Functional Dissection of the Free Fatty Acid-Binding Pocket in the SARS-CoV-2 Spike Protein

Structural investigations on non-enveloped picornaviruses first identified a hydrophobic pocket in the virus particle serving as the binding site for host-derived lipids known as a 'pocket factor'. The pocket was shown to play a pivotal role in virus entry and presented an opportunity for antiviral drug targeting (1-3). We were the first to identify a hydrophobic pocket in the SARS-CoV-2 spike protein, conclusively establishing its interaction with linoleic acid (LA), a fatty acid humans cannot synthetise, as the pocket factor (4). Intriguingly, we could demonstrate that the pocket and LA-binding are strictly conserved in SARS-CoV, MERS-CoV, SARS-CoV-2 and all Variants of Concern (5), indicating a pivotal but yet elusive role of the pocket that is strictly maintained over 20+ years. Recent studies on other enveloped viruses, such as flaviviruses (6,7), alphaviruses (8), and influenza virus (9), unveiled more pocket factors, suggesting shared mechanisms whereby structural rearrangements in the virus particle, essential for infection, are regulated. However, the underlying functional mechanisms remain poorly understood. The combined use of state-of-the-art structural biology, protein engineering, biophysics, computational modelling and virological approaches have placed us at the forefront of the field in defining how the binding of a pocket factor to an enveloped virus can affect spike protein stability, structural rearrangements, virus entry and replication (4,5,10-14).

The pocket in SARS-CoV-2 spike we discovered binds specifically LA, with nanomolar affinity (4). We showed that LA-binding induces a locked spike conformation incompatible with binding to human host cell receptor ACE2, thus inhibiting viral infection. Further, LA-treatment of human cells already infected with SARS-CoV-2 suppresses viral replication and results in deformed virions (5). Here, we aim to elucidate the functional importance of the pocket, and the molecular mechanisms how LA-binding (i) impacts the structural integrity and dynamics of spike and the virion (ii) alters viral infection, and (iii) regulates viral replication. To obtain these fundamental new insights, we will use computational, biophysical and structural approaches to design and characterize SARS-CoV-2 ancestral and variant spike protein mutants that no longer bind LA or any other fatty acid. We will compare wild-type and mutant spike proteins, dissecting the impact of LA-binding to the pocket on spike architecture, dynamics and ACE2-binding. By reverse genetics, we will prepare virus comprising the spike mutants identified. We will elucidate the effect of LA-treatment on viral infection, replication in cells and cell-to-cell spread with mutant virus we prepare, and analyse mutant virion morphology and spike conformation in situ using state-of-the-art imaging approaches.

Our proposal aims at fundamental new understanding to advance the frontiers of bioscience discovery, in line with the BBSRC long-term research and innovation priority 'Understanding the Rules of Life'. Leveraging our interdisciplinary research strategy and utilizing the SARS-CoV-2 interaction with LA as a model, we aim to address essential gaps in understanding the role pocket factors play in the viral lifecycle and explore the evolutionary advantages specific viruses may gain from these interactions, towards a paradigm for pocket factor function.