Structural investigations of coronavirus spike protein

PROJECT SUMMARY The spike (S) protein decorates the surface of coronavirus (CoV) particles such as SARS-CoV-2 and enables CoV's to enter and infect host cells. The S protein is the immunogen in genetic and subunit vaccines against COVID-19, which were critical in controlling the COVID-19 global pandemic. In cells that are infected with CoV's or have received a genetic vaccine, the S protein is synthesized in the mammalian cell endoplasmic reticulum (ER), and then trafficking via the secretory pathway consisting of ER, Golgi network, and plasma membrane (PM). This secretory trafficking of S is bidirectional between ER and Golgi, which are the secretory organelles that provide the enzymatic machinery for post-translational modifications, remodeling, and maturation. This includes enzymes for N-glycosylation, which modulates S folding, viral entry, and interactions with the host immune system. Structure-function investigations have shown that N-glycans modulate conformations of S domains, such as the immunogenic receptor binding domain (RBD). Therefore, S N-glycans play a direct role in immune response modulation. As such, N-glycan maturation changes in S due to tissue-, population-, ethnicity- , and age-specific differences in biosynthetic machinery have the potential to dramatically alter infection CoV outcomes and genetic vaccine immunogenicity. Yet little is known about the atomic-level consequences of N- glycan maturation on S structure-function. This is mainly due to the inability to arrest S in various stages of trafficking and N-glycan maturation. In this grant application, we will use an innovative new methodology to control S trafficking. Purified samples of these novel S constructs will be structurally characterized using latest cryoEM and computational tools and assayed for interactions with highly potent conformation-sensitive antibodies. This will generate unprecedented and the first insights into structural modulation of S conformations and epitopes by N-glycan maturation. Collectively, these investigations will be highly significant in providing fundamental insights into secretory trafficking and the role of bidirectional trafficking of S in modulating N-glycan maturation and RBD conformations. These data will open avenues for the design of a new generation of S-based vaccines with improved immunogenicity. In the future, the insights from this research will serve as a platform to inform the design of vaccines against enveloped viruses that pose global health challenges such as HIV and influenza, which utilize the host cell ER-Golgi-PM secretory pathway to acquire immunity evading glycans.