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Ubiquitin Signalling Pathways Required for Influenza Virus Replication: Biological Characterisation and Identification of Novel Drug Targets

Final Report Summary - UBIFLU (Ubiquitin Signalling Pathways Required for Influenza Virus Replication: Biological Characterisation and Identification of Novel Drug Targets)

1. Background.

Influenza viruses are a significant seasonal disease burden, and provide an ever-present threat of causing severe pandemics with potentially devastating clinical, social, and economic consequences. Vaccines and antivirals are available for the prevention and treatment of influenza. However, it often takes too long to manufacture, distribute, and administer an effective strain-matched vaccine under pandemic circumstances, while drug-resistant viruses often emerge against the approved antivirals. Thus, there is urgent need to develop new antivirals with lower chances of selecting drug-resistant strains. Influenza viruses rely extensively on host cell functions, therefore one way to minimise resistance is to target novel antivirals against host proteins required for virus replication. This project was focused on identifying and characterizing cellular E3 ligases with druggable qualities that are host factors modulating influenza virus replication. The project sought to establish functional and mechanistic links between E3 ligases modulating virus replication and the ubiquitin-like modifications they cause. The central hypothesis was that there are specific modifications on cellular or viral proteins that are essential for virus replication. Understanding the mechanisms underlying these modifications will provide insights into the interplay between influenza viruses and their hosts, and could represent potential new therapeutic targets.

2. Results & Conclusions.

The ubiquitin-like modifiers relevant to this study are SUMO1 and SUMO2/3. Like other ubiquitin-like modifiers, SUMO proteins are important mediators of cell signalling, and are covalently linked to other proteins post-translationally in order to alter substrate function. Thus, dynamic protein modification by SUMOs contributes to an array of nuclear biology, notably cellular stress responses. As such, SUMO modification is a key player in the replication of many viruses. As part of this Marie-Curie Career Integration Grant study, we observed that influenza virus infection causes a dramatic redistribution of intra-nuclear SUMO which parallels a global increase in SUMO conjugates. This induction of SUMOylation is dependent on virus genome replication and protein synthesis, but is independent of several canonical virus-activated stress pathways. We also generated evidence that active viral RNA polymerase activity is a major trigger for SUMOylation during influenza virus infection, and this may be part of a novel, previously uncharacterised, host-cell stress response. SILAC-based affinity proteomics allowed the system-wide identification of the SUMO1- and SUMO2- modified proteome in human lung epithelial cells, as well as the quantification of specific changes that occur in SUMOylation during influenza virus infection. Combined with results from large-scale shRNA-depletion screening, the data suggested that influenza virus infection re-targets SUMO proteins to a multitude of host factors that either promote or restrict virus replication. We hypothesize that a component of virus-triggered SUMOylation is an intrinsic cellular response to virus insult that modulates protection against infection. Understanding the molecular basis of such a response, and the E3 ligases involved, will be key to identifying natural mechanisms of protection that could be harnessed in novel anti-influenza therapeutic strategies. Overall, our analysis of SUMO posttranslational modifications established a new framework for understanding the way in which the host-cell nucleus is ‘re-wired’ during influenza virus infection. The major findings from this work were published in Domingues, Golebiowski et al., Cell Reports, 2015.

3. Impact.

(a) For fundamental research.

Prior to our work, only very limited knowledge was available on ubiquitin-like modifications that occur during influenza virus infection. Specifically to SUMO, a few viral proteins, but no cellular proteins, were identified as targets during influenza virus infection. The proteomic methodologies we have now successfully developed have expanded on this by defining >800 cellular SUMO targets in human lung epithelial cells. We have further identified approximately 100 cellular targets that increase in SUMO modification upon influenza virus infection, and validated 3 viral proteins that become SUMO modified, one of which is a novel finding. Our use of shRNA screening to determine the impact of the newly-identified cellular targets on influenza virus replication has defined new host factors that are either required for virus replication, or which act as potential restriction factors. Our work therefore opens up new opportunities for understanding influenza virus replication and interactions with the host cell.

(b) Possible translational opportunities.

Our SUMO proteomics work has given us new fundamental insights into the cellular pathways and protein-modification cascades that influenza virus infection modulates. In the future, this will help us define the basic mechanisms of how influenza viruses replicate, and may uncover new host pathways that contribute to influenza disease. Understanding the principles underlying viral activation of these pathways will aid in better defining whether they may be exploitable in the future as novel therapeutic targets. Given increasing resistance of influenza viruses to currently available antivirals, such as oseltamivir, the identification of new drug targets is of critical importance. There is urgent need to develop new antivirals with lower chances of selecting resistance, and one way to minimize resistance is to target new antivirals against host factors. In this regard, cellular signalling pathways under investigation as potential therapeutic targets for other human diseases (e.g. E3 ligases and cancer) might be good candidates, as existing inhibitors could be ‘repurposed’ for influenza treatment.