SARS-CoV-2 Omicron Specific Mutations Affecting Infectivity, Fusogenicity, and Partial TMPRSS2-Independency

Abstract

The COVID-19 pandemic resulted from the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since its first appearance in 2019, new SARS-CoV-2 variants of concern (VOCs) have emerged frequently, changing the infection’s dynamic. SARS-CoV-2 infects cells via two distinct entry routes; receptor-mediated endocytosis or membrane fusion, depending on the absence or presence of transmembrane serine protease 2 (TMPRSS2), respectively. In laboratory conditions, the Omicron SARS-CoV-2 strain inefficiently infects cells predominantly via endocytosis and is phenotypically characterized by decreased syncytia formation compared to the earlier Delta variant. Thus, it is important to characterize Omicron’s unique mutations and their phenotypic manifestations. Here, by utilizing SARS-CoV-2 pseudovirions, we report that the specific Omicron Spike F375 residue decreases infectivity, and its conversion to the Delta S375 sequence significantly increases Omicron infectivity. Further, we identified that residue Y655 decreases Omicron’s TMPRSS2 dependency and entry via membrane fusion. The Y655H, K764N, K856N and K969N Omicron revertant mutations, bearing the Delta variant sequence, increased the cytopathic effect of cell–cell fusion, suggesting these Omicron-specific residues reduced the severity of SARS-CoV-2. This study of the correlation of the mutational profile with the phenotypic outcome should sensitize our alertness towards emerging VOCs.

The Transmembrane Protease Serine 2 (TMPRSS2) Non-Protease Domains Regulating Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike-Mediated Virus Entry

“…Omicron exhibits reduced TMPRSS2-dependency, enabling infection of TMPRSS2-positive cells through both entry pathways, facilitated by the presence of Omicron-specific Spike Y655 residue ( Figure 5 a). However, the precise underlining mechanism remains elusive [ 13 , 24 , 40 ]. To investigate whether the diminished Omicrons reduced TMPRSS2-dependency might stem from a decreased binding between Omicron-Spike and TMPRSS2 due to the Y655 residue, HEK293T were transfected with Omicron-Spike wt and Y655H mutant, along with a reciprocal experiment involving Delta-Spike wt and H655Y.…”

“…A mediator of TMPRSS2 interaction with SARS-CoV-2 Spike protein is the H655 residue. The observation that Omicron infection is only partially dependent on TMPRSS2 might be explained by the weakened TMPRSS2 interaction due to the Omicron Spike Y655 residue [ 13 , 24 , 53 ]. Considering the observed correlation between TMPRSS2 affinity for Spike and the infection pathway via the cell membrane, we hypothesize that the physical interaction between these two proteins reduces the utilization of the endosomal route for SARS-CoV-2 Spike-mediated entry ( Figure 6 ).…”

“…From Anthony Koleske, we received pDsRed-Abl2 plasmid, and we replaced the C-terminal RFP reporter with a stop-codon via the NotI and AgeI restriction sites. The procedures for cloning pBiFC-Jun-YFPn, pBiFC-Fos-YFPc, pCDNA-Δ81-Abl1, pCDNA3.1-Delta-Spike, and pCDNA3.1-Omicron-Spike, and respective aa 655 mutants were previously documented [ 22 , 23 , 24 ]. All used oligos can be found in Supplement Table S1 .…”

Impacts of the SARS-CoV-2 Spike Protein on the Innate Immune System: A Review

“…The Spike protein in Omicron strains may be less sensitive to the action of transmembrane serine proteases, notably TMPRSS2, TMPRSS11d, and TMPRSS13, due to mutations, and their fusogenic capacities therefore appear impaired [137][138][139]. Studies tend to show that Omicron variants prefer the endosomal entry route to that of the fusion at the plasma membrane [139,140], although the independence of their Spike protein from TTSPs may be questioned, particularly for BA.2.86 [141,142].…”

Pseudovirus-Based Systems for Screening Natural Antiviral Agents: A Comprehensive Review

“…It has been demonstrated that SARS-CoV-2 can use both Cathepsin B/L and TMPRSS2 for cell entry, particularly in human lung cells. Otherwise, the omicron variant of SARS-CoV-2 enters cells by endo/lysosomal cysteine protease Cathepsin L [44].…”