CYTOMEGALOVIRUS GENE REGULATION IN IMMUNODEFICIENCY

该基金支持鉴定具有巨噬细胞和树突状细胞祖细胞特征的骨髓来源细胞类型作为CMV潜伏位点，提供证据证明潜伏病毒基因组构型类似于质粒，构建了许多病毒突变体以解决病毒功能在潜伏中的作用，并建立了病毒基因组与潜伏转录阳性细胞之间的关系。 我们建议继续调查，寻求解开病毒控制的潜伏期和再激活的遗传和分子基础，使用粒细胞-巨噬细胞祖细胞培养物作为实验系统。 病毒基因在潜伏期和再激活中的生理作用将通过一组突变病毒和检测潜伏期相关基因功能的试验进行研究。 首先，我们将完成先前表征的潜伏和立即早期（a）基因产物在粒细胞-巨噬细胞祖细胞培养物中潜伏期的建立、维持和再激活中的功能评价。 第二，我们将调查的细胞定位和功能的ORF已确定的ie 1/ie 2区域的潜伏转录本。 在这里，我们将研究定位于细胞核和核仁的ORF 94，并确定其在核改组、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PCR引物来鉴定在潜伏期和再激活期间表达的其他病毒基因。 这将完成病毒基因组上潜伏基因的鉴定。 在这里，我们将集中在UL 112 -113区域的转录本，已检测到的CMV株托莱多感染的GM-Ps。 我们还将进行系统的RT-PCR扩增的所有病毒的ORF使用一套CMV ORF特异性PCR引物，我们已经组装。 这项工作将继续建立对这种重要病原体潜伏期期间表达和参与的病毒功能的理解，并将继续提供可以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.