Functional analysis of molecular interactions between West Nile virus and its human and avian hosts

"West Nile Virus (WNV) is an Orthoflavivirus that is responsible for neurological diseases and death in humans. The virus is transmitted through the bite of infected mosquitoes. It circulates between birds, which serve as amplifying hosts, and humans and other mammals may accidentally become infected. Due mainly to climate changes, its geographical range is expanding into new areas, including Europe. The aim of our project is to provide new insights into the molecular mechanisms regulating WNV infection in human and bird cells, in hope to understand better the interactions between the virus and 2 of his hosts, and thus accelerate discovery of novel compounds for disease treatment. We propose to first characterize the interactions between the 2 WNV enzymes, which are key viral proteins and represent thus virus Achilles heel, and cellular proteins using affinity purification-mass spectrometry strategies. The 2 viral proteins (NS2B3 and NS5) will be expressed in human and avian cells. We will compare the list of human and bird cellular partners and select conserved ones. We will pick ~15 conserved candidates with high confident score per viral protein for further investigations. We will then perform functional screens to determine whether reduced expression of these candidates affect viral replication in human and bird cells. Viral replication will be estimated using a panel of virological assays. We will then pick the 2-3 candidates that are required for WNV replication to perform in-depth mechanistic studies. We will use a combination of microscopy, cell biology, biochemistry, proteomic and molecular virology assays to determine by which mechanisms the candidate proteins modulate the replication of WNV in several cellular models, including primary human cells. Finally, we will use Alphafold to model the interaction between WNV proteins and their selected cellular partners. We will use these models, as well as computational studies and analysis of open-source chemical databases, to identify molecules that potentially disrupt these viral-dependency interactions in human cells. Our project will uncover the first complete set of molecular interactions between 2 key WNV enzymes and human and bird proteins. It will provide a better understanding of the molecular interplays between these viruses and two of his hosts. It will enable the characterization of key host pathways that serve as critical proviral factors that are conserved. Finally, it will identify compounds that disrupt NS2B3 and NS5 interactomes and could led to the development of novel therapeutic intervention."