Deciphering cellular decluttering The impact of autophagy mechanisms in host-virus immune interactions

Within this thesis, we connect in-depth molecular investigation of innate intracellular immune mechanisms with the practical study of repurposing host-targeting pharmaceuticals for antiviral defense. These dual aims are underpinned by the use of human-relevant, animal-free models. Throughout, we focus on autophagy, a host system of ‘decluttering’ and defense in which cytoplasmic material can be (selectively) targeted for lysosomal degradation, or alternatively for extracellular secretion. The work herein spans the contexts of three viruses that each represent major global health concerns, namely HIV-1, SARS-CoV-2, and dengue virus (DENV). Our work on HIV-1 includes a novel conceptualization of the autophagy receptor TRIM5α as a cell-specific HIV-1 restriction factor with a varied portfolio antiviral functions, the discovery of a multivesicular body (MVB)-mediated transcellular pathway by which HIV-1 traverses epithelial cells of the human intestinal mucosa, and the identification of several autophagy-enhancing pharmaceuticals that prevent HIV-1 acquisition or intervene in ongoing HIV-1 infections. This work was performed in novel physiological, animal-free models showcased herein, including an ex vivo human skin model encompassing relevant immune cell types such as dendritic cells (DCs) and CD4+ T cells, and a human 2D intestinal epithelial monolayer model with or without the immune compartment. Our work on SARS-CoV-2 identified novel roles for autophagy molecules in viral entry, highlighting human BNIP3 as a relevant molecular target to block intestinal SARS-CoV-2 entry and virus-induced tissue damage. Our human 2D intestinal epithelial monolayer model, which closely recapitulates in vivo gut polarization and barrier function, permitted pre-clinical drug screening and identification of the autophagy-blocking molecule berbamine dihydrochloride as having pan-SARS-CoV-2 antiviral activity with nanomolar potency. Finally, we investigated degradative and secretory functions of autophagy in the context of DENV infection, underlining that DENV exploits early stages of autophagy in primary human DCs and is targeted into LC3+ autophagosomes. We present the first ever characterization of LC3+ extracellular vesicles (EVs) released by primary human DCs, and demonstrate a role for DC-derived EVs in DENV dissemination, thereby highlighting intersections between autophagy and secretory pathways in the context of DENV infection. In summary, this thesis presents molecular insights, pre-clinical drug screenings, and novel animal-free models in the context of HIV-1, SARS-CoV-2, and DENV infections –contributing to theoretical knowledge, translational research, and practical toolkits at the intersection of immunology and virology.