Cholesterol transporters as gatekeepers of viral infection

Many viruses, including human pathogens like the bunyavirus Crimean Congo hemorrhagic fever virus (CCHFV), the only BSL4 virus endemic in Europe, utilize the endocytic pathway to infect their target cells. During this trafficking, changes in K+ concentration are required for effective infection. In addition to K+ changes, cholesterol levels in endocytic vesicles,believed to be mainly regulated by the lysosomal NPC1/NPC2 shuttling system, are critical for cell infection by those and other pathogenic viruses. We have recently shown that the Hedgehog receptor PTCH1 is a cholesterol/K+ antiporter that localizes to lysosomes and regulates degradation of the content of autophagic vesicles. Given this novel information, we propose that PTCH1 might play a significant role in viral entry through the endocytic pathway. This will be the first time that a role for the Hedgehog receptor PTCH1 on viral infection will be investigated. This study will consolidate ongoing recent observations that PTCH1 has Hedgehog- independent, primitive functions in the endocytic pathway, and the requirement of K+ for bunyavirus entry. We will compare the infectivity of BSL2 model bunyaviruses (Bunyamwera virus, a model of the whole order; and Hazara virus, a model for CCHFV) in WT cells and CRISPR-engineered cells deficient in PTCH1, NPC1 or its partner NPC2 by western blot and immunofluorescence imaging. The effect of the NPC1 inhibitor U-18666A in NPC1 KO cells with overexpression or silencing of PTCH1 will be investigated in terms of cholesterol and K+ accumulation in intracellular compartments and viral infectivity. Infection studies will also be extended to PTCH1 KO cells rescued with PTCH1 mutants deficient in cholesterol transport or cation transport. Finally, we will investigate the potential antiviral effect of the nanobody TI23, which prevents PTCH1 cholesterol transport.