Defining the landscape of human genes regulating coronavirus replication in primary airway epithelia and elucidating their mechanisms of action

"Respiratory virus have the potential to spread rapidly and to lead to widespread epidemics, as demonstrated by the COVID-19 pandemic. Coronaviruses widely circulate in animals and possess the notorious ability to cross the species barrier. Over the past 25 years, three pathogenic coronaviruses have emerged in humans: SARS-CoV in 2003, MERS-CoV in 2012, and SARS-CoV-2 in 2019, the latter being the causative agent of COVID-19. Additionally, four human coronaviruses contribute to seasonal outbreaks each year. Like all viruses, coronaviruses are obligate intracellular parasites that rely on host cell resources for replication. In response, host cells have evolved various defence mechanisms, including some intrinsically-expressed or interferon-induced antiviral proteins. A key challenge in virology is to identify the human genes that positively or negatively regulate viral replication. Despite significant advances in high-throughput genetic screening tools such as CRISPR, our understanding of host-pathogen interactions remains relatively limited. Most genetic screens have indeed been conducted in cancer cell lines grown in 2D cultures, which do not adequately replicate the natural environment of human cells and may introduce biases due to their cancerous origin. Studies have shown that genes identified in these screens vary significantly depending on the cellular model used, underscoring the absolute necessity to perform similar studies in a more physiologically relevant context. The PhD thesis project aims to elucidate the landscape of human genes that regulate coronavirus replication using pertinent primary models, namely genetically modified human airway epithelia (HAE) cultured at the air-liquid interface (ALI). This 3D model includes the key epithelial cell types found in vivo (i.e. basal, ciliated, and secretory club and goblet cells) thereby mimicking the pseudostratified architecture and mucociliary functions of the respiratory tract. The host team has developed effective CRISPR methods for editing the progenitor stem cells of the human respiratory epithelium (the basal cells) and is able to generate genetically modified, differentiated and functional HAE-ALI. The PhD thesis project will be structured around three main objectives. Firstly, it will involve conducting rationalised CRISPR screens in an arrayed format within primary HAE-ALI cultures, assessing the regulatory roles of previously identified human genes believed to influence coronavirus replication from cancer cell line data. Secondly, the research will focus on analysing single-cell RNA sequencing (scRNA-seq) data to compile a list of genes specifically expressed in primary HAE-ALI cultures. The impact on coronavirus replication of selected candidate genes from this list will be performed as in the first objective. Lastly, the project aims to elucidate the molecular mechanisms of action a a couple of newly identified genes, through a combination of approaches, including structure-function analyses, interactomics, and microscopy. By leveraging advanced CRISPR technologies in physiologically relevant primary cell models, this doctoral project aspires to enhance our understanding of the interactions between coronaviruses and their human target cells. Such insights could pave the way for developing antiviral strategies that target key host factors. Consequently, this project aligns well with the objectives of this proposal call, which seeks to address the challenges posed by emerging infectious diseases and strengthen our capacity to respond effectively to future pandemics."