Significant Redox Insensitivity of the Functions of the SARS-CoV Spike Glycoprotein

Abstract

The capacity of the surface glycoproteins of enveloped viruses to mediate virus/cell binding and membrane fusion requires a proper thiol/disulfide balance. Chemical manipulation of their redox state using reducing agents or free sulfhydryl reagents affects virus/cell interaction. Conversely, natural thiol/disulfide rearrangements often occur during the cell interaction to trigger fusogenicity, hence the virus entry. We examined the relationship between the redox state of the 20 cysteine residues of the SARS-CoV (severe acute respiratory syndrome coronavirus) Spike glycoprotein S1 subdomain and its functional properties. Mature S1 exhibited ϳ4 unpaired cysteines, and chemically reduced S1 displaying up to ϳ6 additional unpaired cysteines still bound ACE2 and enabled fusion. In addition, virus/cell membrane fusion occurred in the presence of sulfhydryl-blocking reagents and oxidoreductase inhibitors. Thus, in contrast to various viruses including HIV (human immunodeficiency virus) examined in parallel, the functions of the SARS-CoV Spike glycoprotein exhibit a significant and surprising independence of redox state, which may contribute to the wide host range of the virus. These data suggest clues for molecularly engineering vaccine immunogens.SARS 3 -CoV is a new human coronavirus that causes a severe acute respiratory syndrome, sometimes with a fatal outcome. Its Spike glycoprotein is the major surface antigen and induces potent neutralizing antibodies (1-8). It is made up of S1 and S2 subdomains (2, 3). The S1 region interacts with angiotensin-converting enzyme 2 (ACE2), the primary cellular receptor (9 -12), L-SIGN (13), and L-SECtin (14), whereas S2 mediates membrane fusion (2, 3). Here, we investigated the functional role of the 20 conserved cysteine residues of mature S1.A variety of evidence has shown that the capacity of the viral envelope glycoproteins to mediate virus/cell membrane fusion depends on a precise thiol/disulfide balance within the viral surface complex (15-29). On one hand, manipulation of the native disulfide network of mature envelopes at the virus surface using reducing or alkylating reagents has important consequences for their subsequent capacity to carry out the virus/cell interaction process (15, 16, 19, 20, 23, 24, 26, and 28). On the other hand, natural and specific thiol/disulfide rearrangements occur within several viral envelopes to trigger conformational changes that insert the fusion peptide into the cell surface leading to virus entry (20,21,23,(25)(26)(27). For HIV, gentle chemical reduction of Env efficiently prevents viral infectivity by inhibiting the capacity of the surface subunit (SU) to bind its ligands on the target cell surface (15,19). In contrast, after receptor binding, fusion mediated by the transmembrane subunit occurs solely following reduction of SU by a cell-surface protein-disulfide isomerase (PDI) activity (19,(25)(26)(27). For these reasons, reducing or free sulfhydryl reagents and nonpermeant inhibitors of PDI (e.g. the thiol reagent DTNB and bacitracin, ...

Contribution of Redox Status to Hepatitis C Virus E2 Envelope Protein Function and Antigenicity

“…In support of this hypothesis, incubation with iodoacetamide following DTT treatment to prevent reoxidation prevented the recovery of infection following dilution. We also examined whether disulfide reshuffling occurs during entry; 5,5Ј-dithiobis(2-nitrobenzoic acid) (DTNB) concentrations (1-2 mM) used to block free thiols on protein (22,23,33), hence the activity of oxidoreductases, had no effect (Fig. 5), suggesting that free sulfhydryls and redox isomerases are not involved during cell entry.…”

“…We confirmed inhibition of infection by Bafilomycin A1, which could not be rescued by pH 3-7 (34), and also found that Bafilomycin A1 treatment and virus binding at 4°C followed by mild reduction (0.04 -0.2 mM) at 37°C and acidic pH levels did not improve infection (not shown). Controls were performed; preincubation of MLVpps (33) with DTNB or DTT prevented infection in a parallel assay, as expected of the inhibition of the redox activity of the envelope endogenous CXXC motif and the reported dependence of MLV entry on the integrity of the envelope redox state (32,33). RSD within each condition was Յ8 and 6% for HCVpps and MLVpps, respectively.…”

“…7) and is intriguing because the many viruses displaying a strong dependence on redox state for function of their envelopes and the very few that are poorly sensitive appear to fuse virus cell membranes by similar mechanisms. Severe acute respiratory syndrome coronavirus is an example of a virus whose envelope function is poorly sensitive to reduction (33). However, its FIGURE 7.…”

Computational simulations reveal the binding dynamics between human ACE2 and the receptor binding domain of SARS-CoV-2 spike protein

“…In the crystal structure, the C480 and C488 of the RBD are crosslinked via a disulfide bond that reduces the flexibility of the L 67 region, limiting its access to the ACE2. On the other hand, it has been reported that the binding of SARS-RBD to ACE2 is insensitive to the redox states of the cysteines to a high extend 26 . Based on the simulation results, we hypothesize that the reduced form of C480 and C488 can also exist during the virus invasion to host cells, and the reduced cysteines can potentially enhance the binding to ACE2.…”

Supramolecular Architecture of the Coronavirus Particle

“…In the model of coronavirus spike protein-mediated fusion, receptor binding triggers conformational changes including disulfide reshuffling (Gallagher, 1996;Lavillette et al, 2006) that release HR1 and HR2 to form the coiled-coil fusion motor structure, thereby driving fusion of the viral and host cell membranes and release of the viral ribonucleoprotein into the cytosol (Bosch et al, 2003). The fusion motor complex of S2 consisting of two hydrophobic amino acid 4-3 heptad repeat regions (HR1 and HR2) which form amphipathic helices of a coiled-coil structure has also been solved in several forms (Bosch et al, 2003;Duquerroy et al, 2005;Liu et al, 2004;Tripet et al, 2004;Xu et al, 2004a,b).…”