Research uncovers mechanisms governing SARS-CoV-2 Omicron subvariant entry

By Bhavana KunkalikarOct 20 2022Reviewed by Aimee Molineux

In a recent study posted to the bioRxiv* preprint server, researchers at Ohio State University and the University of Texas Medical Branch assessed the underlying mechanisms involved in the fusogenicity and endosomal entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants.

Serious concerns have been voiced regarding the global coronavirus disease 2019 (COVID-19) pandemic due to the rapid transmission and effective immune evasion displayed by the SARS-CoV-2 Omicron subvariants. Compared to the ancestor D614G variation, these novel variants show decreased fusogenicity and greater endosomal entry route use. However, the underlying processes of the altered manifestations are still unknown.

Study: Determinants and Mechanisms of the Low Fusogenicity and Endosomal Entry of Omicron Subvariants. Image Credit: Adao / Shutterstock

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

About the study

In the present study, researchers demonstrated that the SARS-CoV-2 Omicron BA.1.1 subvariant spike (S)-1 mutations at the C-terminal, namely H655Y and T547K, are essential regulators of Omicron's low fusogenicity.

The team proposed that S1's endosomal entry may be affected by mutations at the C-terminus or close to the furin cleavage site of the Omicron S protein. With the previously described human immunodeficiency virus (HIV) lentiviral pseudotyping technique, reversion mutations targeted to the Omicron subvariant BA.1.1's residues T547K, H655Y, N679K, and P681H were made and tested for their effects on BA.1.1's entry into HEK293T-angiotensin-converting enzyme-2 (ACE-2), HEK293T-ACE2-transmembrane serine protease 2 (TMPRSS2), and Calu-3 cells. In parallel, mutations such as H655Y, T547K, N679K, and P681H that arose in the ancestral D614G construct were also tested for entry into these cell types.

To ascertain the effect of these BA.1.1 mutants on viral entrance when the TMPRSS2 inhibitor Camostat or the endosomal Cat L/B inhibitor E64d is present, the team employed HEK293T-ACE2-TMPRSS2 cells, which permitted entry through both endosomal and plasma membrane routes.

The function of these two mutants, together with N679K, P681H, and parental BA.1.1, concerning S expression and S-mediated membrane fusion, was assessed. This allowed the researchers to understand the underlying process by which T547K and H655Y mutations affect BA.1.1 entry preference. The effects of the forward mutants were concurrently assessed. The surface expression of S in HEK293T cells utilized to develop pseudotyped lentiviruses was analyzed using flow cytometry.

Hence, the team predicted that the H655Y mutation also controlled other variants' entrance preferences and low fusogenicity. This prediction was examined using the HIV lentiviral pseudotyping technique to introduce the H655Y reversion mutation in the Omicron subvariants, namely, BA.1, BA.2, BA.2.12.1, BA.4/5, and BA.2.75. In addition, their effects on the entrance of these Omicron subvariants in HEK293T-ACE2 and Calu-3 cells were also assessed.

Results

The study findings showed that compared to SARS-CoV-2 Omicron BA.1.1 variant, the reversion mutation Y655H displayed a significant reduction in entry efficiency in HEK293T-ACE2-TMPRSS2 and HEK93T-ACE2 cells. However, the team noted improved entry of BA.1.1 in Calu-3 cells. This finding suggested that H655Y is the most important change in BA.1.1 S that differentiated its entry in different cell types. The forward mutation H655Y showed decreased entry efficiency in Calu-3 cells, while there was an increase in entry efficiency in 293T-ACE2 and 293T ACE2-TMPRSS2 cells. Similar outcomes were also reported for the BA.1.1 K547T mutation; however, the effect on entry was not as significant as that for the H655Y mutant.

BA.1.1-Y655H showed less sensitivity to E64d than BA.1.1. Compared to D614G, BA.1.1 showed higher sensitivity to treatment by E64d but significantly lesser sensitivity to treatment by Camostat. Furthermore, BA.1.1-Y655H demonstrated higher Camostat sensitivity than BA.1.1. Furthermore, the forward mutant D614G-H655Y showed less sensitivity to Camostat and more sensitivity to E64d.

With the exception of the reversion mutant K547T, which exhibited slightly reduced surface expression, all of these reversion mutants were roughly comparable in terms of expression to each other and the original BA.1.1. On the other hand, all of the forward mutants were comparable in terms of expression to D614G. The team observed that K679N and H681P had no impact, whereas K547T and Y655H significantly increased the S-mediated cell-cell fusion. Surprisingly, two other mutations, N679K and P681H, increased D614G S-mediated fusion, similar to some prior studies, while forward mutations H655Y and T547K marginally decreased the D614G S-induced syncytia.

The team found that all of the entry efficiency displayed by the Omicron subvariants in HEK293T-ACE2 cells was significantly decreased by the reversion mutation Y655H. However, their entry in Calu-3 cells was significantly promoted, even though the upsurge in Calu-3 cells was typically modest or not present in some cases. The team also discovered that, like BA.1.1, Y655H greatly aided the development of S-mediated syncytia in BA.1, BA.2, BA.2.12.1, BA.4/5, and BA.2.75.

Overall, the study findings indicated that it is vital to closely monitor the mutations occurring at position 655 in the SARS-CoV-2 spike protein of existing and future variants. The H655Y mutation's effects on virus tropism and pathogenicity must also be examined in-vivo since any reversion of the mutation could raise novel questions about the COVID-19 pandemic's trajectory as new Omicron subvariants continue to evolve.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources