Sec61-gerichte remmers van virale eiwitimport in het ER als antivirale middelen tegen orthoflavivirussen

The genus of Orthoflaviviruses includes mosquito-borne RNA viruses like dengue, Zika and yellow fever virus which impose a significant threat to global health. Dengue virus (DENV) is currently the most widespread arbovirus, endemic in more than 100 countries. Due to the spread of its Aedes vector, DENV is further expanding to new areas in Europe and the Americas. Infections by one of the four DENV serotypes manifest across a broad clinical spectrum, ranging from subclinical or self-limiting febrile illness to life-threatening severe dengue. At present, no FDA-approved specific antiviral treatment is available, and the safety of a vaccine against DENV is still on debate. The first chapter gives an overview of the role of the ER in DENV replication and describes the different ER translocation inhibitors. The endoplasmic reticulum (ER) plays a central role in orthoflaviviral infections, supporting multiple stages of the viral life cycle, including translation, post-translational modification, genome replication, and assembly. Integration of the orthoflaviviral polyprotein into the ER membrane is proposed to occur through the Sec61-dependent co-translational translocation pathway. Although inhibitors of Sec61-dependent translocation are considered as promising candidates for anti-cancer therapies or to treat several human diseases, their potential in the context of antiviral treatment has received limited attention. In the Vermeire lab, the small molecule inhibitors cyclotriazadisulfonamides (CADAs) have been discovered as a new class of Sec61 translocon inhibitors with high substrate selectivity. CADA interferes with Sec61-mediated co-translational protein translocation in a signal peptide (SP)-dependent manner. In this PhD project, we explored the antiviral potential of CADA derivatives against DENV and other orthoflaviviruses and studied the mode-of-action of CADAs as translocation inhibitors of DENV. In the first part of this project (chapter 4), we describe the antiviral activity for the CADA derivative Flavitransin (FT), previously named VGD020, against DENV serotype 2 (DENV2) in various cell lines and primary immune cells at sub-micromolar concentrations. Moreover, FT showed consistent antiviral activity against all four DENV serotypes, and also against Zika and yellow fever virus. In the second part of the project (chapter 5), we focus on the mode-of-action of FT. A time-of-drug addition assay suggested that FT interferes with a post-entry event of virus replication taking place between 4-8 hours post infection, in line with its anticipated Sec61 inhibition. Through a set of cell-transfection experiments and complimentary cell-free in vitro translation/translocation assays we could irrefutably demonstrate that FT specifically and selectively inhibits the first translocation step of the DENV polyprotein, i.e., the translocation of the pre-membrane protein. Additional cell-transfection experiments indicated that only the first (N-terminal) transmembrane domain (TMD), which serves as the main Sec61 targeting sequence of the viral nascent chain, conferred sensitivity to FT. In contrast, the more downstream SP-like TMD of the Envelope and Non-Structural protein 1 showed full resistance to FT. Combinational treatment of transfected cells with a proteasome inhibitor and FT, showed that upon FT treatment, a small fraction of the non-translocated protein is re-routed to the cytosol for proteasomal degradation. In the third and fourth part of this project (chapter 6 and 7), we mapped the complex interplay between FT, the Sec61 translocon (host), and the DENV targeting sequence. Selection and genotyping of FT-resistant HCT116 cells revealed a unique set of mutations in the Sec61α subunit e.g. A70V, E78A and L449F. In the presence of these mutations, FT's antiviral activity was diminished or completely abolished as verified in DENV-infected cells, transfected cells with DENV-reporter constructs, but also in a cell-free in vitro translation/translocation system. In addition, FT exerted a high barrier to the development of viral resistance. FT-desensitized DENV, obtained after a high number of passages under increasing FT pressure, had acquired multiple AA substitutions at different sites (also at the first TMD) throughout the polyprotein, but remained fully sensitive to high concentrations of FT. In the final chapter, we explored the antiviral activity of FT in the mosquito vector context. FT preserved its antiviral activity in the Aedes albopictus mosquito cell line and blocked the translocation of the DENV pre-membrane protein in a cell-free translation/translocation assay with insect microsomes. In an ex vivo DENV2 infected midgut model of Aedes aegypti mosquitoes FT preserved its antiviral activity, although in the micromolar range and with some related toxicity. In conclusion, this PhD project characterised the pan-DENV serotype and pan-orthoflaviviral potency of the CADA derivative FT. The antiviral activity of FT could be clearly attributed to selective inhibition of Sec61-directed translocation of the first N-terminal SP-like TMD of the viral polyprotein into the ER membrane. This confirms the critical role of the ER in orthoflavivirus replication and sets the base for selective Sec61 inhibitors as an alternative antiviral strategy against orthoflaviviruses.