Viruses in the Sarbecovirus subgenus have given rise to two highly transmissible coronaviruses in recent human history: severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Both of these viruses enter human cells through the interaction of viral spike (S) glycoprotein and human angiotensin-converting enzyme 2 (hACE2). The SARS-CoV-2 virus that emerged in late 2019-early 2020 led to far more extensive spread than the 2002-2003 outbreak caused by SARS-CoV. It is of fundamental interest to understand the molecular basis for its increased transmissibility as well as the effect of mutations identified in variants of SARS-CoV-2. One hypothesis that we are testing is whether differences in structural and conformational dynamics in SARS-CoV and SARS-CoV-2 S trimers influence their ability to bind and be activated by the ACE2 receptor. To probe the structural and dynamic differences amongst SARS-CoV-1, SARS-CoV-2 and a D614G variant that increased SARS-CoV-2 transmissibility, we performed hydrogen deuterium exchange mass spectrometry (HDX-MS) on prefusion spike proteins in both apo and hACE2 bound states. In unliganded S trimers, bimodal mass spectral envelopes were detected for several peptides throughout the trimeric spike, indicative of conformational sampling in trimers. The extent of these bimodal spectra were strain-specific. Notable differences in the apparent stability of the S2, fusion subunits, were also observed between SARS-CoV, SARS-CoV-2 and the D versus G614 variants. ACE2 binding led to more prominent bimodal deuterium uptake behavior reflecting ACE2-induced trimer activation. Lastly, we observed that in response to hACE2 binding, SARS-CoV and SARS-CoV-2 spikes exhibited different allosteric responses at sites distal to the receptor binding domain (RBD) at the trimer apex. These results provide insights relevant to understanding the impact of functional mutations on spike fusion protein activation and their impact on transmission phenotypes.
2214-Pos