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. 2003 Jan;77(2):830-40.
doi: 10.1128/jvi.77.2.830-840.2003.

Conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees C either by soluble murine CEACAM1 receptors or by pH 8

Bruce D Zelus  1 , Jeanne H Schickli, Dianna M Blau, Susan R Weiss, Kathryn V Holmes
Affiliations

Conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees C either by soluble murine CEACAM1 receptors or by pH 8

Bruce D Zelus et al. J Virol. 2003 Jan.

Abstract

The spike glycoprotein (S) of the murine coronavirus mouse hepatitis virus (MHV) binds to viral murine CEACAM receptor glycoproteins and causes membrane fusion. On virions, the 180-kDa S glycoprotein of the MHV-A59 strain can be cleaved by trypsin to form the 90-kDa N-terminal receptor-binding subunit (S1) and the 90-kDa membrane-anchored fusion subunit (S2). Incubation of virions with purified, soluble CEACAM1a receptor proteins at 37 degrees C and pH 6.5 neutralizes virus infectivity (B. D. Zelus, D. R. Wessner, R. K. Williams, M. N. Pensiero, F. T. Phibbs, M. deSouza, G. S. Dveksler, and K. V. Holmes, J. Virol. 72:7237-7244, 1998). We used liposome flotation and protease sensitivity assays to investigate the mechanism of receptor-induced, temperature-dependent virus neutralization. After incubation with soluble receptor at 37 degrees C and pH 6.5, virions became hydrophobic and bound to liposomes. Receptor binding induced a profound, apparently irreversible conformational change in S on the viral envelope that allowed S2, but not S1, to be degraded by trypsin at 4 degrees C. Various murine CEACAM proteins triggered conformational changes in S on recombinant MHV strains expressing S glycoproteins of MHV-A59 or MHV-4 (MHV-JHM) with the same specificities as seen for virus neutralization and virus-receptor activities. Increased hydrophobicity of virions and conformational change in S2 of MHV-A59 could also be induced by incubating virions at pH 8 and 37 degrees C, without soluble receptor. Surprisingly, the S protein of recombinant MHV-A59 virions with a mutation, H716D, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37 degrees C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptor-triggered S(A59)H716D virions with trypsin at 4 degrees C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37 degrees C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2.

