Cooperative involvement of the S1 and S2 subunits of the murine coronavirus spike protein in receptor binding and extended host range

Abstract

To study the process of spike (S)-receptor interaction during coronavirus entry, we evaluated the contributions of mutations in different regions of the murine hepatitis virus (MHV) S protein to natural receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (CEACAM1a) dependence and to the acquisition of extended host range. Extended-host-range variants of MHV strain A59 were previously obtained from persistently infected cells (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9504, 1997). These variant viruses contain several mutations in the S protein that confer to the viruses the ability to enter cells in a heparan sulfate-dependent manner (C. A. de Haan, Z. Li, E. te Lintelo, B. J. Bosch, B. J. Haijema, and P. J. M. Rottier, J. Virol. 79:14451-14456, 2005). While the parental MHV-A59 is fully dependent on murine CEACAM1a for its entry, viruses carrying the variant mutations in the amino-terminal part of their S protein had become dependent on both CEACAM1a and heparan sulfate. Substitutions in a restricted, downstream part of the S protein encompassing heptad repeat region 1 (HR1) and putative fusion peptide (FP) did not alter the CEACAM1a dependence. However, when the mutations in both parts of the S protein were combined, the resulting viruses became independent of CEACAM1a and acquired the extended host range. In addition, these viruses showed a decreased binding to and inhibition by soluble CEACAM1a. The observations suggest that the amino-terminal region of the S protein, including the receptor-binding domain, and a region in the central part of the S protein containing HR1 and FP, i.e., regions far apart in the linear sequence, communicate and may even interact physically in the higher-order structure of the spike.

FIG. 1.

Cloning strategy and organization of the chimeric S proteins. (A) The spike protein is depicted as an elongated box. Each vertical line in this box indicates an amino acid substitution in the MHV/BHK S protein relative to the parental MHV-A59 spike protein. The triangle indicates a 7-amino-acid (aa) insertion. The position of the arrow marks the position where the MHV-A59 S protein can be cleaved into an amino-terminal S1 and a carboxy-terminal S2 subunit. Horizontal lines designate the approximate locations of the receptor-binding domain (RBD), the putative fusion peptide (FP), heptad repeat regions 1 (HR1) and 2 (HR2), and the transmembrane domain (TM). The circled numbers indicate the locations of the heparan sulfate-binding consensus sequences, which are marked by gray boxes and triangles. Fragments A, B, C, c, and D of the MHV/BHK S protein are indicated. Restriction enzymes used to clone the PCR fragments encoding these fragments are shown. (B) The chimeric S proteins are also depicted as elongated boxes, while the recombinant viruses containing these proteins are specified on the right side. White and gray boxes indicate the parts encoded by MHV-A59 and MHV/BHK, respectively. The numbers in the boxes indicate the number of amino acid differences in this part of the MHV/BHK S protein compared to the MHV-A59 S protein.

FIG. 2.

Infection of murine cells. (A) LR7 cells were inoculated with the indicated recombinant viruses (MHV-2aFLS WT, rec, A, etc.) at an MOI of 1. At the indicated time points postinfection, the cells were lysed and the intracellular luciferase expression was determined by using a luminometer (values are expressed in relative light units [RLU]). Standard deviations (error bars) are indicated. (B) Plaque phenotypes of the recombinant viruses on LR7 cells. At 18 h postinfection, cells were fixed with a 3% formaldehyde solution after which the agar overlay was removed. After permeabilization with 1% Triton X-100 in PBS, viral antigen was detected with the anti-MHV serum k134. Peroxidase-conjugated swine immunoglobulins to rabbit immunoglobulins (Dakopatts) were used as secondary antibodies.

FIG. 3.

Infection of human cells. (A) HeLa cells were inoculated with the indicated recombinant viruses (MHV-2aFLS WT, rec, A, etc.), and the intracellular luciferase expression was determined as described in the legend to Fig. 2A. Values for recombinant viruses entering HeLa cells 8.5 and 18 hours postinfection are shown. (B) HeLa cells were infected with the recombinant viruses as described in the legend to Fig. 2B. At 18 h postinfection, the cells were fixed, and viral antigen was detected as described in the legend to Fig. 2B.

FIG. 4.

Inhibition of MHV infection by anti-mCEACAM1a antibodies. (A) LR7 and HeLa cells were inoculated with the recombinant viruses (MHV-2aFLS WT, rec, A, etc.) as described in the legend to Fig. 2A, except the cells had been pretreated with the polyclonal (41) (A) or CC1 monoclonal (18) (B) antibody against mCEACAM1a (or control antibody) for 1 h prior to the inoculation. The control antibodies used were a polyclonal rabbit antiserum against Semliki Forest virus (anti-SFV) and a mouse immunoglobulin G1 (IgG1) isotype control antibody. The relative luciferase expression levels in the cultures are shown. For each individual recombinant virus, the luciferase expression level after pretreatment with the control antibodies was set at 100%. Standard deviations (error bars) are indicated.

FIG. 5.

Interaction of recombinant viruses with soluble mCEACAM1a. (A) Recombinant viruses (MHV-2aFLS WT, rec, A, etc.) were incubated with different concentrations of soluble mCEACAM1a (19), after which the virus-soluble receptor mixtures were used to inoculate LR7 cells. The relative luciferase expression levels in the cultures measured at 5 h postinfection are shown. For each individual recombinant virus, the luciferase expression level observed after mock incubation with soluble mCEACAM was set at 100%. Standard deviations (error bars) are indicated. (B) Radiolabeled virions were incubated with different concentrations of soluble mCEACAM1a after which the amounts of bound virus were determined. The relative amounts of affinity-purified virions are shown. The amount of radioactivity affinity purified from each virion preparation using the MHV antiserum K134 was defined as 100%.

FIG. 6.

Temperature- and pH-dependent stability. Recombinant viruses (MHV-2aFLS WT, rec, A, etc.) were incubated at 4 or 37°C at pH 6.5 or 8.2 prior to inoculation of LR7 cells. The relative luciferase expression levels in the cultures measured at 5 h postinfection are shown. For each individual recombinant virus, the luciferase expression level observed after incubation at 4°C for each pH was set at 100%.

FIG. 7.

Heparan sulfate-dependent entry. LR7 cells were inoculated with the recombinant viruses (MHV-2aFLS WT, rec, A, etc.) as described in the legend to Fig. 2A, except the cells had been pretreated with heparinase I for 1.5 h before the inoculation (A) or the recombinant viruses had been incubated with different concentrations of heparin for 1 h at 4°C (B). At 5 h postinfection, the FL activity in the cultures was determined. For each individual recombinant virus, the luciferase expression level observed after mock treatment was set at 100%. Standard deviations (error bars) are indicated.