Loss-of-function mutation in Omicron variants reduces spike protein expression and attenuates SARS-CoV-2 infection.

SARS-CoV-2 Omicron variants emerged in 2022 with >30 novel mutations in the spike alone. While most studies focus on receptor binding domain changes, mutations in the C-terminus of S1 (CTS1), adjacent to the furin cleavage site, have largely been ignored. Here, we examine three Omicron mutations in CTS1: H655Y, N679K, and P681H. Generating a SARS-CoV-2 triple mutant (YKH), we find that the mutant increases spike processing, consistent with prior reports for H655Y/P681H. In addition, the YKH mutant induces attenuated disease, but augments viral loads in male golden Syrian hamsters. Next, we generate a single N679K mutant, finding it reduces viral replication in Calu3 human respiratory cells and induces less disease in male golden Syrian hamsters. Mechanistically, the N679K mutant has increased spike processing but also reduces spike in purified virions; spike decreases are further exacerbated in infected Calu3 cell lysates. Importantly, exogenous spike expression reveals that N679K reduces overall spike protein in the context of the epidemic strain. Although a loss-of-function mutation, transmission competition demonstrates that N679K confers a replication advantage in the upper airway, potentially impacting transmissibility. Together, the data show that N679K reduces overall spike protein during Omicron infection, which has implications for infection, immunity, and transmission.