Defining the role of src family kinases in cytomegalovirus reactivation

人巨细胞病毒（HCMV），像所有疱疹病毒，可以建立终身感染的主机。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子代病毒。尽管健康个体中HCMV潜伏和再活化的这种循环很少有问题，并且可能有助于传播，但在免疫应答受损的患者（例如移植受体和AIDS患者）中，这通常导致大量发病，导致移植后不良结果，并且如果不治疗，可能导致死亡。目前的策略依赖于使用具有显著相关毒性的抗病毒药物，其作用是抑制病毒复制-HCMV疾病的主要驱动因素。另一种方法是防止病毒再激活潜伏期，以限制宿主中病毒复制的水平。细胞利用细胞表面受体检测细胞外的分子（配体）。这些受体：配体相互作用然后转化为细胞内的生物反应。这是通过激活信号传导通路来实现的，该信号传导通路将信号从细胞外翻译到细胞内。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。由白细胞介素-6（IL-6）驱动的炎症反应激活关键的细胞通路（ERK-MAPK），该细胞通路继而促进称为树突状细胞（DC）的特定细胞类型中的病毒基因表达。IL-6可以在许多细胞类型中激活这一通路，因此为什么它只促进DC中的再激活尚不清楚。我们将检验这样的假设，即当用IL-6刺激时，DC中的额外途径被激活，所述额外途径与IL-6/ERK途径协同作用以产生由于DC中存在关键效应分子而重新激活HCMV所需的特异性输出。6可以观察到，我们确定，在可以支持HCMV再活化的细胞中，有证据表明，与ERK-MAPK一起的第二种特异性信号传导途径- src家族激酶（SFKs）-的活化相关增加。然后，我们表明，化学抑制这一途径足以防止病毒再活化。在这些研究中，我们将：1）确定这第二个信号通路对HCMV再活化的具体贡献2）确定SFK家族的特定成员是否对HCMV再活化至关重要3）证明这两个通路协同作用以促进DC中的再活化，并为这种相互作用提供机制基础4）研究抑制该通路的一个组分的新策略，该组分是HCMV再活化的必要条件这些研究的结果将是对DC中HCMV再活化过程的更详细的理解，以及两个中心信号通路之间发生串扰以促进再活化的机制基础。此外，我们建议，了解这两种途径如何相互作用，可能会提供一种新的策略，通过靶向宿主依赖性反应来抑制HCMV再激活。这变得很有吸引力，因为靶向宿主反应可能会提高产生耐药病毒的屏障，而耐药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.