Van Berwaer, R. | Search bioRxiv

bioRxiv2022

Effect of SARS-CoV-2 spike mutations on its activation by TMPRSS2 and TMPRSS13

The continuous emergence of new SARS-CoV-2 variants urges better understanding of the functional motifs in the spike (S) protein and their tolerance towards mutations. We here focus on the S2 motif which, during virus entry, requires cleavage by a cell surface 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 S-pseudotyped viruses, we show that K814, which precedes the scissile R815 residue, is dispensable for SARS-CoV-2 spike activation by TMPRSS2 but not TMPRSS13. The latter protease lost its activity towards SARS-CoV-2 S when the 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 TMPRSS2 activation. Also in the MERS-CoV spike, introducing a dibasic scissile motif was fully accepted by TMPRSS13 but less so by 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 TMPRSS13 exhibiting higher preference for K/R rich motifs than TMPRSS2. This preemptive insight can help to estimate the impact of S2 motif changes as they may appear in new SARS-CoV-2 variants. IMPORTANCESince the start of the COVID-19 pandemic, SARS-CoV-2 is undergoing worldwide selection with frequent appearance of new variants. The surveillance would benefit from proactive characterization of the functional motifs in the spike protein, the most variable viral factor. This is linked to immune evasion but also influences spike functioning in a direct manner. Remarkably, though located in a strong immunogenic region, the S2 cleavage motif has, thus far, remained highly conserved. This suggests that its amino acid sequence is critical for spike activation by airway proteases. To investigate this, we assessed which S2 site mutations affect processing by TMPRSS2 and TMPRSS13, two main activators of the SARS-CoV-2 spike. Being the first in its kind, our study will help to assess the biological impact of S2 site variations as soon as they are detected during variant surveillance.

microbiology↗

bioRxiv2020

The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV-1 and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. First, the SARS-CoV-2 spike (SARS-2-S) reached higher levels in pseudoparticles when produced at 33{degrees}C instead of 37{degrees}C. Even stronger preference for the upper airway temperature of 33{degrees}C was evident for the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV-1 and MERS-CoV favored 37{degrees}C, in accordance with their preference for the lower airways. Next, SARS-2-S proved efficiently activated by TMPRSS13, besides the previously identified host cell protease TMPRSS2, which may broaden the cell tropism of SARS-CoV-2. TMPRSS13 was found to be an effective spike activator for the virulent coronaviruses but not the common cold HCoV-229E virus. Activation by these proteases requires pre-cleavage of the SARS-2-S S1/S2 cleavage loop, and both its furin motif and extended loop length proved critical to achieve virus entry into airway epithelial cells. Finally, we show that the D614G mutation in SARS-2-S increases S protein stability and expression at 37{degrees}C, and promotes virus entry via cathepsin B/L activation. These spike properties might promote virus spread, potentially explaining why the G614 variant is currently predominating worldwide. Collectively, our findings indicate how the coronavirus spike protein is fine-tuned towards the temperature and protease conditions of the airways, to enhance virus transmission and pathology. SIGNIFICANCE STATEMENTThe rapid spread of SARS-CoV-2, the cause of COVID-19, is related to abundant replication in the upper airways, which is not observed for other highly pathogenic human coronaviruses. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. Coronavirus spikes exhibit distinct temperature preference to precisely match the upper (~33{degrees}C) or lower (37{degrees}C) airways. We identified airway proteases that activate the spike for virus entry into cells, including one protease that may mediate coronavirus virulence. Also, a link was seen between spike stability and entry via endosomal proteases. This mechanism of spike fine-tuning could explain why the SARS-CoV-2 spike-D614G mutant is more transmissible and therefore globally predominant.

microbiology↗