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Characterization of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein.

ABSTRACT: SARS-CoV-2 is of zoonotic origin and contains a PRRA polybasic cleavage motif which is considered critical for efficient infection and transmission in humans. We previously reported on a panel of attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction of the spike protein. Here, we characterize pathogenicity, immunogenicity, and protective ability of a further cell-adapted SARS-CoV-2 variant, Ca-DelMut, in in vitro and in vivo systems. Ca-DelMut replicates more efficiently than wild type or parental virus in Vero E6 cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut causes no obvious pathological changes and does not induce elevation of proinflammatory cytokines, but still triggers a strong neutralizing antibody and T cell response in hamsters and mice. Ca-DelMut immunized hamsters challenged with wild type SARS-CoV-2 are fully protected, with little sign of virus replication in the upper or lower respiratory tract, demonstrating sterilizing immunity.

SUBMITTER: Wang P

PROVIDER: S-EPMC8119425 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction.

Project description:The emergence of SARS-CoV-2 has led to the current global coronavirus pandemic and more than one million infections since December 2019. The exact origin of SARS-CoV-2 remains elusive, but the presence of a distinct motif in the S1/S2 junction region suggests the possible acquisition of cleavage site(s) in the spike protein that promoted cross-species transmission. Through plaque purification of Vero-E6 cultured SARS-CoV-2, we found a series of variants which contain 15-30-bp deletions (Del-mut) or point mutations respectively at the S1/S2 junction. Examination of the original clinical specimen from which the isolate was derived, and 26 additional SARS-CoV-2 positive clinical specimens, failed to detect these variants. Infection of hamsters shows that one of the variants (Del-mut-1) which carries deletion of 10 amino acids (30bp) does not cause the body weight loss or more severe pathological changes in the lungs that is associated with wild type virus infection. We suggest that the unique cleavage motif promoting SARS-CoV-2 infection in humans may be under strong selective pressure, given that replication in permissive Vero-E6 cells leads to the loss of this adaptive function. It would be important to screen the prevalence of these variants in asymptomatic infected cases. The potential of the Del-mut variants as an attenuated vaccine or laboratory tool should be evaluated.

| S-EPMC7241555 | biostudies-literature

D614G Mutation Alters SARS-CoV-2 Spike Conformation and Enhances Protease Cleavage at the S1/S2 Junction.

Project description:The severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein is the target of vaccine design efforts to end the coronavirus disease 2019 (COVID-19) pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic and are now the dominant form worldwide. Here, we explore S conformational changes and the effects of the D614G mutation on a soluble S ectodomain construct. Cryoelectron microscopy (cryo-EM) structures reveal altered receptor binding domain (RBD) disposition; antigenicity and proteolysis experiments reveal structural changes and enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the up/down ratio of the RBDs in the G614 S ectodomain, demonstrating an allosteric effect on RBD positioning triggered by changes in the SD2 region, which harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 S conformational landscape and allostery and have implications for vaccine design.

| S-EPMC7762703 | biostudies-literature

Functional analysis of polymorphisms at the S1/S2 site of SARS-CoV-2 spike protein.

Project description:Several SARS-CoV-2 variants emerged that harbor mutations in the surface unit of the viral spike (S) protein that enhance infectivity and transmissibility. Here, we analyzed whether ten naturally-occurring mutations found within the extended loop harboring the S1/S2 cleavage site of the S protein, a determinant of SARS-CoV-2 cell tropism and pathogenicity, impact S protein processing and function. None of the mutations increased but several decreased S protein cleavage at the S1/S2 site, including S686G and P681H, the latter of which is found in variants of concern B.1.1.7 (Alpha variant) and B.1.1.529 (Omicron variant). None of the mutations reduced ACE2 binding and cell-cell fusion although several modulated the efficiency of host cell entry. The effects of mutation S686G on viral entry were cell-type dependent and could be linked to the availability of cathepsin L for S protein activation. These results show that polymorphisms at the S1/S2 site can modulate S protein processing and host cell entry.

| S-EPMC8956166 | biostudies-literature

SARS-CoV-2 spike protein S:D614G + S:A222V variant

Project description: Not available

| EMPIAR-10891 | biostudies-other

Humoral response to spike S1 and S2 and nucleocapsid proteins on microarray after SARS-CoV-2 infection.

Project description:Many aspects of the humoral immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as its role in protection after natural infection, are still unclear. We evaluated IgA and IgG response to spike subunits 1 and 2 (S1 and S2) and Nucleocapsid proteins of SARS-COV-2 in serum samples of 109 volunteers with viral RNA detected or seroconversion with different clinical evolution (asymptomatic, mild, moderate, and severe coronavirus disease 2019), using the ViraChip® Test Kit. We observed that the quantification of antibodies to all antigens had a positive correlation to disease severity, which was strongly associated with the presence of comorbidities. Seroreversion was not uncommon even during the short (median of 77 days) observation, occurring in 15% of mild-asymptomatic cases at a median of 55 days for IgG and 46 days for IgA. The time to reach the maximal antibody response did not differ significantly among recovered and deceased volunteers. Our study illustrated the dynamic of anti-S1, anti-N, and anti-S2 IgA and IgG antibodies, and suggests that high production of IgG and IgA does not guarantee protection to disease severity and that functional responses that have been studied by other groups, such as antibody avidity, need further attention.

| S-EPMC8661980 | biostudies-literature

Neuropilin-1 assists SARS-CoV-2 infection by stimulating the separation of Spike protein S1 and S2.

