SARS-CoV-2 Spike N-Terminal Domain modulates TMPRSS2-dependent viral entry and fusogenicity

Abstract

Over 20 mutations have been identified in the N-Terminal Domain (NTD) of SARS-CoV-2 spike and yet few of them are fully characterised. Here we first examined the contribution of the NTD to infection and cell-cell fusion by constructing different VOC-based chimeric spikes bearing B.1617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus (PV). We found the Delta NTD on a Kappa or WT background increased spike S1/S2 cleavage efficiency and virus entry, specifically in Calu-3 lung cells and airway organoids, through use of TMPRSS2. Delta was previously shown to have fast cell-cell fusion kinetics and increased fusogenicity that could be conferred to WT and Kappa variant spikes by transfer of the Delta NTD. Moving to contemporary variants, we found that BA.2 had higher entry efficiency in a range of cell types as compared to BA.1. BA.2 showed higher fusogenic activity than BA.1, but the BA.2 NTD could not confer higher fusion to BA.1 spike. There was low efficiency of TMPRSS2 usage by both BA.1 and BA.2, and chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD did not result in more efficient use of TMRPSS2 or cell-cell fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions such as entry and cell-cell fusion in a spike context dependent manner, and allosteric interactions may be lost when combining regions from more distantly related spike proteins. These data may explain the lack of dominant SARS-CoV-2 inter-variant recombinants bearing breakpoints within spike.

Evolutionary remodelling of N‐terminal domain loops fine‐tunes SARS‐CoV‐2 spike

“…Secondly, the NTD influenced spike S1/S2 preprocessing with Wu‐Hu‐1 + BA.1 NTD exhibiting enhanced proteolysis and vice versa (compare intensities/quantification of S/S2 bands in Figs 2N and EV1C ). This observation is broadly consistent with recent reports suggesting allosteric linkage between the NTD and distant protease target sites in spike (Meng et al , 2022 ; Qing et al , 2022 ).…”

Therapeutic and vaccine-induced cross-reactive antibodies with effector function against emerging Omicron variants

“…SARS-CoV-2 evolution is driven by viral immune escape and transmissibility 60,61 . The Omicron BA.1, BA.2 and BA.5 lineages, which led to successive waves of infection over the past year, were characterized by immune evasion from Wu S-elicited neutralizing antibodies, higher ACE2-binding affinity, impaired S-mediated fusogenicity and a change in entry pathway due to reduced TMPRSS2-dependence [3][4][5][6][7][8][9][10][11][12][13][14][15]39 . Here, we report that the recently emerged SARS-CoV-2 Omicron variants have an unprecedented level of immune evasion with reduction of neutralizing antibody titers reaching up to 100-fold for XBB.1.…”

“…We and others previously showed that BA.1, BA.2 and BA.5 had an altered cell entry pathway relative to previous SARS-CoV-2 strains, with Omicron variants entering preferentially through the endosomal entry route (cathepsin-mediated) as opposed to plasma membrane fusion (TMPRSS2-mediated) [6][7][8]19,39 . To assess the preferred cell entry route of the emerging Omicron variants, we investigated the impact of protease inhibitors on entry of non-replicative vesicular stomatitis virus (VSV) pseudotyped with S glycoproteins into VeroE6-TMPRSS2 cells (enabling both plasma membrane and endosomal entry routes) and HEK293T-ACE2 cells (enabling endosomal entry only).…”

Receptor binding domain (RBD) antibodies contribute more to SARS-CoV-2 neutralization when target cells express high levels of ACE2

“…Therefore, in the context of polyclonal serum, the relative neutralization contributions of antibodies targeting different epitopes shifts with target cell ACE2 expression. Why the neutralization potency of different antibodies differs based on ACE2 expression remains unclear, in part because the neutralization mechanisms of non-ACE2-competing antibodies are still incompletely understood [1,[20][21][22][23][24] . We also note that there is literature suggesting neutralization of other viruses by antibodies and drugs sometimes depends on target-cell receptor expression [25,26].…”