Utilization of DC-SIGN for entry of feline coronaviruses into host cells - PubMed (original) (raw)

Utilization of DC-SIGN for entry of feline coronaviruses into host cells

Andrew D Regan et al. J Virol. 2008 Dec.

Abstract

The entry and dissemination of viruses in several families can be mediated by C-type lectins such as DC-SIGN. We showed that entry of the serotype II feline coronavirus strains feline infectious peritonitis virus (FIPV) WSU 79-1146 and DF2 into nonpermissive mouse 3T3 cells can be rescued by the expression of human DC-SIGN (hDC-SIGN) and that infection of a permissive feline cell line (Crandall-Reese feline kidney) was markedly enhanced by the overexpression of hDC-SIGN. Treatment with mannan considerably reduced infection of feline monocyte-derived cells expressing DC-SIGN, indicating a role for FIPV infection in vivo.

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Figures

FIG. 1.

FIG. 1.

Expression of hDC-SIGN rescues FCoV infection of nonpermissive cells. (A) 3T3, 3T3-DCSIGN, or CRFK cells were infected with FIPV-1146 or FIPV-DF2 at a MOI of 10. Cells were rinsed and placed in fresh media after 2 h. At 24 h, cells and the supernatants were collected. The production of virus was determined from at least three independent replicates of the experiment by 50% tissue culture infective dose (TCID50) assay. Error bars represent the standard deviations of the means. (B) 3T3 cells were transfected with pCDNA3-hDCSIGN. Twenty-four hours posttransfection, cells were infected with virus at a MOI of 10. Cells were fixed 9 h postinfection and stained for immunofluorescence microscopy with the IgG2a anti-hDC-SIGN MAb 120526 and the IgG2b anti-FCoV nucleocapsid (anti-FIPV N) MAb 17B7.1.

FIG. 2.

FIG. 2.

Expression of hDC-SIGN enhances FCoV infection of permissive cells. (A) CRFK or CRFK-hDCSIGN cells were infected with FIPV-1146 or FIPV-DF2 at a MOI of 0.01. Six hours postinfection, cells were fixed and stained for immunofluorescence microscopy with the IgG2a anti-hDC-SIGN MAb 120526 and the IgG2b anti-FCoV nucleocapsid (anti-FIPV N) MAb 17B7.1. (B) CRFK or CRFK-hDCSIGN cells were infected with FIPV-1146 or FIPV-DF2 at the indicated concentrations. Six hours postinfection, cells were fixed and stained for immunofluorescence microscopy with the anti-FCoV nucleocapsid MAb 17B7.1. Images from at least three independent experiments were captured and quantified. For quantification, >1,000 cells from three independent replicates of each experimental condition were scored. Error bars represent the standard deviations of the means. TCID50, 50% tissue culture infective dose.

FIG. 3.

FIG. 3.

Mannan and anti-hDC-SIGN MAbs block the enhancement of infection by FCoVs. CRFK, CRFK-hDCSIGN, or CRFK-hDCSIGN cells pretreated with either mannan (50 μg/ml), anti-hDC-SIGN MAb 9E9A8 (20 μg/ml), or anti-hDC-SIGN MAb 120526 (20 μg/ml) were infected with FIPV-1146 or FIPV-DF2 at a MOI of 0.01. Cells were fixed at 6 h postinfection and stained for immunofluorescence microscopy with the anti-FCoV nucleocapsid MAb 17B7.1. Images from at least three independent experiments were captured and quantified. For quantification, >1,000 cells from three independent replicates of each experimental condition were scored. Error bars represent the standard deviations of the means.

FIG. 4.

FIG. 4.

Mannan inhibits FCoV infection of primary feline monocyte-derived cells expressing DC-SIGN. (A) Primary feline cells expressing DC-SIGN were pretreated with mannan (50 μg/ml), anti-fAPN MAb R-G-4 (200 μg/ml), or both in combination for 60 min before being infected with FIPV-1146 or FIPV-DF2 at a MOI of 10. Cells were fixed 12 h postinfection and stained for immunofluorescence microscopy with the anti-FCoV nucleocapsid (anti-FCoV N) MAb 17B7.1. (B) Images from at least three independent experiments were captured and quantified. For quantification, >250 cells from three independent replicates of each experimental condition were scored. Error bars represent the standard deviations of the means.

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