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. 2006 May;80(9):4482-90.
doi: 10.1128/JVI.80.9.4482-4490.2006.

Junctional adhesion molecule 1 is a functional receptor for feline calicivirus

Affiliations

Junctional adhesion molecule 1 is a functional receptor for feline calicivirus

Akiko Makino et al. J Virol. 2006 May.

Abstract

The life cycle of calicivirus is not fully understood because most of the viruses cannot be propagated in tissue culture cells. We studied the mechanism of calicivirus entry into cells using feline calicivirus (FCV), a cultivable calicivirus. From the cDNA library of Crandell-Rees feline kidney (CRFK) cells, feline junctional adhesion molecule 1 (JAM-1), an immunoglobulin-like protein present in tight junctions, was identified as a cellular-binding molecule of the FCV F4 strain, a prototype strain in Japan. Feline JAM-1 expression in nonpermissive hamster lung cells led to binding and infection by F4 and all other strains tested. An anti-feline JAM-1 antibody reduced the binding of FCV to permissive CRFK cells and strongly suppressed the cytopathic effect (CPE) and FCV progeny production in infected cells. Some strains of FCV, such as F4 and F25, have the ability to replicate in Vero cells. We found that regardless of replication ability, FCV bound to Vero and 293T cells via simian and human JAM-1, respectively. In Vero cells, an anti-human JAM-1 antibody inhibited binding, CPE, and progeny production by F4 and F25. In addition, feline JAM-1 expression permitted FCV infection in 293T cells. Taken together, our results demonstrate that feline JAM-1 is a functional receptor for FCV, simian JAM-1 also functions as a receptor for some strains of FCV, and the interaction between FCV and JAM-1 molecules may be a determinant of viral tropism. This is the first report concerning a functional receptor for the viruses in the family Caliciviridae.

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Figures

FIG. 1.
FIG. 1.
Nucleotide and deduced amino acid sequences of fJAM-1 cDNA. The putative hydrophobic signal peptide and transmembrane sequences are underlined. Four cystein residues that are predicted to form Ig-like domains are boxed. The putative N-linked glycosylation site is marked in boldface and underlined.
FIG. 2.
FIG. 2.
Detection of JAM-1 molecules on the cell surface by flow cytometry using anti-JAM-1 antibody. Cells used in this assay were Vero, 293T, CRFK, and HmLu-1 cells stably transduced with pcDNA3.1-fJAM-1 (HmLu-1-fJAM-1) or vector alone (HmLu-1-pcDNA). Cells were incubated with anti-JAM-1 antibody (shaded area), control antibody (thick line), or buffer only (thin line). Cells were incubated with a second antibody and analyzed by flow cytometry. (A) Detection of JAM-1 on cells using anti-fJAM-1 antibody. (B) Detection of JAM-1 on cells using anti-hJAM-1 antibody.
FIG. 3.
FIG. 3.
FCV-binding assay by flow cytometry. HmLu-1-fJAM-1 or HmLu-1-pcDNA cells were incubated with four strains of FCV (thick line) or with buffer only (thin line). Viral antigens on cells were detected with an anti-capsid MAb by flow cytometry.
FIG. 4.
FIG. 4.
Binding inhibition assay by flow cytometry using anti-fJAM-1 and anti-hJAM-1 antibodies. (A) CRFK cells were incubated with anti-fJAM-1 antibody (shaded area) or control normal mouse serum (thick line) prior to incubation with FCV. In parallel, cells were incubated with buffer only (thin line). Vero (B) and 293T (C) cells were preincubated with anti-hJAM-1 antibody (shaded area) or normal goat IgG (thick line). FCV capsids were detected on the cells by flow cytometry.
FIG.5.
FIG.5.
FCV infection in nonpermissive cells expressing fJAM-1. (A) HmLu-1-fJAM-1 or HmLu-1-pcDNA cells were inoculated with FCV F4 at a multiplicity of infection of 10 and fixed with acetone at 1, 12, 24, and 48 h postinfection. FCV antigens were detected by IFA. (B) 293T cells transduced with pcDNA3.1-fJAM-1 (293T-fJAM-1) or vector alone (293T-pcDNA) were inoculated with FCV F4 and fixed with acetone at 24 h postinfection. FCV antigens were detected by IFA. (C) Viral titers in supernatants of HmLu-1-fJAM-1 and HmLu-1-pcDNA cells at 24 h postinfection. Viral titers are expressed as the mean of three independent experiments. *, P < 0.05, and **, P < 0.01, compared to the supernatants of control cells.
FIG.5.
FIG.5.
FCV infection in nonpermissive cells expressing fJAM-1. (A) HmLu-1-fJAM-1 or HmLu-1-pcDNA cells were inoculated with FCV F4 at a multiplicity of infection of 10 and fixed with acetone at 1, 12, 24, and 48 h postinfection. FCV antigens were detected by IFA. (B) 293T cells transduced with pcDNA3.1-fJAM-1 (293T-fJAM-1) or vector alone (293T-pcDNA) were inoculated with FCV F4 and fixed with acetone at 24 h postinfection. FCV antigens were detected by IFA. (C) Viral titers in supernatants of HmLu-1-fJAM-1 and HmLu-1-pcDNA cells at 24 h postinfection. Viral titers are expressed as the mean of three independent experiments. *, P < 0.05, and **, P < 0.01, compared to the supernatants of control cells.
FIG. 6.
FIG. 6.
Effect of anti-fJAM-1 antibody on FCV infection in CRFK cells. CRFK cells were incubated with the anti-fJAM-1 antibody or control normal mouse serum prior to inoculation with FCV at a multiplicity of infection of 0.1. After antibody and inocula were removed, cells were added to fresh medium and incubated for 0 h or 2 days. Cells were observed, and virus titration was performed. (A) CRFK cells at 2 days postinfection or mock infection. (B) Viral titers in supernatants of CRFK cells at 0 h or 2 days postinfection. Viral titers are expressed as the mean of three independent experiments. **, P < 0.01, compared to the supernatants of CRFK cells incubated with anti-fJAM-1 antibody at 2 days postinfection.
FIG. 7.
FIG. 7.
Effect of anti-hJAM-1 antibody on FCV infection in Vero cells. Vero cells were incubated with anti-hJAM-1 antibody or normal goat IgG prior to inoculation with FCV F4 and F25 strains at a multiplicity of infection of 1. After antibody and inocula were removed, cells were added to fresh medium and incubated for 0 h or 4 days. Cells were observed, and virus titration was performed. (A) Vero cells at 4 days postinfection or mock infection. (B) Viral titers in supernatants of Vero cells at 0 h or 4 days postinfection. Viral titers are expressed as the mean of three independent experiments. **, P < 0.01, compared to the supernatants of Vero cells incubated with anti-hJAM-1 antibody at 4 days postinfection.
FIG. 8.
FIG. 8.
A comparison of JAM-1 amino acid sequences of cats, monkeys, and humans. The putative signal peptide and transmembrane domain are underlined. Simian and human JAM-1 had a high identity of 96.7%, while fJAM-1 had 75.6% sequence identity with hJAM-1 and 76.6% sequence identity with sJAM-1. Two amino acid residues (S91 and K155) in extracellular domains of JAM-1 that were conserved between cats and monkeys, but not in humans, are boxed.

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