Foot-and-mouth disease virus virulent for cattle utilizes the integrin alpha(v)beta3 as its receptor

Abstract

Adsorption and plaque formation of foot-and-mouth disease virus (FMDV) serotype A12 are inhibited by antibodies to the integrin alpha(v)beta3 (A. Berinstein et al., J. Virol. 69:2664-2666, 1995). A human cell line, K562, which does not normally express alpha(v)beta3 cannot replicate this serotype unless cells are transfected with cDNAs encoding this integrin (K562-alpha(v)beta3 cells). In contrast, we found that a tissue culture-propagated FMDV, type O1BFS, was able to replicate in nontransfected K562 cells, and replication was not inhibited by antibodies to the endogenously expressed integrin alpha5beta1. A recent report indicating that cell surface heparan sulfate (HS) was required for efficient infection of type O1 (T. Jackson et al., J. Virol. 70:5282-5287, 1996) led us to examine the role of HS and alpha(v)beta3 in FMDV infection. We transfected normal CHO cells, which express HS but not alpha(v)beta3, and two HS-deficient CHO cell lines with cDNAs encoding human alpha(v)beta3, producing a panel of cells that expressed one or both receptors. In these cells, type A12 replication was dependent on expression of alpha(v)beta3, whereas type O1BFS replicated to high titer in normal CHO cells but could not replicate in HS-deficient cells even when they expressed alpha(v)beta3. We have also analyzed two genetically engineered variants of type O1Campos, vCRM4, which has greatly reduced virulence in cattle and can bind to heparin-Sepharose columns, and vCRM8, which is highly virulent in cattle and cannot bind to heparin-Sepharose. vCRM4 replicated in wild-type K562 cells and normal, nontransfected CHO (HS+ alpha(v)beta3-) cells, whereas vCRM8 replicated only in K562 and CHO cells transfected with alpha(v)beta3 cDNAs. A similar result was also obtained in assays using a vCRM4 virus with an engineered RGD-->KGE mutation. These results indicate that virulent FMDV utilizes the alpha(v)beta3 integrin as a primary receptor for infection and that adaptation of type O1 virus to cell culture results in the ability of the virus to utilize HS as a receptor and a concomitant loss of virulence.

FIG. 1

Analysis of viral proteins synthesized in FMDV-infected K562 cells transfected with α_vβ₃ cDNAs. K562-pRSV (C) or K562-α_vβ₃ (T) cells were infected with type A₁₂, O₁BFS, vCRM4, or vCRM8 at an MOI of 10 PFU/cell (a) or type A₁₂ or vRM-SSP at an MOI of 1 PFU/cell (b). Cells were labeled between 3 and 24 h after infection with [³⁵S]methionine, and extracts were analyzed by RIP and SDS-PAGE as described in Materials and Methods. Viral proteins synthesized in infected and labeled BHK-21 cells are included as markers (M), and the positions of major viral proteins are indicated on the left. Type A₁₂ was used as the marker for panel b.

FIG. 2

Analysis of CHO cells transfected with α_vβ₃ cDNAs. CHO-K1 cells and the two GAG-deficient mutants pgsA-745 and pgsD-677 were transfected with α_vβ₃ cDNAs and selected as described in Materials and Methods. After two to three passages, transfected and nontransfected cells were incubated with MAb LM609 for 30 min at 4°C in phosphate-buffered saline, washed, and incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG for an additional 30 min at 4°C. After being washed with phosphate-buffered saline, cells were analyzed on a Becton Dickson FACSCaliber analyzer. Nontransfected cells are represented by open curves, and transfected cells are represented by closed curves.

FIG. 3

Analysis of viral proteins synthesized in FMDV-infected CHO cells transfected with α_vβ₃ cDNAs. Transfected (T) or nontransfected (C) CHO-K1, pgsA-745, or pgsD-677 cells were infected with FMDV type A₁₂, O₁BFS, vCRM4, or vCRM48-KGE at an MOI of 10 PFU/cell. Cells were labeled between 3 and 24 h after infection with [³⁵S]methionine, and extracts were analyzed by RIP and SDS-PAGE as described in Materials and Methods. Viral proteins synthesized in infected and labeled BHK-21 cells are included as markers (M), and the positions of major viral proteins are indicated on the left.

FIG. 4

Analysis of viral proteins synthesized in FMDV-infected CHO cells transfected with α_vβ₃ cDNAs. Transfected (T) or nontransfected (C) CHO-K1, pgsA-745, or pgsD-677 cells were infected with FMDV type vCRM8 or O₁BFS at an MOI of 10 PFU/cell (a) or vRM-SSP at an MOI of 1 PFU/cell (b). Cells were labeled between 24 and 48 h after infection with [³⁵S]methionine, and extracts were analyzed by RIP and SDS-PAGE as described in Materials and Methods. Viral proteins synthesized in infected and labeled BHK-21 cells are included as markers (M), and the positions of major viral proteins are indicated on the left.

FIG. 5

Heparin neutralization of FMDV. Different concentrations of heparin (Sigma) were mixed with 35 to 100 PFU of the indicated viruses in basal medium Eagle with 25 mM HEPES (pH 7.5) and 0.05% bovine serum albumin and incubated for 20 min at room temperature. Aliquots (200 μl) were adsorbed to BHK-21 cells for 1 h at 37°C. The inoculum was aspirated, and the plates overlaid with a mixture of MEM and 0.6% gum tragacanth and incubated at 37°C. Plates were stained with crystal violet-formalin at 48 h (vCRM8) or 72 h (vCRM4 and vCRM48-KGE). Results are expressed as plaque numbers relative to the number of plaques appearing in virus incubated under the same conditions without heparin.