Final Report Summary - HCV-AKAP (The role of PKA in the Hepatitis C virus life cycle) Hepatitis C virus (HCV) is a globally important human pathogen with approximately 170 million infected individuals. HCV establishes a persistent infection in the majority of infected subjects, leading to liver disease including fibrosis, steatosis and hepatocellular carcinoma. HCV is an enveloped positive-stranded RNA virus and the sole member of the genus Hepacivirus within the Flaviviridae. The recent development of an infectious system allowing the generation of HCV particles in cell culture has enabled the complete viral life cycle to be explored. Multiple steps in the viral life cycle including entry, internalization, replication, virus assembly and release depend on host cellular signaling pathways.We recently discovered a role of the cAMP-dependent protein kinase (PKA) in the assembly of infectious hepatitis C virus particles (1). During a short stay in 2009 funded by a Boehringer Ingelheim travel grant Dr Diskar showed that HCV encoded Core protein negatively regulates PKA dynamics, suggesting a novel mechanism for the virus to perturb PKA signaling. Based on these data we hypothesized that the HCV Core protein is a potential A-kinase anchoring protein (AKAP). AKAPs are scaffolding proteins that tether PKA to target membranes and organelles. PKA plays an important role in the regulation of protein trafficking along the constitutive secretory pathways. By supplying an aberrant anchoring signal to PKA, HCV may promote its localization to lipid droplets where virus assembly and egress of infectious particles occurs from the cell. This is the first observation that a virus can hijack PKA signaling and relocalize PKA to defined cellular locations.Core is the central component of the virus particle and comprises three domains: domain D1, the N-terminal 120 amino acids (aa), involved in the recruitment of viral RNA to the viral particle; domain D2, a hydrophobic region (aa 120-175) consisting of two conserved helices, involved in ER and lipid droplet association; and domain D3, the C-terminal 20 aa, responsible for targeting E1 and E2 glycoproteins to the ER. Our newly discovered PKA-R binding sequence lies within the first of the two α-helices in D2, an area known to be important for core targeting to lipid droplets (2). The project aimed to (i) investigate the nature and regulation of the PKA-HCV Core interaction by applying a cell-based assay to monitor the interaction in living cells in real-time; (ii) to identify the amino acid sequence involved in the interaction and find substances that specifically disrupt this interaction to provide lead compounds for anti-viral drug design (small molecules and peptide disruptors); and (iii) to study the role of this interaction in the development of steatosis and insulin resistance. Dr Diskar´s data that are in preparation for publication show that (i) HCV infection redirects PKA-RI and -RII from the cytoplasm to lipid droplets; (ii) HCV Core binds PKA-RII engaging the minimal binding sequence S116RNVGKVIDTLTC in the D2 domain; (iii) the Core-PKA RII interaction can be successfully reconstituted in a cell-based assay using truncated versions of Core and the N-terminal part of PKA-RII to study its regulation in more detail, and (iv) that this assay was exploited for a high-throughput screening campaign to detect small molecule disruptors. Importantly, these findings were confirmed by an external collaborator using independent experimental approaches (Prof K. Tasken and Anna Mari Lone, Oslo, Norway). Interestingly, the viral encoded Core protein is not acting as an AKAP in the classical sense since it doesn´t involve the amphipathic groove of the PKA-R dimerization/docking domain and cannot be competed with conventional AKAP disruptors. In summary, we have found a completely different binding mode unique to Hepacivirus Core proteins. Thus, Dr Diskar´s work provides conclusive evidence of a novel mechanism for HCV interfering with the cellular machinery, representing a major advance in our understanding HCV infection. The next important step is the synthesis of a tailor-made peptide that inhibits the core-PKA RII interaction and to examine its impact on HCV assembly and infectivity. Alongside her own research project Dr Diskar initiated a new collaboration with Dr Sally Roberts (UoB) to study the role of PKA in the Human Papillomavirus (HPV) life cycle. Several critical HPV functions are regulated by PKA phosphorylation and PKA signalling may play a role in HPV oncoprotein-induced transformation (3,4). However, the nature of the interaction between HPV and PKA is not known. By using Dr Diskar´s PKA sensors based on bioluminescence resonance energy transfer (BRET) in human keratinocyte models of the oncogenic HPV life cycle we have shown that HPV18 up-regulates PKA activity. The mechanism underpinning this change in activity of the kinase cannot be explained by an increase in cAMP levels and thus remains to be resolved. HPV replication switches to vegetative genome amplification upon differentiation of the infected cells; this phase of the life cycle is recapitulated in the keratinocyte-HPV models following stratification in organotypic raft culture. Remarkably, in this phase of the life cycle, is a marked decrease in RI protein levels compared to the HPV-negative donor keratinocytes, suggesting differential engagement with the PKA pathway. We hypothesize, that HPV engages PKA signalling to facilitate the HPV life cycle and this virus-host interaction contributes to the progression of HPV infections.Furthermore, Dr Diskar was awarded a Wellcome Trust ISSF grant from the University of Birmingham to perform a compound screening with the Fraunhofer IME ScreeningPort in Hamburg, Germany to identify small molecules that disrupt the PKA-HCV Core interaction. This has provided her with a unique opportunity to acquire work experience in an industrial environment. As this project is still ongoing Dr Diskar will still be involved in this work after the end of her fellowship and several publications from this project are anticipated upon final analysis by the project end in 2015.