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Abstract

Dengue virus (DENV) is a highly prevalent pathogen of the Flaviviridae family that can be transmitted to humans by the bite of female Aedes mosquitoes. Although only a small proportion of infections result in a potentially life-threatening disease characterized by haemorrhagic fever and shock, even the majority of the approximately 100 million annual symptomatic cases, which typically present with a debilitating but self-limiting febrile infection, collectively cause an enormous medical and socioeconomic burden, particularly in tropical countries where most infections occur. Despite the great need, antiviral drugs for prevention or treatment of the disease are not yet available. As part of my PhD research, I have been working on elucidating the mechanism-of-action of two novel DENV-specific drug candidates named JNJ-A07 and JNJ-1802. JNJ-A07 is the first compound for which nano- to picomolar activity against all available genotypes within the four DENV serotypes has been demonstrated in vitro. JNJ-1802 is from the same chemical series of compounds and was selected for the clinical development process based on improved pharmacological properties. Using expression-based systems, I uncovered that JNJ-A07 blocks the formation of a previously unknown interaction between the viral protease-helicase complex NS2B/NS3 and another viral component, the NS4A-2K-NS4B cleavage intermediate. The same inhibition of interaction was found for JNJ-1802. Electron microscopy studies showed that blocking NS2B/NS3 interaction with NS4A-2K-NS4B by JNJ-A07 is functionally linked to a halt in the assembly of vesicle packets (VPs), which are membrane invaginations on the endoplasmic reticulum. As revealed by pulse-chase analysis in the context of the viral polyprotein, JNJ-A07 does not affect the cleavage kinetics of NS4A-2K-NS4B, suggesting that delayed protein maturation is unlikely to be a mediating effect and that instead JNJ-A07 inhibits the assembly of a multi-protein complex around NS4A-2K-NS4B and NS2B/NS3, which is structurally important for the biogenesis of vesicle packets. In any case, their loss explains the antiviral activity of JNJ-A07 since VPs are essential for viral replication processes. In support, I demonstrated that resistance mutations, which remarkably all accumulated in the sequence of NS4B, rendered both the NS4A-2K-NS4B interaction with NS2B/NS3 and de novo formation of VPs less sensitive to JNJ-A07. Furthermore, these mutations caused a reduced level of co-localization between NS4B-containing structures and a photoaffinity derivative of JNJ-A07, implying diminished compound binding as an important component of the resistance mechanism. Surprisingly, I found that some of the mutants had little or no fitness deficits in mammalian cells. In fact, the strongest resistance mutant, NS4B L94F, showed a pronounced fitness advantage in this system; however, virus mutants containing L94F were unable to replicate in mosquito cells. In conclusion, the results presented in this dissertation provide important insights into novel DENV-specific inhibitors with unprecedented activity. Moreover, the discovered NS4A-2K-NS4B interaction with NS2B/NS3 delivers new evidence that DENV uses short-lived cleavage intermediates to carry out early steps in the viral replication cycle.