Mechanisms of Human Cytomegalovirus Latency

描述（由申请方提供）：人巨细胞病毒（HCMV）在宿主中建立终身潜伏感染，并在免疫抑制患者中重新激活，造成毁灭性后果，强调需要确定决定HCMV潜伏期的条件。为此，我们已经开发并报道了一种新的HCMV潜伏期模型系统，该系统利用Kasumi 3细胞（K3 s）概括了HCMV再活化的所有关键阶段，使我们能够通过实验测试HCMV潜伏期的关键条件，这在以前是不可能的。该提议旨在检验以下假设：miRNA诱导的HCMV IE基因抑制与马槟榔碱介导的IE基因诱导之间的拮抗关系是从潜伏性感染转变为裂解性感染的主要决定因素。我们将这一概念基于我们令人兴奋的观察结果，即细胞编码的hsa-miR-200家族成员靶向必需的HCMV立即早期（IE）2蛋白。此外，我们表明，IE 2可以诱导病毒早期（E）启动子的基因组内的背景下，独立于其他病毒蛋白，提供了第一个确认IE 2是HCMV病毒裂解转录的关键调节因子。此外，我们发现，炎症细胞因子有效地启动我们的K3细胞中的HCMV裂解再活化，这可以通过用NF-κ B抑制剂治疗来阻断。基于这些观察结果，我们倾向于一种模型（来源于已发表的对单纯疱疹病毒的观察结果），其中通过病毒基因组的染色化建立潜伏期，从而抑制病毒转录。然而，由靶向IE转录物的miRNA介导的额外水平的抑制将抑制病毒翻译，确保潜伏期维持。我们提出了新的概念，即HCMV已经增选细胞miRNA来额外限制病毒再激活。 我们寻求扩大这些研究的机制，潜在的HCMV潜伏期和再激活。在目标1中，我们将确定细胞编码的miRNA在限制HCMV IE翻译方面的生物学作用，以及评估病毒编码的miRNA对HCMV潜伏期的需求。目的2将阐明炎性细胞因子激活HCMV的机制，并确定细胞转录因子对促进潜伏性细胞裂解转换的要求。上述目标的完成将有助于我们对病毒潜伏和裂解再激活机制的认识。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.