CYTOMEGALOVIRUS GENE REGULATION IN IMMUNODEFICIENCY

这笔赠款支持将具有巨噬细胞和树突状细胞特征的骨髓来源的细胞类型确定为CMV潜伏期的部位，提供证据表明潜伏期病毒基因组构型类似于质粒，构建了一些病毒突变体以解决病毒功能在潜伏期中的作用，并建立了病毒基因组和潜伏期转录阳性细胞之间的关系。我们建议继续进行研究，试图揭开病毒控制潜伏期和再激活的遗传和分子基础，使用粒细胞-巨噬细胞前体培养作为实验系统。病毒基因在潜伏期和重新激活中的生理作用将通过一组突变病毒和测试潜伏期相关基因功能的测试来研究。首先，我们将完成先前表征的潜伏期和即刻早期(A)基因产物在粒细胞-巨噬细胞前体细胞培养中潜伏期的建立、维持和重新激活方面的功能评估。其次，我们将研究已在IE1/IE2区潜在转录本上发现的ORF的细胞定位和功能。在这里，我们将研究定位于细胞核和核仁的ORF94，并确定其在核洗牌、RNA结合和RNA剪接中的作用。第三，我们将使用一组针对所有病毒ORF的聚合酶链式反应(PCR)引物，识别在潜伏期和重新激活期间表达的其他病毒基因。这将完成对病毒基因组上潜在基因的识别。在这里，我们将重点关注在感染GM-Ps的CMV托莱多株中检测到的UL112-113区域转录本。我们还将使用我们组装的一套CMV ORF特异的PCR引物，对所有病毒的ORF进行系统的RT-PCR扩增。这项工作将继续建立对病毒功能在这种重要病原体潜伏期间表达和参与的理解，并将继续提供可在自然感染个体中评估的见解。

This grant has supported the identification of bone marrow-derived cell types with characteristics of macrophage and dendritic cell progenitors as sites of CMV latency, provided evidence that the latent viral genome configuration circular similar to a plasmid, constructed a number of viral mutants to address the role of viral functions in latency and established the relationship between viral genome and latent transcript positive cells. We propose to continue investigations that seek to unravel the genetic and molecular basis of viral control of latency and reactivation, using granulocyte-macrophage progenitor cultures as the experimental system. The physiological role of viral genes in latency and reactivation will be investigated with a panel of mutant viruses and assays to test latency-associated gene functions. First, we will complete a function evaluation of previously characterized latent and immediate early (a) gene products in the establishment, maintenance and reactivation of latency in granulocyte-macrophage progenitor cultures. Second, we will investigate the cellular localization and function of ORFs that have been identified on ie1/ie2 region latent transcripts. Here we will investigate ORF94 which localizes to the nucleus and nucleolus and determine its role in nuclear shuffling, RNA binding and RNA splicing. Third, we will identify additional viral genes that are expressed during latency and reactivation using a set of PCR primers that have been made to all viral ORFs. This will complete the identification of latent genes on the viral genome. Here we will focus on UL112-113 region transcripts that have been detected in CMV strain Toledo infected GM-Ps. We will also undertake systematic RT-PCR amplification of all viral ORFs using a set of CMV ORF-specific PCR primers we have assembled. This work will continue to build an understanding of viral functions expressed during and involved in latency of this important pathogen and will continue to provide insights that can be evaluated in naturally infected individuals.