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The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urges better understanding of the functional motifs in the spike (S) protein and their tolerance to mutations. Here, we focused on the S2' motif, which, during virus entry, requires cleavage by a host cell protease to release the fusion peptide. Though belonging to an immunogenic region, the SARS-CoV-2 S2' motif (811-KPSKR-815) has shown hardly any variation, with its three basic (K/R) residues being >99.99% conserved thus far. By creating a series of mutant pseudoviruses bearing the spikes of Wuhan-Hu-1, its G614 mutant or the Delta and Omicron variants, we show that residue K 814 (preceding the scissile R 815 ) is dispensable for TMPRSS2 yet favored by the alternative TMPRSS13 protease. Activation by TMPRSS13 was drastically reduced when the SARS-CoV-2 S2' motif was swapped with that of the low pathogenic 229E coronavirus (685-RVAGR-689), and also, the reverse effect was seen. This swap had no impact on recognition by TMPRSS2. In the Middle East respiratory syndrome coronavirus (MERS-CoV) spike, introducing a dibasic scissile motif was easily accepted by TMPRSS13 but less so by TMPRSS2, confirming that TMPRSS13 favors a sequence rich in K/R residues. Pseudovirus entry experiments in Calu-3 cells confirmed that the S2' mutations have minor impact on TMPRSS2. Our findings are the first to demonstrate which S2' residues are important for SARS-CoV-2 spike activation by these two airway proteases, with TMPRSS2 being more tolerant to variation than TMPRSS13. This preemptive insight will help to estimate the impact of S2' motif changes as they appear in new SARS-CoV-2 variants. IMPORTANCE Since its introduction in humans, SARS-CoV-2 is evolving with frequent appearance of new variants. The surveillance would benefit from proactive characterization of the functional motifs in the spike (S) protein, the most variable viral factor. This is linked to immune evasion but also influences spike functioning. Remarkably, though located in a strongly immunogenic region, the S2' cleavage motif has, thus far, remained highly conserved. This suggests that its sequence is critical for spike activation by airway proteases. To investigate this, we assessed how pseudovirus entry is affected by changes in the S2' motif. We demonstrate that TMPRSS2 readily accepts variations in this motif, whereas the alternative TMPRSS13 protease is more fastidious. The Wuhan-Hu-1, G614, Delta and Omicron spikes showed no difference in this regard. Being the first in its kind, our study will help to assess the impact of S2' variations as soon as they are detected during variant surveillance.

Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population. IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses., (Copyright © 2019 American Society for Microbiology.)