Project description:The cell surface receptor Neuropilin-1 (Nrp1) was recently identified as a host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry. The Spike protein of SARS-CoV-2 is cleaved into two segments, the S1 (residues (res.) 1-685) and the S2 (res. 686-1273) domains by furin protease. Nrp1 predominantly binds to the C-terminal RRAR amino acid motif (res. 682-685) of the S1 domain. In this study, we firstly modeled the association of an Nrp1 protein (consisting of domains a2-b1-b2) with the Spike protein. Next, we studied the separation of S2 from the S1 domain, with and without Nrp1 bound, by utilizing molecular dynamics pulling simulations. During the separation, Nrp1 stabilizes the S1 C-terminal region (res. 640-685) and thereby assists the detachment of S2 N-terminal region (res. 686-700). Without Nrp1 bound, S1 tends to become stretched, whereas the bound Nrp1 stimulates an earlier separation of S2 from the S1 domain. The liberated S2 domain is known to mediate the fusion of virus and host membranes; thus, Nrp1 likely increases virus infectivity by facilitating the S1 and S2 separation. We further analyzed the possible topological structure of the SARS-CoV-2 Spike protein when bound with Nrp1 and angiotensin-converting enzyme 2 (ACE2). Understanding of such an Nrp1-assisted viral infection opens the gate for the generation of protein-protein inhibitors, such as antibodies, which could attenuate the infection mechanism and protect certain cells in a future Nrp1-ACE2 targeted combination therapy.

| S-EPMC8169233 | biostudies-literature

Molecular modelling of S1 and S2 subunits of SARS coronavirus spike glycoprotein.

Project description:The S1 and S2 subunits of the spike glycoprotein of the coronavirus which is responsible for the severe acute respiratory syndrome (SARS) have been modelled, even though the corresponding amino acid sequences were not suitable for tertiary structure predictions with conventional homology and/or threading procedures. An indirect search for a protein structure to be used as a template for 3D modelling has been performed on the basis of the genomic organisation similarity generally exhibited by coronaviruses. The crystal structure of Clostridium botulinum neurotoxin B appeared to be structurally adaptable to human and canine coronavirus spike protein sequences and it was successfully used to model the two subunits of SARS coronavirus spike glycoprotein. The overall shape and the surface hydrophobicity of the two subunits in the obtained models suggest the localisation of the most relevant regions for their activity.

| S-EPMC7110993 | biostudies-literature

Proteolytic Activation of SARS-CoV-2 Spike at the S1/S2 Boundary: Potential Role of Proteases beyond Furin.

Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2' site(s) is associated with viral entry, which can occur at either the cell plasma membrane (early pathway) or the endosomal membrane (late pathway), depending on the cell type. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which appears to expand viral tropism to cells with suitable protease and receptor expression. Here, we utilize viral pseudoparticles and protease inhibitors to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 cleavage is essential for early pathway entry into Calu-3 cells, a model lung epithelial cell line, but not for late pathway entry into Vero E6 cells, a model cell line. The S1/S2 cleavage was found to be processed by other proteases beyond furin. Using bioinformatic tools, we also analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the origin of the insertion of the furin-like cleavage site in SARS-CoV-2.

| S-EPMC7839419 | biostudies-literature

Proteolytic Cleavage of the SARS-CoV-2 Spike Protein and the Role of the Novel S1/S2 Site.

Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19) has rapidly spread to the entire world within a few months. The origin of SARS-CoV-2 has been related to the lineage B Betacoronavirus SARS-CoV and SARS-related coronaviruses found in bats. Early characterizations of the SARS-CoV-2 genome revealed the existence of a distinct four amino acid insert within the spike (S) protein (underlined, SPRRAR↓S), at the S1/S2 site located at the interface between the S1 receptor binding subunit and the S2 fusion subunit. Notably, this insert appears to be a distinguishing feature among SARS-related sequences and introduces a potential cleavage site for the protease furin. Here, we investigate the potential role of this novel S1/S2 cleavage site and present direct biochemical evidence for proteolytic processing by a variety of proteases. We discuss these findings in the context of the origin of SARS-CoV-2, viral stability, and transmission.

| S-EPMC7255728 | biostudies-literature

Neuropilin-1 Assists SARS-CoV-2 Infection by Stimulating the Separation of Spike Protein Domains S1 and S2.

Project description:The cell surface receptor Neuropilin-1 (Nrp1) was recently identified as a host factor for SARS-CoV-2 entry. As the Spike protein of SARS-CoV-2 is cleaved into the S1 and the S2 domain by furin protease, Nrp1 binds to the newly created C-terminal RRAR amino acid sequence of the S1 domain. In this study, we model the association of a Nrp1 (a2-b1-b2) protein with the Spike protein computationally and analyze the topological constraints in the SARS-CoV-2 Spike protein for binding with Nrp1 and ACE2. Importantly, we study the exit mechanism of S2 from the S1 domain with the assistance of ACE2 as well as Nrp1 by molecular dynamics pulling simulations. In the presence of Nrp1, by binding the S1 more strongly to the host membrane, there is a high probability of S2 being pulled out, rather than S1 being stretched. Thus, Nrp1 binding could stimulate the exit of S2 from the S1 domain, which will likely increase virus infectivity as the liberated S2 domain mediates the fusion of virus and host membranes. Understanding of such a Nrp1-assisted viral infection opens the gate for the generation of protein-protein inhibitors, such as antibodies, which could attenuate the infection mechanism and protect certain cells in a future combination therapy.

| S-EPMC7805474 | biostudies-literature

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