Defining the role of src family kinases in cytomegalovirus reactivation

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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.