Defining the role of src family kinases in cytomegalovirus reactivation

人类巨细胞病毒(HCMV)和所有疱疹病毒一样，可以建立对宿主的终生感染。这些潜伏感染可以周期性地重新进入完整的裂解病毒生命周期(重新激活)，以在宿主的整个生命周期中产生新的后代病毒。虽然健康人巨细胞病毒潜伏期和重新激活的这一周期很少有问题，而且可能导致传播，但在免疫反应受损的患者(例如移植受者和艾滋病患者)中，这往往会导致大量发病率，导致移植后预后不良，而且如果不进行治疗，可能会导致死亡。目前的战略依赖于使用具有显著相关毒性的抗病毒药物，这些药物可以抑制病毒复制--这是导致巨细胞病毒病的主要原因。另一种方法是防止病毒从潜伏期重新激活，以限制宿主中的病毒复制水平。细胞利用细胞表面受体检测细胞外的分子(配体)。这些受体与配体的相互作用随后被转化为细胞内的生物反应。这是通过激活信号通路来实现的，信号通路将信号从细胞外转换到细胞内。我们现在知道，HCMV利用这些基本的信号通路从潜伏期重新激活。由白介素6(IL-6)驱动的炎症反应激活了一个关键的细胞通路(ERK-MAPK)，而ERK-MAPK反过来又促进了病毒基因在一种称为树突状细胞(DC)的特定细胞类型中的表达。IL-6可以在许多细胞类型中激活这一途径，因此，为什么它只促进DC的重新激活尚不清楚。我们将测试假设，当IL-6刺激DC时，额外的通路被激活，该通路与IL-6/ERK通路协同作用，由于DC中存在关键效应分子，从而产生重新激活HCMV所需的特定输出。使用一种允许我们调查是否可以观察到任何细胞类型特异性效应的方法，我们发现在可以支持HCMV重新激活的细胞中，有证据表明第二个特定的信号通路-src家族激酶(SFKs)-与ERK-MAPK一起被相关地增强激活。然后我们表明，化学抑制这一途径足以防止病毒重新激活。在这些研究中，我们将：1)确定这第二个信号通路对HCMV再激活的具体贡献2)确定SFK家族的某个特定成员是否对HCMV再激活起关键作用3)证明这两个通路协同作用促进DC的再激活并为这种相互作用提供机制基础4)研究一种新的策略来抑制这一通路的一个组成部分，这是HCMV再激活所必需的。这些研究的结果将是更详细地了解DC中的HCMV再激活过程，以及两个中央信号通路之间发生的促进再激活的串扰的机制基础。此外，我们认为，了解这两条通路是如何相互作用的，可能会提供一种新的策略，通过靶向宿主依赖的反应来抑制HCMV的重新激活。这变得很有吸引力，因为针对宿主的反应可能会增加产生抗药性病毒的障碍，而抗药性病毒在临床上可能是严重的疾病来源。

Human cytomegalovirus (HCMV), like all herpes viruses, can establish lifelong infections of the host. These latent infections can periodically re-enter the full lytic viral lifecycle (reactivate) to produce new progeny viruses throughout the lifetime of the host. Although this cycle of HCMV latency and reactivation in healthy individuals is rarely problematic and likely contributes to transmission, in patients with impaired immune responses (e.g. transplant recipients and AIDS patients) this often results in substantial morbidity contributing to poor outcomes post transplant, and without treatment, can lead to death. Current strategies rely on the use of anti-viral drugs with significant associated toxicities which act to inhibit viral replication - the major driver of HCMV disease. An alternative approach is to prevent viral reactivation from latency to limit the level of viral replication in the host. Cells detect molecules (ligands) outside of the cell using cell surface receptors. These receptor:ligand interactions are then translated into biological responses within the cell. This is achieved via the activation of signalling pathways which translate the signal from outside the cell to inside the cell. We now know that HCMV utilises these fundamental signalling pathways to reactivate from latency. Inflammatory responses driven by interleukin-6 (IL-6) activate a key cellular pathway (ERK-MAPK) which in turn promotes viral gene expression in a specific cell type called dendritic cells (DCs). IL-6 can activate this pathway in many cell types and thus why it only promotes reactivation in DCs is not clear. We will test the hypothesis that additional pathways are activated in DCs when stimulated with IL-6 that act in concert with the IL-6/ERK pathway to generate the specific output required to reactivate HCMV due to the presence of key effector molecules in DCs.Using an approach which allowed us to investigate if any cell type specific effects of IL-6 could be observed we identified that in cells that could support HCMV reactivation there was evidence of a correlative increased activation of a second specific signalling pathway - src family kinases (SFKs) - alongside ERK-MAPK. We then showed than chemical inhibition of this pathway was sufficient to prevent viral reactivation. In these studies, we will:1) Define the specific contribution this second signalling pathway makes to HCMV reactivation2) Define whether a specific member of the SFK family is critical for HCMV reactivation3) Demonstrate that these two pathways are acting co-operatively to promote reactivation in DCs and provide a mechanistic basis for that interaction4) Investigate a novel strategy to inhibit a component of this pathway that is requirement for HCMV reactivation The outcomes of these studies will be a more detailed understanding of the process of HCMV reactivation in DCs, and a mechanistic basis for the cross-talk occurring between two central signalling pathways to promote reactivation. Furthermore, we propose that understanding how these two pathways interact may provide a novel strategy to inhibit HCMV reactivation via the targeting of a host-dependent response. This becomes attractive as targeting a host response potentially raises the barrier to the generation of resistant viruses which, clinically, can be a serious source of disease.