Characterisation and therapeutic manipulation of Flaviviridae innate immune evasion

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.

We have developed three series of novel cyclophilin inhibitors (CypI) and demonstrated that specific compounds activate innate immunity against hepatitis C virus (HCV) and are potent inhibitors of dengue virus (DV) and Zika virus (ZV). We will now measure replication of defined HCV/DV/ZV subgenomic replicon systems encoding luciferase and full length viruses in Cyp manipulated cells (RNAi/CRISPR or CypI) to identify the Cyps involved and the active CypI, and to define the role of innate immunity in the inhibitory process (by manipulating sensors and adapters to rescue inhibited infection). We will explain CypI antiviral specificity by correlating Cyp specificity (measured in recombinant protein binding assays) with antiviral specificity. We will examine the effect of Cyps on the viral replication compartments (confocal/electron microscopy), testing whether sensors colocalise with viral RNA/proteins on Cyp manipulation, and how host and viral proteins are relocalised when sensing is activated. We will discover which viral proteins are targeted by Cyps using binding assays between candidate proteins as well as by immunoprecipitating viral proteins and examining copurifying cellular proteins using mass spectrometry. We will determine how Cyp recruitment changes viral proteins using X-ray crystallography of Cyp-viral protein complexes. We will select virus CypI resistant mutants and characterise their sensitivity to innate immunity. We will identify the sensors and innate immune pathways activated by wild type and mutant viruses in Cyp manipulated cells. Overall, this work will define the Cyps acting as cofactors for Flaviviridae, explain how they aid in evasion of innate immune sensing, and produce a series of specific and potent inhibitors that disturb innate evasion to unleash the innate immune system against the virus as a new paradigm for antiviral prophylaxis and therapy.

Here we will demonstrate how the innate immune system can be harnessed for antiviral therapy by drugging the protective cloaking mechanisms viruses use to hide from innate immune sensors. We have already developed host-targeting cyclophilin inhibitors (CypI), which effectively inhibit emerging viruses dengue virus (DV) and Zika virus (ZV), as well as hepatitis C virus (HCV), an established and worldwide human infection. Critically, our preliminary data illustrate the involvement of innate immunity in anti-HCV activity of these molecules and we anticipate innate immune involvement for DV/ZV too. Detailing how innate immunity is activated against infection and how these molecules can be developed as effective antivirals provides a new paradigm for anti-infectives. We expect this strategy to be widely applicable, as all infectious agents must evade host defences through related evasion and cloaking mechanisms. The generality of our approach is illustrated by the literature that demonstrates that CypI act against a huge range of untreatable viruses.

Importantly, our preliminary data demonstrate some compounds with broad antiviral specificity inhibiting HCV, DV and ZV and other molecules with antiviral specificity inhibiting only a single virus. By explaining this specificity, through the experiments proposed, this work could facilitate the development of broad and narrow specificity inhibitors. This is important because broad specificity inhibitors are not always preferable due to the risk of inadvertently selecting resistance against a different drug/pathogen combination. Our results will detail mechanistically how specificity is determined, allowing its management. Our work is also expected to explain drug resistance, its likelihood and its mechanisms. Beneficiaries will therefore include all those who benefit from the increased understanding of development of specific and broad specificity antivirals.

Should our research lead to the establishment of CypI as effective antivirals, then the beneficiaries will include two large populations (i) everybody who takes the new drugs to treat infection and (ii) everyone who uses them as prophylactics. DV currently infects 60 millions people per year and kills 10,000. Given the range of viruses inhibited by CypI, the numbers of individuals eventually treated with inhibitors developed based on the science discoveries proposed herein could be enormous. As our inhibitory strategy targets innate cloaking mechanisms, our molecules only activate innate sensing in the presence of the virus. This has the effect of making the virus behave like a virus that is not adapted to evade innate sensing, and therefore replicate, in that particular host. Thus, our antiviral strategy could protect treated individuals from becoming infected in the first place, through a similar mechanism that protects hosts from infection naturally. By describing a mechanism that is different to the classical approach of developing inhibitors of viral enzymes, we will open new avenues of research that could lead to enormous steps forward in inhibiting pathogenic infection in humans and animals as well as developing effective prophylactics for infection.

Our work will also benefit those studying and developing antivirals in general. Our observation that antivirals work more effectively in cells that are competent for innate immune sensing will be an important consideration, even when developing direct acting antivirals. This is because viral enzymes also have roles in evasion of innate immunity. Thus, their inhibition can make inhibited virus sensitive to innate immune sensing, meaning that antiviral effects are best studied in systems where innate immunity is active. Our work will detail how this can be achieved and demonstrate the difference in the inhibitory effects in innate competent and innate defective systems. In this way, our work will contribute to the improvement of antiviral discovery in general.