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Abstract

Arboviruses are one of the greatest threats to human health since they cause millions of infections each year. The mosquito-borne flavivirus Zika virus (ZIKV), which is listed as a priority disease, has been declared a public health emergency of global concern by the World Health Organization (WHO) in 2016. Even though most infections are asymptomatic or manifest only moderate symptoms, infections have been related to neurological disorders such as the Guillain-Barré syndrome and microcephaly. Despite years of intensive research, there are presently no effective antiviral therapies or vaccines. Their development requires further studies of the fundamental principles of the ZIKV life cycle to provide the necessary knowledge for the development of effective therapeutic measures. Upon ZIKV infection, the cell’s endoplasmic reticulum (ER) is significantly remodeled resulting in the formation of defined ER membrane invaginations termed vesicle packets (VPs), which are thought to be the sites where viral RNA replication takes place. Since their formation is coupled to viral replication, it has been challenging to retrieve mechanistic information on VP biogenesis thus far, as perturbations that reduce viral replication inevitably impact VP formation. To overcome this hurdle, my first project was to develop an expression system supporting the formation of ZIKV VPs to enable research focusing entirely on their biogenesis and the factors involved. I was able to demonstrate that ZIKV VPs are forming upon expression of the viral replicase (NS1-NS5) via a construct comprising in addition the 5’ and 3’ untranslated region (UTRs). Ultrastructural characterization of VPs in transfected cells revealed that the VPs are morphologically identical to those produced in infected cells. This newly created system was given the name pIRO (plasmid-induced replication organelle). A thorough deletion mutagenesis study focusing on RNA elements contained in the 5’ and 3’UTR revealed that for VP formation the 5’UTR is largely dispensable. In contrast, the 3’UTR was required as deletion of distinct RNA elements contained therein decreased the efficiency of VP induction. In conclusion, I was able to show that ZIKV VP formation occurs independent of RNA elements with the size and morphology of VPs being independent of the length of the viral RNA. The second part of my PhD thesis was devoted to characterizing the mode of action of a novel ZIKV NS4A inhibitor. This topic was a perfect fit to the first part of my thesis, given the essential role of NS4A in VP formation. The inhibitor was developed by a collaborator who did a large-scale cell-based high-content screen, resulting in the identification of (2E)-N-benzyl-3-(4-butoxyphenyl) prop-2-enamide (SBI-0090799). This compound exhibits potent and wide- spread antiviral activity against numerous ZIKV strains in vitro. Using a combination of biochemical, virological, and imaging-based techniques, I confirmed that SBI-0090799 inhibits ZIKV replication by blocking the de novo formation of ZIKV VPs. Resistance mutations mapping to NS4A rescued viral RNA replication and restored VP formation in cells treated with high concentrations of the compound. These findings suggest that SBI-0090799 perturbs VP formation by interfering with NS4A, either by preventing it from inducing ER membrane curvature or inhibiting it from binding viral or cellular factors contributing to VP formation. Thus, the mechanism of action of SBI-0090799 is comparable to the one of hepatitis C virus NS5A inhibitors that are in clinical use. The third part of my PhD study aimed to unravel the role of cholesterol in the viral replication cycle. Although it has already been shown that cholesterol plays a critical role in virus entry, particularly at the stage of viral envelope fusion with the endosome, as well as virion assembly, direct viral protein-cholesterol interactions have not been determined thus far. To fill this gap in knowledge I performed chemo-proteomics in ZIKV-infected human cells, using a photoactivatable and clickable cholesterol probe, to identify cholesterol binding viral proteins. I discovered that both the structural protein prM and its cleavage product, the M protein, can be efficiently cross-linked to cholesterol. Combining bioinformatics analyses and site-directed mutagenesis, alongside with cholesterol binding assays, I was able to show that the M protein has two functional cholesterol binding domains (CRAC motifs) in its transmembrane domains (TMD) 2 and 3 (CRAC2 and CRAC3, respectively). Using reverse genetics studies, I could show that the M protein’s ability to bind cholesterol is not required for the processing of the viral polyprotein, viral RNA replication, and subcellular localization of the uncleaved prM protein. However, mutations affecting the cholesterol binding motif 2 (CRAC2) significantly impaired viruses in their ability to infect cells having low cholesterol levels. Furthermore, I was able to show that complete exchange of the cholesterol binding motif 3 (CRAC3) severely affected virus particle assembly. In line with these results, atomistic molecular dynamics simulations confirmed cholesterol binding to membrane-associated wild type M protein, whereas M proteins containing mutations in CRAC2 and CRAC3 lost cholesterol interactions. In conclusion, I was able to uncover a bifunctional role for the cholesterol interaction of the M protein in the ZIKV life cycle: facilitating virus entry requiring CRAC2 and virus particle assembly requiring CRAC3.