SARS-CoV-2 N-terminal domain modulates S1/S2 cleavage and spike-mediated functions

In a recent study published on bioRxiv* preprint server, researchers explored the role of the N-terminal domain (NTD) of the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) spike protein (S) in the fusion of SARS-CoV- 2 with host membranes and coronavirus disease 2019 (COVID-19).

Study: N-terminal domain of SARS-CoV-2 spike modulates TMPRSS2-dependent viral entry and fusogenicity. Image Credit: Dotted Yeti / Shutterstock

SARS-CoV-2 binds to host angiotensin-converting enzyme 2 (ACE2) to initiate viral entry based on expression of transmembrane serine protease 2 (TMPRSS2) and cleavage S. In the NTD of the SARS-CoV-2 spike, more than 20 mutations have been identified, but very few have been characterized. Therefore, the impact of mutations on viral infectivity and vaccine immunogenicity is unclear.

The authors of the present study had previously shown that Delta S and Kappa S demonstrated high S1/S2 cleavage efficiency compared to strain 614G WT (wild-type) and the infectivity of the correlated SARS-CoV-2 strain to the S-cleavage efficiency.

About the study

In this new study, researchers assessed the importance of NTD in SARS-CoV-2 S cleavage and fusion of the virus to host cells leading to subsequent SARS-CoV-2 infections.

The team constructed a panel of chimeric S including Kappa NTD or Delta NTD with their corresponding parental strain origins. Subsequently, pseudoviruses (PV) expressing S of several variants of concern (VOC) were used to transduce TMPRSS2-expressing 293T cells and parental 293T cells to assess the invasion efficiency of SARS-CoV- 2. In addition, the difference in fusogenicity of the Delta and Kappa strains was evaluated.

In further analysis, WT S carrying the Delta NTD or Kappa NTD was expressed. Additionally, Delta NTD was introduced into the Omicron BA.1 and BA.2 sublines to determine if Delta NTD could increase the fusogenicity of Omicron. In addition, the impact of BA.2 NTD on the fusogenicity of BA.1 and the reverse were evaluated.

The contribution of NTD in S cleavage was assessed in purified PVs by Western blot analysis. Additionally, the dependency of SARS-CoV-2 on TMPRSS2 for host invasion was assessed by pretreating A549-ACE2/TMPRSS2 cells or airway organoids with E64D (a cathepsin inhibitor) or camostat (a TMPRSS2 inhibitor). Impairment of ACE2 accessibility in chimeric S was assessed by transducing parental 293T cells with abolished expression of TMPRSS2 and overexpression of ACE2.

The contribution of Delta NTD mutations to increased viral infectivity was assessed in cultured human airway epithelial cells (Calu3) treated with S-expressing PVs of different COVs and amino acid cluster reversion (156G , 142D, del158R and del157F) to the WT Wuhan -1 strain.

To assess the effect of reversion of mutations on vaccine susceptibility, PVs were used to transduce HeLa-ACE2 cells with serial dilution of serum samples obtained from individuals doubly vaccinated with the BNT162b2 vaccine. . In addition, virus neutralization tests were performed.

SARS-CoV-2 Delta NTD increases Kappa and WT spike fusion kinetics.  (A): A schematic diagram showing the split GFP system for spike-ACE2-mediated cell fusion.  (B): 681R or 681H is required for increased fusogenicity in Delta and its chimera carrying Kappa NTD.  (C): Delta NTD fused in Kappa and WT increased the fusion kinetics of their counterparts, respectively.  The line graphs on the right show the percentage of the GFP positive area at 12, 14, 16, 20, 22 and 23 h after transfection.  Data showing the SEM at each point were averaged from two experiments.  The heat map at each time point shows the average of the GFP positive area over the field of view of two experiments.

SARS-CoV-2 Delta NTD increases Kappa and WT spike fusion kinetics. (A): A schematic diagram showing the split GFP system for spike-ACE2-mediated cell fusion. (B): 681R or 681H is required for increased fusogenicity in Delta and its chimera carrying Kappa NTD. (C): Delta NTD fused in Kappa and WT increased the fusion kinetics of their counterparts, respectively. The line graphs on the right show the percentage of the GFP positive area at 12, 14, 16, 20, 22 and 23 h after transfection. Data showing the SEM at each point were averaged from two experiments. The heat map at each time point shows the average of the GFP positive area over the field of view of two experiments.

Results

Consistent with the team’s previous findings on the association of increased S cleavage with increased fusogenicity and infectivity, enhanced TMPRSS2-mediated S1/S2 cleavage and increased infectivity were observed among Kappa and Delta chimeras in the airway organoids and Calu3 cells. Additionally, Kappa S was prone to SARS-CoV-2 S1 shedding with a higher S2/S1 ratio compared to the WT strain, while Delta S demonstrated increased stability. Additionally, Delta showed the highest efficiency of TMPRSS2-mediated SARS-CoV-2 entry and fusogenicity. Interestingly, an increased entry efficiency of chimeric S Kappa was observed, indicating that the Delta NTD enabled Kappa entry into the host using TMPRSS2.

In contrast, the Omicron sublines carrying the Delta NTD demonstrated no increase in fusogenicity, indicating that the Delta NTD could not increase the efficiency of using Omicron’s TMPRSS2. In the extended analysis, Delta NTD and Kappa NTD enhanced S cleavage in WT chimeras and thus increased WT infectivity in Calu3 cells.

In pretreated cells and airway organoids, the half-maximal inhibitory concentration (IC50) camostat value was twice as high for Delta compared to Kappa, while E46D showed negligible effects on SARS-CoV-2. Delta showed increased resistance to camostat and therefore demonstrated greater efficiency in using TMPRSS2 to initiate virus entry.

Upon overexpression of ACE2, a 30- and four-fold increase in Delta and Kappa efficiency, respectively, was observed. Of note, the chimeric S Kappa bearing Delta NTD demonstrated a similar degree of increased sensitivity to Delta. On the other hand, reduced dependence on ACE2 was observed in the Delta chimera carrying Kappa NTD. This indicated that Delta NTD modulated TMPRSS2-mediated S-cleavage and allosterically regulated RBD for increased ACE2 utilization efficiency

Each reverted mutant showed reduced infectivity in Calu3 cells, with the highest reduction (three-fold) seen after 157R/158R reinsertion. It should be noted that the reduction in infectivity was only observed in Calu3 cells and not in Hela-ACE2 cells, indicating the specificity of SARS-CoV-2 conferred by the NTD.

In neutralization assays, the presence of G156E and D142G and repair of the 157F/158R deletion doubled the susceptibility to neutralization of SARS-CoV-2, underpinning the importance of NTD for evasion of host immune responses and SARS-CoV-2 infectivity.

In fusion assays, upon fusion of Kappa NTD into Delta, the fusion phenotype shifted to Delta’s Kappa, with a delay in kinetics. On the other hand, a fast-melting Delta phenotype was observed upon the exchange of Delta NTD by Kappa. Additionally, a faster fusion phenotype was observed when fusing the Delta NTD into the WT backbone. This indicates that Delta NTD could improve the fusion kinetics of WT and Kappa strains.

Overall, the study results highlighted the variant-specific allosteric modulation of SARS-CoV-2 S cleavage and infectivity by NTD. The increased efficiency of S cleavage warranted a cognate NTD since S carrying only receptor binding domain (RBD) mutations could not be cleaved efficiently.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.

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