Characterisation and therapeutic manipulation of Flaviviridae innate immune evasion

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Flavirviridae include hepatitis C virus (HCV) and the insect transmitted viruses dengue virus (DV) and Zika virus (ZV). DV and ZV are emerging infections, spreading to new regions of the world, in part due to climate change expanding insect vector ranges. Currently around 60 million people suffer symptomatic DV infection each year with 10,000 deaths. It will be increasingly important to understand how to develop new antivirals as new infectious agents emerge and old agents spread. Cyclophilins (Cyps) are host enzymes that alter the shape of target proteins, but have poorly understood cellular functions. Critically, Cyps are widely found to act as cofactors for viral infection and HCV, DV and ZV all depend on Cyps for replication. Cells are very good at protecting themselves from infection using a variety of mechanisms termed cell autonomous innate immunity. This complex system detects incoming and replicating viruses using sensors that detect molecular patterns that are specific to the pathogen. Sensor triggering activates production interferon, which is secreted and induces expression of a whole host of antiviral proteins and pathways that potently suppress infection in nearby cells. Successful viruses must evade or antagonise these protective systems. Typically viruses hide from the sensors in a process we term cloaking. Flaviviridae hide by subverting the intracellular membrane system, cloaking their replication complexes in a membranous web. This excludes key sensors allowing unhindered RNA and viral protein production and viral assembly. Our preliminary data demonstrate that inhibiting Cyps uncloaks HCV revealing it to sensors including the RNA sensor RIG-I. This is evidenced by production of interferon when Cyp use by the virus is disturbed. Cyp inhibition is a powerful approach to developing antivirals because the antiviral effect is mediated by the host innate immune system, which is potent and difficult for the virus to escape. Targeting host factors makes the virus behave like a virus which doesn't infect humans because it activates and succumbs to innate immune defences.Our key discovery was to find that HCV doesn't require Cyps if its target cells have a defective innate immune system. This revealed that the virus uses Cyps to evade innate immunity. Here we aim to understand exactly how Flaviviridae use Cyps to evade innate immune protective systems. We will identify the specific Cyps used and test whether our novel Cyp inhibitors can suppress replication. We will identify the viral proteins targeted by Cyps and work out what Cyps do structurally to the virus proteins to help the virus hide. Our drug series distinguishes between different viruses and we will work out how. We will test which Cyps are targeted by the active drugs to explain the antiviral specificity that we see. We will study the membranous web and viral replication compartment and see how it changes when we disturb Cyp activity. Do the sensors enter the replication compartment and get activated when Cyps are disturbed? We will also study the innate immune system and identify the active sensors that detect the virus when we reveal it. All of these aims use tried and tested techniques in use in our or collaborators' laboratories. Our work will generate critical understanding of how host factors can protect viruses from innate immunity and how this interaction can be disturbed as a novel therapeutic or prophylactic strategy. Our goal is to generate new knowledge detailing the molecular mechanisms of the process of innate immune evasion by Flaviviridae and the consequences of disturbing it. We propose that this new knowledge and the demonstration of the tractability of host targeting strategies will be necessary before such strategies can be taken to the clinic as effective therapeutics.