Mechanisms of MCMV reactivation in immunodeficient transplant recipients

描述(由申请人提供)：尽管使用了抗病毒药物，潜伏的人类巨细胞病毒(HCMV)的重新激活仍然是移植受者发病和死亡的重要原因。因此，需要新的方法来减少这种病原体的并发症。由于巨细胞病毒的种属特异性，我们建立了一种新的移植模型，利用高度相关的小鼠巨细胞病毒(MCMV)作为模型来研究器官移植中巨细胞病毒的潜伏期和再激活。在该模型中，将MCMV潜伏感染的肾脏移植到T、B和NK细胞缺陷的免疫低下的NOD.Cg-PrkdcsordIL2rgtm1Wjl/Szj(NSG)小鼠体内。将潜伏感染的肾脏移植到NSG受体体内会导致供体肾脏中潜伏病毒的重新激活，这种病毒会系统性传播。人巨细胞病毒的重新激活与各种炎性损伤有关。我们的初步数据显示，IL-6、可溶性CD40L、IL-18和LIF在受体小鼠的血浆中升高，其时间与重新激活的因果作用一致。IL-6在造血细胞模型中诱导人巨细胞病毒的再激活。这一建议的中心假设是，移植肾引发的炎症反应导致受体小鼠体内介质的释放。由此产生的信号级联刺激潜伏病毒基因组的表观遗传重编程，转录重新激活病毒即刻早期(IE)基因表达，并在免疫受损的受体中，潜伏病毒重新进入裂解复制程序。为了验证这一假设，在目标1中，我们将调查在我们的初步研究中确定的候选因子及其下游信号中间体在诱导NSG模型中重新激活方面的需求。我们以前的研究和其他人的研究表明，由于病毒基因组的异染色质，裂解病毒基因的表达在CMV潜伏期受到抑制。此外，我们已经证明，移植诱导的MCMV即刻早期基因表达的重新激活与表观遗传重新编程有关。我们的合作者Thomas Kristie博士之前的研究已经确定了抑制潜伏的单纯疱疹病毒重新激活的表观遗传抑制剂。在目标2中，我们将研究这些有希望的治疗干预措施在NSG模型中防止潜伏的MCMV重新激活的能力。最后，在目标3中，我们将研究小鼠造血祖细胞中MCMV潜伏期和分化诱导重新激活的新的体外模型。在没有生物体复杂性的情况下，这种互补的体外模型对于确定分子机制将特别有用。完成后，我们的研究将确定导致病毒染色质表观遗传重新编程的信号通路，以重新激活潜伏的MCMV，以响应移植和潜在的治疗靶点。

DESCRIPTION (provided by applicant): Reactivation of latent human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in transplant recipients, despite the use of antiviral drugs. Therefore, new approaches are required to reduce the complications from this pathogen. Due to the species specificity of HCMV, we have developed a novel transplant model using the highly related murine CMV (MCMV) as a model to study CMV latency and reactivation in the context of organ transplantation. In this model, MCMV latently infected kidneys are transplanted into immunocompromised NOD.Cg-PrkdcscidIL2rgtm1Wjl/Szj (NSG) mice, which are deficient in T, B, and NK cells. Transplantation of latently infected kidneys into NSG recipients results in reactivation of latent virus in the donor kidney, which disseminates systemically. Reactivation of HCMV is associated with various inflammatory insults. Our preliminary data shows that IL-6, soluble CD40L, IL-18, and LIF are elevated in the plasma of recipient mice with timing consistent with a causal role in reactivation. IL-6 induces reactivation of HCMV in hematopoietic cell models. The central hypothesis of this proposal is that the inflammatory response elicited by the transplanted kidney results in the release of mediators in the recipient mice. The resulting signaling cascade stimulates epigenetic reprogramming of latent viral genomes, transcriptional reactivation of viral immediate early (IE) gene expression, and in immunocompromised recipients, re-entry of latent virus into the lytic replication program. To test this hypothesis, in Aim 1 we will investigate the requirement for candidate factors identified in our preliminary studies, and their downstream signaling intermediates, in inducing reactivation in the NSG model. Our previous studies and those of others show that lytic viral gene expression is repressed in CMV latency due to heterochromatinization of viral genomes. In addition, we have shown that transplant-induced reactivation of MCMV immediate early gene expression is associated with epigenetic reprogramming. Previous studies by our collaborator, Dr. Thomas Kristie, have identified epigenetic inhibitors that suppress reactivation of latent herpes simplex virus. In Aim 2 we will investigate these promising therapeutic interventions for their ability to prevent reactivation of latent MCMV in the NSG model. Finally, in Aim 3 we will investigate new in vitro models for MCMV latency and differentiation-induced reactivation in murine hematopoietic progenitor cells. This complementary in vitro model would be exceptionally useful for defining molecular mechanisms in the absence of the complexity of the organism. Upon completion, our studies will have identified signaling pathways that lead to epigenetic reprogramming of viral chromatin to reactivate latent MCMV in response to transplantation and potential therapeutic targets.