Conformational change of the coronavirus peplomer glycoprotein at pH 8.0 and 37 degrees C correlates with virus aggregation and virus-induced cell fusion

Abstract

We have obtained biochemical and electron microscopic evidence of conformational changes at pH 8.0 and 37°C in the coronavirus spike glycoprotein E2 (S). The importance of these changes is reflected in the loss of virus infectivity, the aggregation of virions, and increased virus-induced cell fusion at the same pH. Coronavirus (MHV-A59) infectivity is exquisitely sensitive to pH. The virus was quite stable at pH 6.0 and 37°C (half-life,-24 h) but was rapidly and irreversibly inactivated by brief treatment at pH 8.0 and 37°C (half-life,-30 min). Virions treated at pH 8.0 and 37°C formed clumps and large aggregates. With virions treated at pH 8.0 and 37°C, the amino-terminal peptide E2N (or Sl) was released from virions and the remaining peptide, E2C (S2), was aggregated. Viral spikes isolated from detergent-treated virions also aggregated at pH 8.0 and 37°C. Loss of virus infectivity and E2 (S) aggregation at pH 8.0 and 37°C were markedly enhanced in the presence of dithiothreitol. On the basis of the effects of dithiothreitol on the reactions of the peplomer, we propose that release of E2N (S1) and aggregation of E2c (S2) may be triggered by rearrangement of intramolecular disulfide bonds. The aggregation of virions and the isolated E2 (S) glycoprotein at pH 8.0 and 37°C or following treatment with guanidine and urea at pH 6.0 and 37°C indicate that an irreversible conformational change has been induced in the peplomer glycoprotein by these conditions. It is interesting that coronavirus-induced cell fusion also occurred under mildly alkaline conditions and at 37°C. Some enveloped viruses, including influenza viruses and alphaviruses, show conformational changes of spike glycoproteins at a low pH, which correlates with fusion and penetration of those viruses in acidified endocytic vesicles. For coronavirus MHV-A59, comparable conformational change of the spike glycoprotein E2 (S) and cell fusion occurred at a mildly alkaline condition, suggesting that coronavirus infection-penetration, like that of paramyxoviruses and lentiviruses, may occur at the plasma membrane, rather than within endocytic vesicles. * Corresponding author. binding to receptors on murine cells (16; S. R. Compton and K. V. Holmes, unpublished data), induction of neutralizing antibodies (7) and cell-mediated cytotoxicity (17, 42), and virus-induced cell fusion (11, 39). The induction of cell fusion by MHV-A59 requires cleavage of the 180,000-molecular weight E2 glycoprotein (180K E2 [or S] glycoprotein)

A new Sephadex™-based method for removing microbicidal and cytotoxic residues when testing antiseptics against viruses: Experiments with a human coronavirus as a model

“…This could be due to a high sensitivity of the HCoV 229E to the pH. Rapid loss of infectivity and an aggregation of virions have been shown at pH 8 and 37 • C, whereas the virus was quite stable at pH 6 and 37 • C (Sturman et al, 1990). So pH should be verified and adjusted to the neutrality to allow a standardization of the protocol.…”

The Molecular Biology of Coronaviruses

“…Such treatm ent caused the diss ociation and relea se of th e cleaved S1 subunit and th e aggre gation of S2 subunits ; the accompan ying confor mational changes in S1 were monitored by differential access of a panel of monoclonal antibodies at neutral and alkaline pH (Weismiller et al, 1990). Disulfide bond formation plays an important role in S protein folding, and disulfides in S1 may become rearranged during the conformational transitions of S1 following receptor binding (Lewicki and Gallagher, 2002;Opstelten et al, 1993;Sturman et al, 1990). The S protein of the highly virulent MHV strain 4 (JHM) has been shown to exist in a particularly metastable configuration.…”

Molecular Interactions in the Assembly of Coronaviruses

“…A fusion peptide has not yet been identified in any of the coronavirus spike proteins, but is predicted to be located at (Bosch et al, 2004b;Chambers et al, 1990) or within (Luo and Weiss, 1998) the amino terminus of the first HR region. Binding of the S1 subunit to the (soluble) receptor, or exposure to 37 C and an elevated pH, has been shown to trigger conformational changes that are supposed to facilitate virus entry by activation of the fusion function of the S2 subunit (Breslin et al, 2003;Gallagher, 1997;Lewicki and Gallagher, 2002;Matsuyama and Taguchi, 2002;Miura et al, 2004;Sturman et al, 1990;Taguchi and Matsuyama, 2002;Zelus et al, 2003). This conformational change is thought to lead to exposure of the fusion peptide and its interaction with the target membrane, further changes resulting in the formation of a heterotrimeric six-helix bundle, characteristic of class I viral fusion proteins, during the membrane fusion process.…”