Novel fusion inhibitor blocks SARS-CoV-2 spike-mediated cell-cell fusion

In a recent study published in the journal Viruses, researchers constructed a novel fusion inhibitor−based recombinant protein, referred to as 5−Helix, to block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) mediated cell-cell fusion.

Study: A Five-Helix-Based SARS-CoV-2 Fusion Inhibitor Targeting Heptad Repeat 2 Domain against SARS-CoV-2 and Its Variants of Concern. Image Credit: CKA/Shutterstock
Study: A Five-Helix-Based SARS-CoV-2 Fusion Inhibitor Targeting Heptad Repeat 2 Domain against SARS-CoV-2 and Its Variants of Concern. Image Credit: CKA/Shutterstock

It found that 5−Helix potently inhibited infection by SARS−CoV−2 pseudoviruses (PsV) and its variants of concern (VOCs), including Omicron; additionally, it inhibited infection by authentic SARS−CoV−2 wild−type (wt) nCoV−SH01 strain and Delta VOC.


Amid the continuous emergence of SARS-CoV-2 VOCs and variants of interest (VOIs, e.g., Lambda), there is an urgent need to develop effective, broader−spectrum antivirals against SARS−CoV−2, the causative agent of the deadly coronavirus disease 2019 (COVID-19).

To date, neutralizing antibody (NAb) therapeutics and vaccines against COVID-19 have primarily targeted the receptor-binding domain (RBD) and N-terminal domain (NTD) of the SARS-CoV-2 S1 subunit.

However, the researchers have now shifted focus to heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains of the S2 subunit as targets for viral fusion inhibitors. These domains in the S2 subunit are only transiently exposed immediately after binding between the RBD in S1 and the host cell receptor angiotensin-converting enzyme 2 (ACE2).

Due to space constraints during the fusion-intermediate state, HR1 and HR2 domains are not accessible to immunoglobulin G (IgG) molecules with a molecular weight of 150 kDa. Moreover, they are the most conserved regions of the S protein; consequently, researchers speculated that antivirals targeting these domains will have a higher genetic barrier to resistance.

Among the HR1 and HR2, HR2 is a better target for broad−spectrum viral fusion inhibitors since its amino acid sequences are more conserved than that of HR1. It is worth noting here that the amino acid sequences of HR1 and HR2 domains of SARS−CoV−2 are also 92.6% and 100% similar to those of HR1 and HR2 of SARS−CoV, respectively.

Since 5−Helix is a recombinant protein, it may also elicit antibodies after repeated use in COVID−19 patients. As opposed to anti−human immunodeficiency virus (HIV) therapeutics, which are used for prolonged periods, an antiviral agent against SARS−CoV−2 needs to be used for a few days only. Therefore, antibodies induced against 5−Helix might not hinder its future clinical application.

Previously, the authors had engineered several peptide−based fusion and entry inhibitors targeting the HR1 domain of several viruses, including HIV-1 and SARS-CoV-2.

About the study

In the present study, they engineered the fusion protein 5−Helix and assessed its inhibitory activity on SARS−CoV−2 S−mediated cell-cell fusion. They counted the percentage of fused cells under a fluorescence microscope to compute their half-maximal inhibitory concentration (IC50). They also observed the secondary structure of 5−Helix using circular dichroism (CD) spectroscopy.

They synthesized SARS-CoV-2 PsV and determined their tissue culture infectious dose (TCID50) using the Reed−Muench method. To examine the inhibitory activity of 5−Helix against SARS-CoV-2 PsV infection, they seeded Caco−2 cells in a 96−well plate, added 100 TCID50 of SARS-CoV-2 PsV, mixed with an equal volume of 5−Helix, which was serially diluted with phosphate buffer saline (PBS) at 37 °C for 30 minutes.

The mixture was transferred to the Caco−2 cells, rinsed with a fresh medium after 12 hours, and finally, they measured the luminescence after 48 hours of incubation. A luminescence value ten−fold higher than the control indicated a positive result.

They also performed inhibitory activity assessments of 5−Helix against authentic SARS-CoV-2 infections in a Biosafety Level 3 (BSL−3) laboratory. To this end, they seeded Calu−3 cells into a 96−well plate, added authentic SARS−CoV−2 at 0.01 multiplicity of infection (MOI) mixed with an equal volume of 5−Helix, serially diluted with PBS maintained at 37°C for 30 minutes. The mixture was transferred to the Calu−3 cells, and 48 hours later, they collected the supernatants and quantified SARS−CoV−2 nucleocapsid (N) gene copies using quantitative reverse−transcription polymerase chain reaction (RT−qPCR).

They used the cell counting kit−8 to measure the 5−Helix cytotoxicity to Huh 7 cells, Caco−2 cells, and Calu−3 cells. Lastly, they used the biolayer interferometry (BLI) instrument Octet QK system to record 1:1 real−time binding between 5−Helix and biotinylated SARS−CoV−2 HR2P and compute the equilibrium dissociation constant (KD).

Study findings

5−Helix fusion protein consisted of three copies of HR1P and two copies of HR2P and formed 5−helix−bundle (5−HB) that exposed one hydrophobic groove that could be accessed and bound by the HR2 region in the S2 subunit of the SARS−CoV−2 S protein. The purified 5−Helix protein showed a single band of about 24 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),  similar to its theoretical molecular weight.

As observed during CD spectroscopic examination, it displayed a helical structure with a high α−helicity of almost 100%, and a melting temperature (Tm) of 52 °C, indicating its thermo-stability.

Enzyme-linked immunosorbent assay (ELISA) showed that 5-Helix could bind with SARS−CoV−2 HR2P peptide with high affinity and a KD of 17.3 nM suggested that this binding was specific and strong. Additionally, it could block six-helix bundle (6−HB) formation between the HR1 and HR2 peptides of SARS−CoV−2.

5−Helix effectively inhibited SARS−CoV−2 PsV infection in Caco−2 cells with a mean IC50 value of 243 nM. It inhibited SARS−CoV−2 S−mediated cell-cell fusion, and pseudotyped SARS−CoV−2 wt strain D614G and five VOCs, including Alpha, Beta, Gamma, Delta, and VOI Lambda with IC50 of 364, 170, 142, 369, and 238 nM, respectively. Unexpectedly, 5−Helix displayed the highest inhibitory activity against Omicron PsV infection with IC50 of 141 nM.

Similar to the HR1P peptide, 5−Helix at 30 μM concentration showed no significant cytotoxicity and had a selectivity index (SI) over 80, indicating that it was not cytotoxic in vitro. It was also potent against the authentic SARS−CoV−2 wt strain, nCoV−SH01, and the Delta VOC.


The study findings confirmed that 5−Helix had a lower production cost and longer half−life than a pan−CoV fusion inhibitor EK1. Moreover, it exhibited about two− to three−fold higher antiviral activity against SARS−CoV−2 and its VOCs than EK1. Furthermore, since 5−Helix targeted the more conserved HR2 domain, it had a higher genetic barrier to resistance than the EK1 peptide and lipopeptides. More importantly, it was possible to synthesize 5−Helix on a large scale in yeast or Escherichia coli expression systems.

Taken together, these findings suggest that the 5−Helix fusion protein is a potent inhibitor of all currently circulating and emerging SARS−CoV−2 variants thus, an ideal candidate for development as a COVID-19 prophylactic or therapeutic agent.    

Journal reference:
Neha Mathur

Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.


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