为模型，开发了功能分析方法。突变的小鼠伽马疱疹病毒68病毒文库在RTA基因座上随机插入15个碱基，在小鼠成纤维细胞中传代进行多轮筛选。用荧光-聚合酶链式反应分析每个15个碱基插入的效果。我们鉴定了3845bpRTA基因座上的1229个插入，其中393个、282个和554个分别是病毒生长的严重受损、减毒和耐受。通过检测RTA启动子的反式激活功能和RTA缺失病毒的反式互补功能的几个单独的RTA突变克隆，验证了功能图谱的表型。因此，图谱分析方法使我们能够在疱疹病毒基因组的背景下识别RTA基因座中的几个新的功能结构域。重要的是，我们的研究展示了一种新的系统来进行高密度功能遗传图谱。基因组规模的扩大将加速疱疹病毒的功能基因组学研究。
Gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus are associated with multiple human cancers. Our goal was to develop a quantitative, high-throughput functional profiling system to identify viral cis-elements and protein subdomains critical for virus replication in the context of the herpesvirus genome. In gamma-2 herpesviruses, the transactivating factor RTA is essential for initiation of lytic gene expression and viral reactivation. We used the RTA locus as a model to develop the functional profiling approach. The mutant murine gammaherpesvirus 68 viral library, containing 15-bp random insertions in the RTA locus, was passaged in murine fibroblast cells for multiple rounds of selection. The effect of each 15-bp insertion was characterized using fluorescent-PCR profiling. We identified 1,229 insertions in the 3,845-bp RTA locus, of which 393, 282, and 554 were critically impaired, attenuated, and tolerated, respectively, for viral growth. The functional profiling phenotypes were verified by examining several individual RTA mutant clones for transactivating function of the RTA promoter and transcomplementing function of the RTA-null virus. Thus, the profiling approach enabled us to identify several novel functional domains in the RTA locus in the context of the herpesvirus genome. Importantly, our study has demonstrated a novel system to conduct high-density functional genetic mapping. The genome-scale expansion of the genetic profiling approach will expedite the functional genomics research on herpesvirus.