Understand the Mechanism of SARS-CoV-2 Entry by Single-Molecule Approaches

This research project aims to unravel the complex dynamics of viral entry and immune responses, focusing on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. Viruses constantly mutate to enhance adaptability and transmissibility, leading to the emergence of variants of concern (VOCs). Adaptations of SARS-CoV-2 often increased with higher affinity to host factors, but given the highly dynamic environment in respiratory tract, the binding of spike (S) protein to angiotensin-converting enzyme 2 (ACE2) is constantly challenged by mechanical forces. Therefore, understanding the binding dynamics and stability of virus attachment onto host cell surface under external force is crucial. Also, current investigations have left critical questions about the detachment of S1/S2 subunits, structural changes in S2 and their dynamics to activate the membrane fusion unanswered. In addition, the interaction of neutralizing antibodies (nAbs) with S proteins under force and simultaneous identification of binding sites are crucial for a quantitative understanding of differential immune responses. To address these crucial questions, I propose to study the biomolecular processes associated with the virus entry and immune responses at single molecule level using the novel DNA nanoswitch calipers (DNC) technique in fluorescence imaging integrated optical tweezers, and high-throughput magnetic tweezers. We showed in proof-of-concept examples that the DNC technique can be used to map the distances in peptides and protein complexes for providing identification fingerprints and structural information at angstrom level precision. We also demonstrated high-throughput measurements of DNC in magnetic tweezers to analyze the heterogeneous mixture of peptides. Therefore, DNC is a powerful technique to study multicomponent protein-protein interactions and simultaneously track the structural changes associated with the process in real-time at single-molecule level. First, I will measure binding dynamics of full-length S protein and monomer/dimer ACE2 receptor proteins under force to understand the entire energy landscape governing the interaction of these proteins. Second, I will measure the detachment kinetics of S1/S2 subunits and determine the conformational dynamics of the S2 subunit. Third, I will utilize a similar approach to measure the binding dynamics of nAbs with S protein and spatial mapping of binding sites in S protein. By high-throughput measurements in magnetic tweezers, I will characterize the heterogeneity of nAbs to understand the differential immune responses of the individuals. Hence, the proposed work will provide detailed insight into understanding the mechanism of SARS-CoV-2 entry and quantitative characterization of immune response of the individuals. My quantitative single-molecule approaches will bring new insights into the field of virology and immunology. This study will also provide me a firsthand experience in biochemistry, molecular biology, virology, and immunology, enabling me to better apply my quantitative and physical training to more topics in biological research in the future.