Host-targeted Antiviral strategies for Limiting Transmission of Crimean-Congo hemorrhagic fever virus

"The threat posed by emerging viral infectious diseases lingers heavily on public health. Modern shifts in global dynamics and fluctuation of climate conditions strongly affect the distribution of many arthropods, leading to a rise in the incidence of arthropod-borne infections. The Crimean-Congo Hemorrhagic Fever (CCHF) is a severe tick-borne disease with fatality rates reaching 40%. Its etiological agent is the CCHF virus (CCHFV), an orthonairovirus of the Bunyaviricetes class, which is endemic in Asia, the Middle East and Africa. The expanding geographical distribution of its vector, the Hyalomma spp. ticks, recently led to the detection of the virus in Spain, Corsica and the southern of France. While new populations are at risk of infection, CCHFV remains neglected, with no vaccines or antivirals approved. CCHFV biosafety level 4 (BSL4) classification, its complex genome organization along with the lack of knowledge on viral proteins functions pose significant challenges for research. Nonetheless, recent evidence from my host laboratory indicated a possible intersection between CCHFV life cycle inside the cell and the metabolism of lipoproteins. The first objective (work-package 1, WP1) of my PhD project will exploit this novel finding to assess the putative antiviral function of licenced lipoprotein inhibitors. In specific, I will incubate selected inhibitors with cells producing CCHFV particles (both in BSL-2, using a reverse genetics assay producing defective CCHFV particles, and BSL-4 settings) and will subsequently assess the level of infectivity in Huh7.5 hepatoma cells as well as in human primary hepatocytes (PHH), in order to determine the median inhibitory concentrations (IC50) of the different molecules. This repurposing strategy will be completed within the first year of my PhD and will allow for an efficient screening and prompt identification of needed therapeutic interventions, in line with the rapid emergence of CCHFV. In WP2, the primary aim will be to elucidate the intersection of lipoprotein metabolism, in specific the synthesis of very-low density lipoproteins (VLDLs), with assembly and secretion of CCHFV particles, processes that still remain ill-defined both in terms of location inside the cell and host factors interplay. By analogy with studies on other Bunyaviricetes members, CCHFV assembly has been proposed to occur on Golgi membranes. Yet, recent evidence of my host laboratory indicates the involvement of PACS-2 host connector protein in the assembly process of CCHFV virions, thus pointing to alternative locations linked to the role of PACS-2 in retrograde Golgi-ER trafficking as well as in the regulation of ER-derived mitochondrial associated membranes (MAMs). Exploiting confocal and super-resolution microscopy, and subcellular fractionation assays, I will assess the co-localization of viral elements and proteins involved in VLDLs synthesis on Golgi, Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC), mitochondria and MAMs. In addition, exploiting cell lines with targeted gene silencing and co-immunoprecipitation methods, I will dissect the role and mode of action of host proteins involved in the lipoprotein pathway in the assembly, maturation and secretion of CCHFV particles. WP2 will be carried out over 24-months starting from the second half of the first year of my PhD. Finally, WP3 will be initiated in parallel with WP2 and is expected to be fulfilled at the end of the third year of my PhD. It will assess the conservation of the mechanisms elucidated in the previous WPs across different orthonairoviruses. In addition, I will identify viral determinants involved in the intersection with lipoprotein metabolism through cutting edge biocomputational methods. The findings obtained will advance the knowledge in the rapidly evolving field of emerging pathogens, allowing to anticipate and not only react to these novel threaths. The multidisciplinarity nature of this PhD project will reinforce the capabilities of both the candidate and host laboratory, facilitating collaborative efforts to lay the foundation for the development of novel therapeutics and possible preventive measures."