Mechanisms of Human Cytomegalovirus Latency

描述(由申请人提供)：人类巨细胞病毒(HCMV)在宿主中建立终身潜伏感染，并在免疫抑制的患者中重新激活，造成毁灭性的后果，强调需要确定决定HCMV潜伏期的条件。为此，我们开发并报道了一种利用Kasumi 3细胞(K3)的新型HCMV潜伏期模型系统，该系统概括了HCMV重新激活的所有关键阶段，使我们能够实验测试对HCMV潜伏期至关重要的条件，这是以前不可能的。这一建议旨在验证一种假设，即miRNA诱导的HCMV IE基因抑制和细胞因子介导的IE基因诱导之间的拮抗关系是从潜伏感染转变为裂解感染的主要决定因素。我们的这一概念是基于我们令人兴奋的观察结果，即细胞编码的hsa-miR-200家族成员以必要的HCMV即刻早期(IE)2蛋白为靶标。此外，我们发现IE2可以在基因组范围内诱导病毒早期(E)启动子，并且不依赖于额外的病毒蛋白，这首次证实了IE2是HCMV病毒裂解转录的关键调节因子。此外，我们还发现，炎性细胞因子可以有效地启动K3细胞中HCMV的裂解再激活，而这种激活可以被核因子-kB抑制剂所阻断。基于这些观察，我们倾向于一种模型(源于对单纯疱疹病毒的已发表观察)，其中潜伏期是通过病毒基因组的染色化而建立的，从而导致病毒转录被抑制。然而，通过miRNA靶向IE转录本介导的额外水平的抑制将抑制病毒翻译，从而确保潜伏期的维持。我们提出了一个新的概念，即HCMV已经额外地选择了细胞miRNAs来限制病毒的重新激活。我们试图将这些研究延伸到巨细胞病毒潜伏期和重新激活的机制上。在目标1中，我们将确定细胞编码的miRNAs在限制HCMV IE翻译方面的生物学作用，以及评估病毒编码的miRNAs对HCMV潜伏期的要求。目的2探讨炎性细胞因子重新激活人巨细胞病毒的机制，以及细胞转录因子促进潜伏期至裂解期转换的要求。上述目标的完成将大大有助于我们对病毒潜伏期和裂解再激活机制的了解。这项工作将为确定一种重要的全球病原体病毒的新治疗靶点奠定基础。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) establishes lifelong latent infections in a host and reactivates with devastating consequences within immunosuppressed patients, underscoring the need to identify the conditions that dictate HCMV latency. To this end, we have developed and reported a novel HCMV latency model system utilizing Kasumi 3 cells (K3s) that recapitulates ALL the key stages of HCMV reactivation allowing us to experimentally test conditions critical for HCMV latency that were previously not possible. This proposal aims to test the hypothesis that the antagonistic relationship between miRNA-induced suppression of HCMV IE genes and cytokine-mediated induction of IE genes is a major determinant of the switch from latent to lytic infection. We base this concept on our exciting observations that cellular encoded hsa-miR-200 family members target the essential HCMV Immediate Early (IE) 2 protein. Additionally, we show that IE2 can induce viral Early (E) promoters within the context of the genome and independently of additional viral proteins, providing the first confirmation that IE2 is a key regulator of HCMV viral lytic transcription. In addition, we show that inflammatory cytokines potently initiate lytic reactivation of HCMV in our K3 cels that can be blocked by treatment with an NF-kB inhibitor. Based on these observations we favor a model (derived from published observations with Herpes Simplex Virus) wherein latency is established by chromatization of the viral genome resulting in suppression of viral transcription. However, an added level of suppression mediated by miRNA targeting of IE transcripts would suppress viral translation assuring latency maintenance. We offer the novel concept that HCMV has co-opted cellular miRNAs to additionally restrict viral reactivation. We seek to extend these studies on the mechanisms underlying HCMV latency and reactivation. In Aim 1, we will determine the biological role of cellular encoded miRNAs on restricting HCMV IE translation as well as evaluate the requirement of viral encoded miRNAs on HCMV latency. Aim 2 will address the mechanisms by which inflammatory cytokines function to reactivate HCMV as well as define the requirements of cellular transcription factors on promoting the latent to lytic switch. Completion of the aims above will contribute significantly to our knowledge on the mechanisms of viral latency and lytic reactivation. This work will lay the foundation for identifyig novel therapeutic targets for a virus that is a significant global pathogen.