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Figures

FIG. 1.
FIG. 1.
Soluble CEACAM glycoproteins or alkaline pH at 37°C causes association of virions with liposomes. (A) MHV-A59 virions were incubated with liposomes for 30 min at 4 or 37°C and pH 6.5 with soluble murine CEACAM1a[1-4] or soluble murine CEACAM2[1,4] or with buffer at pH 8.0. The mixtures were chilled to 4°C, loaded into the bottom of sucrose density gradients, and ultracentrifuged as described in Materials and Methods. Gradient fractions were applied to polyvinylidere difluoride membranes; viral spike antigen was detected with goat anti- S antibody, and receptor antigen was detected with goat anti-murine CEACAM1a antibody. (B) In parallel experiments, the association of [3H]uridine-labeled MHV-A59 virions with liposomes after incubation at pH 6.5 for 30 min at 4 or 37°C was determined by measuring the amount of radiolabel in each gradient fraction.
FIG. 2.
FIG. 2.
Binding of MHV-A59 virions to liposomes after incubation at 37°C with soluble CEACAM1a[1-4] glycoprotein. Virions were incubated with soluble receptor protein for 30 min at 37°C and pH 6.5, immediately placed on a carbon-coated Formvar-covered grid, fixed in 1% glutaraldehyde, and negatively stained with 2% phosphotungstic acid. The arrow shows a virion adsorbed to a liposome. Magnification, ×79,400.
FIG. 3.
FIG. 3.
Conformational changes in the S2 domain of MHV-A59 spike glycoprotein induced at 37°C by CEACAM1a at pH 6.5 or by the presence of pH 8.0 without receptor. A trypsin sensitivity assay was used to demonstrate conformational changes in the viral spike protein. Gradient-purified MHV-A59 virions were incubated at 4 or 37°C for 30 min at pH 6.5 with either soluble murine CEACAM1a[1-4] or CEACAM2[1,4] or without receptor proteins at pH 6.5 or 8.0. The samples were then incubated at 4°C for 20 min with trypsin-TPCK, subjected to SDS-PAGE, and immunoblotted with MAbs specific for the receptor binding S1 domain of the spike (MAb A1.9) or for the carboxyl-terminal S2 domain of the spike (MAb 5B93.7) which has been associated with membrane fusion.
FIG. 4.
FIG. 4.
Recombinant MHV-A59 viruses expressing the spike glycoproteins of MHV-A59 or MHV-4 differ in susceptibility to conformational changes in S2 induced by soluble murine CEACAM1a or CEACAM1b glycoproteins. Isogenic recombinant MHV-A59 viruses that express either the spike glycoprotein of MHV-A59 (SA59R) or MHV-4 (S4R) were prepared by targeted RNA recombination (30). The virions were incubated with different concentrations of purified, soluble murine CEACAM1a[1-4] (cloned from MHV-susceptible mice) or CEACAM1b[1-4] receptor glycoproteins (cloned from MHV-resistant SJL mice [10]) at pH 6.5 for 30 min at the temperatures indicated and then incubated for 20 min at 4°C with trypsin. The S2 glycoproteins were detected by immunoblotting with anti-S2 MAb 5B93.7.
FIG. 5.
FIG. 5.
A mutant MHV-A59 strain, SA59H716D, that is not cleaved between S1 and S2 nevertheless exposes a hydrophobic domain and associates with liposomes after incubation at pH 6.5 with soluble CEACAM1a[1-4] glycoprotein or alkaline pH at 37°C but not at 4°C. A mutant MHV-A59 virus with a single mutation, H716D, in the viral spike glycoprotein that prevents protease cleavage at the S1-S2 junction was constructed by targeted RNA recombination (, , , ; Hingley, personal communication). SA59H716D virions were incubated with liposomes for 30 min at 4 or 37°C and pH 6.5 with soluble murine CEACAM1a[1-4] or with buffer at pH 6.5 or 8.0 and then analyzed by sucrose density gradient ultracentrifugation as described in the legend to Fig. 1. Viral spike antigen in each gradient fraction was detected with polyclonal goat A04 anti-S antibody.
FIG. 6.
FIG. 6.
The S2 glycoprotein of mutant MHV-A59 virus SA59H716D undergoes a conformational change and becomes susceptible to a unique trypsin cleavage event after incubation at 37°C with soluble murine CEACAM1a[1-4] at pH 6.5 or at pH 8.0 in the absence of receptor. Gradient purified SA59H716D virions were incubated at 4 or 37°C for 30 min at pH 6.5 with soluble murine CEACAM1a[1-4] or without receptor proteins at pH 6.5 or 8.0. The samples were incubated at 4°C for 20 min with trypsin-TPCK at 10 μg/ml, subjected to SDS-PAGE, and immunoblotted with anti-S2 MAb 5B19.5. Untreated virions contained a novel subunit of S with an apparent molecular mass of 120 kDa. Incubation of the SA59H716D virions with soluble murine CEACAM1a[1-4] at 37°C made the 180-kDa spike glycoprotein susceptible to cleavage by trypsin-TPCK at this novel site. To a lesser degree, incubation of virions at pH 8.0 and 37°C also increased the susceptibility of the SA59H716D spike to trypsin cleavage.
FIG. 7.
FIG. 7.
Comparison of the structures of the fusion domains of spike proteins from several enveloped viruses. The spike glycoproteins of many enveloped viruses have a single protease-susceptible cleavage site (or region) between the N-terminal receptor-binding domain and the carboxyl-terminal fusion domain that is anchored in the viral envelope. Cleavage at this site may activate the membrane-fusing activity of the viral glycoprotein, which is mediated by a hydrophobic fusion peptide that is hidden in the spike on the virion until binding of the RBD to its cognate receptor or a pH change, or both, triggers a specific conformational change in the fusion domain that leads to fusion of the viral envelope with the host plasma membrane or endosomal membrane. The fusion domain of MHV spike (S2) is much larger (90 kDa) than fusion domains of the spikes of many other enveloped viruses such as influenza A virus (Flu), paramyxovirus SV5, Ebola virus, respiratory syncytial virus (HRSV), and HIV-1. The putative fusion peptide of MHV-A59 is not at the N terminus of S2 but lies within the first amphipathic helix. The functions of the large N-terminal region of murine coronavirus S2 are not well understood.
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