Spike protein fragment SF5 exhibits strong binding ability and enhances full-length spike protein binding

In this study, Chinese researchers performed peptide mapping of all regions of the spike (S) protein of SARS-CoV-2 and SARS-CoV and identified a novel fragment SF5 of the SARS-CoV-2 S protein, which exhibits strong binding ability and mediates the binding of the full-length S protein.

The SARS-CoV-2 and SARS-CoV are members of the β-coronaviruses and have a positive-strand RNA genome. The different subgenomic RNAs encode four conserved structural proteins, spike, envelope, membrane, nucleocapsid, and several accessory proteins. It appears that S protein is a major pathogenic factor that contributes to the unique pathogenesis of SARS-CoV-2. The protein is composed of subunits S1 and a S2, which are separated by host cell proteases. S1 is composed of the N-terminal domain (NTD), the receptor binding domain (RBD) with a receptor binding motif (RBM), and two C-terminal domains. Previous studies have shown that RBD in the S1 subunit is responsible for attachment to host cells, and that the S2 subunit is involved in the cell fusion process after initially binding. The S protein interacts not only with the angiotensin-converting enzyme 2 (ACE2) receptor, but also with several other host receptors. The S protein is heavily modified by glycosylation, and this may shield the virus from recognition by the host immune system.

About the study

The peptide mapping showed that the S protein of SARS-CoV-2 consists of 1273 amino acids, whereas the S protein of SARS-CoV consists of 1255 amino acids. These two proteins were divided into subunits S1 and S2. The analysis of homologous S1 and S2 domains in SARS-CoV-2 and SARS-CoV revealed significant linear similarities between these β-coronaviruses.

The authors further subdivided S1 and S2 subunits into fragments. Based on their amino acid structures and their known functions, these fragments of the S protein of SARS-CoV-2 and SARS-CoV were named COVID19-SF1-6 and SARS-SF1-6. Within these 12 fragments, COVID19-SF1 and SARS-SF1 contained the NTD of SARS-CoV-2 and SARS-CoV. The RBD was represented by the COVID19-SF2 or SARS-SF2.

The scientists then used both a flow cytometry and cell-based ELISA analysis to examine the binding activity of six fragments of the SARS-CoV-2 S protein to cell lines, such as VERO-E6 (which is susceptible to SARS-CoV-2 infection) and BEAS-2B (immortalized lung cell line). The cells VERO-E6 and BEAS-2B were incubated with six fragments of the SARS-CoV-2 S protein. The findings revealed that the fragment COVID19-SF5 exhibited the strongest binding ability to both VERO-E6 and BEAS-2B cells, demonstrating a specific binding to both VERO-E6 and BEAS-2B cells. These findings have confirmed that this fragment is a fusion process region. IgGs against COVID19-SF5 were found to inhibit the binding of COVID19-SF5 to the cells in a concentration-dependent manner.

The authors pointed out that COVID19-SF5 is embedded and hidden by trimer S protein or glycosylation, and is not exposed within the full-length glycosylated S protein. As a result, the immune system of the infected individual may fail to recognize it as a foreign invading protein during the initial infection.

The effect of COVID19-SF5 on the binding of the full-length S protein to VERO-E6 cells was further investigated by adding various concentrations of non-His tagged COVID19-SF5. The results showed that COVID19-SF5, a domain that is not exposed in the full-length glycosylated S protein, enhanced the binding of the full-length S protein to VERO-E6 cells in a concentration-dependent manner. The authors noted that enhancement of binding of the full-length glycosylated S protein may facilitate the accessibility of other binding regions of the S protein. This may help the virus to become more transmissible and facilitate infection.

The authors then performed an enzyme-linked immunosorbent assay (ELISA) analysis of serological reactions of the full-length glycosylated S protein with six antisera against fragments COVID19-SF1-6. The results showed that the full-length glycosylated S protein reacted with antisera against fragments COVID19-SF1, 2, 5, and 6, but not with antisera against the fragments COVID19-SF3 and 4.

In addition, ELISA and immunoblotting analysis were used to determine the cross-reactivity of IgGs against each of COVID19-SF1-6 fragments. The results revealed that IgGs against the fragment COVID19-SF5 reacted strongly against this fragment, but also showed a significant cross reactivity with all other 5 fragments of SARS-CoV-2 S protein. Furthermore, specific IgGs against the fragment COVID19-SF5 also strongly cross-reacted with all six recombinant subunit fragments of SARS-CoV S protein.

It is interesting to note that strong cross-reactivity remained detectable with all twelve fragments of SARS-CoV-2 and SARS-CoV S proteins for 3 months. Over time, COVID19-SF5 antiserum reacted with most of the subunit fragments, but the reactivity gradually diminished. According to the authors, these cross-reaction activities showed that fragment SF5 shares a common antigenicity with all twelve fragments of the SARS-CoV-2 and SARS-CoV S protein.

In order to characterize the effect of the COVID19-SF1-6 fragments and their antisera on pseudovirus infection of the cells, the researchers performed neutralization analysis. The six recombinant fragment proteins inhibited SARS-CoV-2 pseudovirus infection, with an infection rate of about 20–50%. The fragment COVID19-SF5 displayed the strongest inhibition of pseudovirus infection to the cells. In addition, the six antisera also showed some neutralization effect on SARS-CoV-2 pseudovirus infection of the cells. These results showed that both proteins and specific IgGs were capable of neutralizing the pseudovirus, thereby preventing the infection of host cells, although the effect between IgGs and fragments varied. According to the authors, this finding strongly suggests that a conserved binding site (epitope) may exist in this region, along with the RBD-ACE-2 site.

In conclusion, this study showed that the fragment COVID19-SF5 may contain a common region(s) for mediating viral binding during infection. The authors noted that this activity likely occurred after ACE2 binding. The findings substantiated their conclusion that its epitopes are somewhat cryptic. Nonetheless, it is intriguing that the fragment COVID19-SF5 enhanced rather than inhibited the recognition of the S protein. Therefore, possible mechanism needs to be further investigated.

This article was published in Scientific Reports.

Journal Reference

Wang, H. et al. Identification and characterization of a novel cell binding and cross-reactive region on spike protein of SARS-CoV-2. Sci Rep 2022; 12, 15668. (Open Access).