Key mutations that govern the Omicron S-mediated low fusogenicity and endosomal entry

A new study by American researchers from Ohio State University-USA and University of Texas Medical Branch-USA has found that newer Omicron variants or sub-lineages possessing the H655Y mutation tend to display lower fusogenicity with possible implications for lower risk of disease severity initially upon infection.

The study findings were published on a preprint printer and are currently being peer reviewed.
https://www.biorxiv.org/content/10.1101/2022.10.15.512322v1

Omicron exhibits exceptional immune evasion (Garcia-Beltran et al., 2022; Kuhlmann et al., 2022; Planas et al., 2022; Pulliam et al., 2022; Viana et al., 2022) because of the presence of large numbers of mutations in the S protein. We and others have reported that Omicron also exhibits a distinct entry preference, substantially impaired furin cleavage, as well as decreased cell-cell fusion (Meng et al., 2022; Qu et al., 2022b; Zeng et al., 2021).

However, the molecular mechanisms of these distinct features of the Omicron subvariants remain elusive. In this study, we interrogated key mutations that govern the Omicron S-mediated low fusogenicity and endosomal entry. We determined H655Y, and to lesser extent T547K, as the key mutation on the S protein of Omicron that critically governs its preferential endosomal entry route and impaired fusion activity, including for the recently emerged BA.4/5, BA.2.12.1 and BA.2.75 subvariants. Moreover, through molecular modelling, we provide evidence that these mutations likely stabilize the S trimer conformation by forming new molecular interactions.

Together, these results provide insights for understanding the altered cellular tropism and pathogenicity for Omicron.

The most important finding of this study is that the altered entry route preference of Omicron is largely determined by the key H655Y mutations. It is well established that SARS-CoV-2 is capable of utilizing either endosomal entry mediated by Cat L/B or plasma membrane entry mediated by TMPRSS2 (Bestle et al., 2020; Hoffmann et al., 2020; Peacock et al., 2021). However, SARS-CoV-2 entry in primary lung epithelial cells and lung-derived cell lines such as Calu-3 cell is largely TMPRSS2 dependent (Peacock et al., 2021), likely occurring on the plasma membrane.

Following its emergence, the Omicron variant BA.1 has been shown to have a distinct entry profile, utilizing predominantly the endosomal entry pathway (Pia and Rowland-Jones, 2022), exhibiting poor replication in lower airway derived primary cells and Calu-3 cells (Gupta, 2022), as well as displaying reduced disease severity (Halfmann et al., 2022).

We found that the H655Y mutation governs BA.1.1 entry through endosomes, as suggested by the significant increase in viral infectivity observed in HEK293T-ACE2 cells but substantial reduction in viral infectivity in Calu-3 cells, and by the increased sensitivity of H655Y bearing variants to E64d, yet with decreased sensitivity to Camostat, which are also supported by other recent studies (Hu et al., 2022; Yamamoto et al., 2022).

However, the impact of H655Y on in vivo virus tropism and pathogenicity remains to be investigated. If the altered entry route preference introduced by the H655Y mutation is responsible for the enhanced nasopharynx tropism and reduced pathogenicity of the Omicron subvariants, any reversion of the H655Y mutation in future variants would be of great concern, as such a variant may exhibit enhanced pathogenicity. Careful monitoring this reversion mutation in the pandemic is warranted.

Membrane fusion is critical for entry of all enveloped viruses. We found that reversion mutations K547T and Y655H significantly promoted BA.1.1 S-mediated cell-cell fusion whereas forward mutations T547K and H655Y slightly impaired the D614G S-mediated cell-cell fusion, indicating that these two residues critically determine the low fusogenicity of BA.1.1.

Quite unexpectedly, we found no evidence that T547K and H655Y affect S processing of BA.1.1 in cell lysates. Rather, reversion mutations K547T and Y655H strongly promote S1 shedding in the presence of sACE2, indicating that T547K and H655Y mutations, especially the latter, critically stabilize the BA.1.1 S conformation.

This result is correlated with our structural modelling that T547K appears to stabilize the close conformation of S protein, which is also supported by a recent study reporting an extra hydrogen bond between the tyrosine residue at position 655 in S1 and the threonine residue at position 696 in S2 of BA.1 (Yamamoto et al., 2022).

Further structural analyses, including comparisons between K547T/Y655H reversion mutants and the parental BA.1.1 or other Omicron subvariants by cryogenic electron microscopy (cryo-EM) or crystallography, are needed to further elucidate the role of T547K and/or H655Y in Omicron subvariant S conformation.

It is important to note that H655Y mutation has been found to be associated with SARS- CoV-2 infection in index cats and minks (BraunID et al., 2021; Escalera et al., 2022). In addition, the H655Y mutation appears to have arisen independently multiple times in human population, and is a lineage-defining mutation for the Gamma (P.1) SARS-CoV-2 variant in addition to the Omicron subvariants (Escalera et al., 2022).

Importantly, H655Y is present in all predominant Omicron sublineages, including BA.1.1, BA.1, BA.2, BA.2.12.1 and more recent BA.4/5 and BA.2.75, indicating that H655Y likely improves fitness and the ability to adapt to new hosts, including humans, cats, minks, and others.

This is supported by a recent report demonstrating an enhancement of virus infectivity in mice for H655Y-containing viruses (Zhu et al., 2022).

Our findings in this work, along with other recent reports, together suggest that the occurrence of mutations at position 655 in S protein of current and future SARS-CoV-2 variants needs to be closely monitored. Additionally, in vivo examinations of the impact of the H655Y mutation on virus tropism and pathogenicity are critical and need to be investigated, as any reversion of the H655Y mutation could generate new concern for the course of the COVID-19 pandemic as novel Omicron subvariants continue to emerge.