Abstract 2416 HCoV-229E infected lung cells reveal key molecular targets using structural proteomic analysis

The emergence of novel viruses from zoonotic reservoirs are on the rise which can lead to outbreaks in humans that severely impact societal functions and the global economy. Indeed, the recent SARS-CoV 2 pandemic exposed vulnerabilities and breakdowns in our response time and approaches to rapidly temper these major threats. Thus, there is a dire need for identification of druggable targets or pathways that limit viral replication, transmission, and/or disease severity. Current systems biology approaches utilize sophisticated mass spectrometer instrumentation that can capture global changes in the abundance of cellular proteins, metabolites, and lipids due to an environmental perturbation such as viral infection. Useful markers that reflect changes in important functional pathways are often missed because they manifest as changes in protein structure in the absence of alterations to protein abundance. These conformational changes are critical for facilitating lock-and-key type interactions with other proteins, lipids and/or metabolites important in, for instance, host-response to infection. To more comprehensively profile host-response to infection at the molecular level, we deployed a novel mass spectrometry-based proteomics assay called limited proteolysis (LiP) which, in addition to protein abundance, identifies proteins undergoing conformational change and the region on proteins that are altered due to infection. Using human coronavirus 229E (HCoV-229E), we infected immortalized human lung epithelial and fibroblast cells for comparison to "mock" infected controls. Nuclear extracts were isolated, and proteins were exposed to a non-specific protease to "nibble" the solvent accessible regions of the proteins. The reactions were rapidly quenched and semi-tryptic peptides were analyzed and quantified by mass spectrometry to create protein "barcodes" revealing conformational changes in proteins due to infection. We identified over 200 different proteins undergoing significant structural changes due to infection by HCoV-229E. Moreover, several of the conformational changes overlapped with documented RNA binding sites on the proteins that could be hijacked by the virus for replication. Pathway enrichment analysis identified multiple macromolecular assemblies to be central in the host-virus interaction including the Drosha complex and NOP56p. These, and other assemblies represent key therapeutic targets for the design of inhibitors or drugs that can prevent viral replication and spread. Our universal workflow shows the power of high-throughput structural proteomics approaches that can be used to identify host-based interventions for pathogenic threats that are likely to emerge in the future.