Defining the role of src family kinases in cytomegalovirus reactivation

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 reactivation
2) Define whether a specific member of the SFK family is critical for HCMV reactivation
3) Demonstrate that these two pathways are acting co-operatively to promote reactivation in DCs and provide a mechanistic basis for that interaction
4) 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.

Human cytomegalovirus (HCMV) reactivation is dependent on the activation of cellular signalling pathways. We have demonstrated that interleukin-6 (IL6) activation of ERK promotes reactivation in dendritic cells (DCs) but not progenitors. Thus why IL-6 activation of ERK only promotes reactivation in DCs is unclear. We hypothesised that the concomitant activation of additional pathways underpinned this DC-specific phenotype. A phospho-proteomic approach revealed that a src family kinase, haematopoietic cell kinase (HCK), was activated more efficiently by IL6 in a DC subtype that supported reactivation. Further evidence for a role was the observation that pharmacological inhibitors of src family kinase signalling inhibited reactivation. Preliminary data suggest that the role of SFK activity is to act in concert with ERK signalling to promote the post-translational modification of histones bound to a key viral promoter to ensure efficient viral gene expression.
In this study we will:
1) Use genetic approaches to delete HCK from DCs to demonstrate a role for HCK in HCMV reactivation focusing on the impact of this deletion of the induction of the viral gene expression
2) Test whether we can rescue HCMV reactivation in cells that do not normally support reactivation through introduction of a constitutively active version of HCK
3) Using Chromatin IP approaches, demonstrate that the role of HCK and SFK activity is to potentiate the chromatin remodelling initiated by ERK-MAPK signalling
4) Demonstrate that a peptide mimic of gp130 (the signalling arm of the IL-6 receptor) inhibits HCMV reactivation through the targeted blockade of IL-6 induced HCK activation

The study will improve our understanding of HCMV reactivation and, furthermore, enhance our understanding how crosstalk between pathways regulates eukaryotic gene expression using HCMV as a paradigm. Ultimately, it may lead to a strategy to target a host response to inhibit HCMV reactivation therapeutically.

Human cytomegalovirus (HCMV) is an opportunistic viral pathogen that causes substantial morbidity in a number of patient groups. A key contributor to this is following the reactivation of lifelong latent infections in patients. The studies proposed herein seek to understand how HCMV is dependent on key host cell functions to reactivate.

An understanding of how HCMV is dependent will have immediate impact on researchers in this area of study through improvement of our understanding of the biology of this pathogen. Furthermore, there is broader impact because the questions being posed are fundamentally a question of how is eukaryotic gene expression regulated? Thus academics working in the field of gene regulation, chromatin biology and host cell signalling will be interested in the outcomes of this research.

Long term impact will be measured through the acquisition and application of new knowledge that will underpin future studies that could lead to more translational outcomes. The primary aim of the project proposed to increase our fundamental understanding of the biology of the virus and its interaction with the host. However, the implications are that the identification of less toxic niche interaction with the host cell could provide a novel strategy to target a host function to control viral replication. Ultimately, we will interact with clinical colleagues at the Royal Free Hospital to ensure that any opportunity for clinical translation is not missed. Successful development of new approaches will have major societal and economic impact. Reducing the effect of HCMV in transplant, where it represents an important cause of morbidity and graft failure in transplants, will improve patient outcomes and reduce the healthcare costs associated with treating the effects of HCMV infection in these patients.

Finally, another important aspect of our impact as researchers is the training of the next cadre of scientists. This work will be undertaken in a university laboratory that hosts and trains undergraduate to post-doctoral research students. It will be given that an aspect of the research proposed will be the training of the PDRA recruited and, in turn, providing the opportunity to train younger students seeking their first exposure to scientific research by immersing them in a research environment (e.g. undergraduate students).