A conserved tyrosine in the S1/S2 loop shapes SARS-CoV-2 spike processing and host protease usage.

Both spike processing efficiency and host protease usage play critical roles in SARS-CoV-2 infection, pathogenesis, and transmission. The SARS-CoV-2 spike contains two motifs, a furin cleavage site (FCS) and an extended S1/S2 loop, which improve both cleavage efficiency and host protease usage. Disruption of these motifs significantly attenuates SARS-CoV-2 infection and pathogenesis. Importantly, modifications and changes within the S1/S2 loop can also shape infection and disease outcomes following infection. In this study, we examined mass spectrometry data generated from purified virions from wild-type (WT) or mutant SARS-CoV-2 infection. We identified phosphorylation occurring at highly conserved residue Y674 and used reverse genetics to generate a mutant that ablates phosphorylation capacity in the S1/ S2 loop (Y674F). Our results show that the Y674F mutant replicated equivalently to wild-type SARS-CoV-2 in Vero cells but had attenuated replication in a human respiratory cell line. Golden Syrian hamsters infected with the Y674F mutant had equivalent weight loss, lung viral titer, and similar immune pathology to WT SARS-CoV-2. However, the mutant had reduced viral load in the nasal wash and reduced antigen staining in the large airways in the lung. Notably, loss of Y674 ablated spike processing to the S1/S2 cleavage product on virions. In addition, Y674F mutant had a diminished capacity to utilize host protease TMPRSS2 for entry. Together, these data indicates that post-translational modification of Y674 impacts SARS-CoV-2 infection of the airway by disrupting both spike processing and host protease function. These results add to data demonstrating the importance of spike S1/S2 loop modifications to SARS-CoV-2 infection, pathogenesis, and transmission.