Abstract
BackgroundOur previous studies have shown that combining the antiviral lectin GRFT and the pan-CoV fusion inhibitory peptide EK1 results in highly potent inhibitory activity against SARS-CoV-2 infection. In this study, we aimed to design and construct a bivalent protein consisting of GRFT and EK1 components and evaluate its inhibitory activity and mechanism of action against infection by SARS-CoV-2 and its mutants, as well as other human coronaviruses (HCoVs).MethodsThe bivalent proteins were expressed in E. coli and purified with Ni-NTA column. HIV backbone-based pseudovirus (PsV) infection and HCoV S-mediated cell–cell fusion assays were performed to test their inhibitory activity. ELISA and Native-PAGE were conducted to illustrate the mechanism of action of these bivalent proteins. Five-day-old newborn mice were intranasally administrated with a selected bivalent protein before or after HCoV-OC43 challenge, and its protective effect was monitored for 14 days.ResultsAmong the three bivalent proteins purified, GL25E exhibited the most potent inhibitory activity against infection of SARS-CoV-2 PsVs expressing wild-type and mutated S protein. GL25E was significantly more effective than GRFT and EK1 alone in inhibiting HCoV S-mediated cell–cell fusion, as well as infection by SARS-CoV-2 and other HCoVs, including SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63 and HCoV-OC43. GL25E could inhibit authentic SASR-CoV-2, HCoV-OC43 and HCoV-229E infection in vitro and prevent newborn mice from authentic HCoV-OC43 infection in vivo. GL25E could bind to glycans in the S1 subunit and HR1 in the S2 subunit in S protein, showing a mechanism of action similar to that of GRFT and EK1 alone.ConclusionsSince GL25E showed highly potent and broad-spectrum inhibitory activity against infection of SARS-CoV-2 and its mutants, as well as other HCoVs, it is a promising candidate for further development as a broad-spectrum anti-HCoV therapeutic and prophylactic to treat and prevent COVID-19 and other emerging HCoV diseases.
Highlights
Our previous studies have shown that combining the antiviral lectin GRFT and the pan-CoV fusion inhibitory peptide EK1 results in highly potent inhibitory activity against SARS-CoV-2 infection
Expression and identification of the bivalent proteins To understand the influence of linker length on the inhibitory activity of the recombinant bivalent proteins, we designed and constructed three pET-28a recombinant plasmids encoding three recombinant bivalent proteins, GRFT-L15-EK1 (GL15E), GRFT-L25-EK1 (GL25E), and GRFT-L35-EK1 (GL35E), containing linkers L15 (GGGGS)3, L25 (GGGGS)5, and L35 (GGGGS)7 between the GRFT and EK1 components, respectively (Fig. 1a and b)
It was concluded that GL25E has much higher potency in inhibiting SARS-CoV-2 infection than either GRFT or EK1 alone, possibly by the synergism of GRFT and EK1 acting together in recombinant bivalent protein GL25E
Summary
Our previous studies have shown that combining the antiviral lectin GRFT and the pan-CoV fusion inhibitory peptide EK1 results in highly potent inhibitory activity against SARS-CoV-2 infection. SARS-CoV-2 binds to its receptor ACE2 on the host cell through the receptor-binding domain (RBD) in S1 subunit of spike (S) protein [3] Such binding triggers conformation changes in the S2 subunit of S protein, resulting in the formation of a six-helix bundle (6-HB) between the heptad repeat 1 and 2 (HR1 and HR2) domains, bringing viral and target cell membranes together for fusion [4]. Both S1 and S2 subunits can serve as important targets for the development of SARS-CoV-2 fusion and entry inhibitors [5]. The sequence of S2 subunit is more conserved than that of S1 subunit, making it a better target for developing broadspectrum SARS-CoV-2 entry inhibitors [6]
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