Junctional adhesion molecule a serves as a receptor for prototype and field-isolate strains of mammalian reovirus

Abstract

Reovirus infections are initiated by the binding of viral attachment protein sigma1 to receptors on the surface of host cells. The sigma1 protein is an elongated fiber comprised of an N-terminal tail that inserts into the virion and a C-terminal head that extends from the virion surface. The prototype reovirus strains type 1 Lang/53 (T1L/53) and type 3 Dearing/55 (T3D/55) use junctional adhesion molecule A (JAM-A) as a receptor. The C-terminal half of the T3D/55 sigma1 protein interacts directly with JAM-A, but the determinants of receptor-binding specificity have not been identified. In this study, we investigated whether JAM-A also mediates the attachment of the prototype reovirus strain type 2 Jones/55 (T2J/55) and a panel of field-isolate strains representing each of the three serotypes. Antibodies specific for JAM-A were capable of inhibiting infections of HeLa cells by T1L/53, T2J/55, and T3D/55, demonstrating that strains of all three serotypes use JAM-A as a receptor. To corroborate these findings, we introduced JAM-A or the structurally related JAM family members JAM-B and JAM-C into Chinese hamster ovary cells, which are poorly permissive for reovirus infection. Both prototype and field-isolate reovirus strains were capable of infecting cells transfected with JAM-A but not those transfected with JAM-B or JAM-C. A sequence analysis of the sigma1-encoding S1 gene segment of the strains chosen for study revealed little conservation in the deduced sigma1 amino acid sequences among the three serotypes. This contrasts markedly with the observed sequence variability within each serotype, which is confined to a small number of amino acids. Mapping of these residues onto the crystal structure of sigma1 identified regions of conservation and variability, suggesting a likely mode of JAM-A binding via a conserved surface at the base of the sigma1 head domain.

FIG. 1.

JAM-A blockade reduces infection of prototype reovirus strains. HeLa cells at equivalent degrees of confluence were pretreated with PBS, an hCAR-specific antiserum as a control, or the hJAM-A-specific MAb J10.4 prior to adsorption with T1L/53, T2J/55, or T3D/55 at an MOI of 0.1 FFU per cell. After incubation for 20 h, the cells were fixed and permeabilized with methanol. Newly synthesized viral proteins were detected by the incubation of cells with a polyclonal rabbit antireovirus serum followed by incubation with an anti-rabbit immunoglobulin-Alexa-546 serum for the visualization of infected cells by indirect immunofluorescence. (A) Representative fields of view. (B) Reovirus-infected cells were quantified by counting fluorescent cells in a minimum of three random fields of view per well for three wells at a magnification of ×20. The results are presented as the mean FFU per field. Error bars indicate standard deviations.

FIG. 2.

CHO cells transfected with hJAM-A support growth of prototype reovirus strains. (A) CHO cells were transiently transfected with a plasmid encoding hCAR, hJAM-A, hJAM-B, or hJAM-C. Following incubation for 24 h to permit receptor expression, cells were incubated with receptor-specific MAbs, and the cell surface expression of receptor constructs was assessed by flow cytometry. (B) Transfected CHO cells at equivalent degrees of confluence were adsorbed with T1L/53, T2J/55, or T3D/55 at an MOI of 0.1 FFU per cell. Reovirus proteins were detected by indirect immunofluorescence at 20 h postinfection. Representative fields of view are shown. Magnification, ×20. (C) Reovirus-infected cells were quantified by counting fluorescent cells in five random fields of view per well for three wells. The results are presented as the mean FFU per field. Error bars indicate standard deviations.

FIG. 3.

Expression of JAM-A confers infectivity on field-isolate reovirus strains. CHO cells were transiently transfected with a plasmid encoding hCAR, hJAM-A, hJAM-B, or hJAM-C. Following incubation for 24 h to permit receptor expression, the cells were adsorbed with the indicated field-isolate strains at an MOI of 1 FFU per cell. Reovirus proteins were detected by indirect immunofluorescence at 20 h postinfection and quantified by counting of the fluorescent cells in three random fields of view per well for three wells. The results are presented as the mean FFU per field. Error bars indicate standard deviations.

FIG. 4.

Phylogenetic relationships among S1 gene nucleotide sequences of 13 reovirus strains. A phylogenetic tree for the σ1-encoding S1 gene sequences of the strains shown in Table 1 was constructed by using the maximum parsimony method as applied in the program PAUP. The tree is rooted at its midpoint. Bootstrap values of >50% (indicated as a percentage of 1,000 repetitions) for major branches are shown at the nodes. Bar, distance resulting from 10 nucleotide changes.

FIG. 5.

Sequence conservation and structural variability within the type 3 σ1 protein. (A) Alignment of deduced amino acid sequences of the σ1 proteins of prototype strain T3D/55 and four type 3 field-isolate strains. The alignment was generated by using the program ALSCRIPT (5), with default conservation parameters applied according to the following color scheme: red, identical residues; orange, conserved residues at 80% conservation; yellow, conserved residues at 60% conservation; white, nonconserved residues. The 80% conservation threshold identifies closely related amino acids (e.g., Ile and Leu), whereas the 60% threshold identifies more distantly related amino acids (e.g., Ser and Ala, both of which have small side chains). The amino acid positions in the alignment are numbered above the sequences. The gray line indicates residues present in the crystallized fragment of T3D/55 σ1 (16). (B) Structure of the σ1 trimer, with residues colored according to the same color code as that used for panel A. Four different views are shown. For each of the views, two σ1 monomers are shown in surface representation, and the other is depicted as a blue ribbon tracing corresponding to the α-carbon backbone. The first three views each differ by 90° along a vertical axis; the fourth view shows the molecule in the third view after rotation by 90° along a horizontal axis. The positions of residues 340 and 419 are marked in the third panel from the left.

FIG. 6.

Sequence conservation and structural variability within the σ1 proteins of the three reovirus serotypes. (A) Alignment of deduced amino acid sequences of the σ1 proteins of 3 prototype and 10 field-isolate reovirus strains. The alignment was generated by using ALSCRIPT and the scheme described in the legend to Fig. 5. Gaps in the aligned sequences are indicated by dots. (B) Mapping of residues onto the σ1 structure, using the same color code as that depicted in Fig. 5. The four views correspond to those in Fig. 5B.

FIG. 6.

Sequence conservation and structural variability within the σ1 proteins of the three reovirus serotypes. (A) Alignment of deduced amino acid sequences of the σ1 proteins of 3 prototype and 10 field-isolate reovirus strains. The alignment was generated by using ALSCRIPT and the scheme described in the legend to Fig. 5. Gaps in the aligned sequences are indicated by dots. (B) Mapping of residues onto the σ1 structure, using the same color code as that depicted in Fig. 5. The four views correspond to those in Fig. 5B.

FIG. 7.

JAM-A is used as a receptor for a neutralization-resistant variant of reovirus T3D/55. HeLa cells at equivalent degrees of confluence were pretreated with PBS, an hCAR-specific antiserum, or the hJAM-A-specific MAb J10.4 prior to adsorption with variant K at an MOI of 1 FFU per cell. Reovirus proteins were detected by indirect immunofluorescence at 20 h postinfection. (A) Representative fields of view. (B) Reovirus-infected cells were quantified by counting fluorescent cells in three random fields of view per well for three wells. The results are presented as the mean FFU per field. Error bars indicate standard deviations.