Cell recognition by foot-and-mouth disease virus that lacks the RGD integrin-binding motif: flexibility in aphthovirus receptor usage

Abstract

Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-143-->Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin alpha(v)beta(3). In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type.

FIG. 1

Schematic representation of the capsid-coding region of chimeric FMDVs. Genomic regions of FMDV of serotype C (white boxes) are inserted in the full-length cDNA of type O1K FMDV (3, 65). Origins of the viruses used for construction of chimeric DNA and procedures employed for the preparation of plasmids are detailed in Materials and Methods. Restriction sites and numbering refer to the C-S8c1 genome (21). Positions at which amino acid residues differ among the compared chimeric genomes are indicated at the bottom. The amino acid sequence of protein VP4 is conserved among the FMDVs of serotypes O and C analyzed here. Boldface letters correspond to amino acid residues which differ from the parental C-S8c1.

FIG. 2

Replication of recombinant FMDV variants in BHK-21 cells. Monolayers of BHK-21 cells were infected with chimeric viruses harboring the capsid of C-S8c1 (A), C-S8c1p100 and its RGG derivative (B), or C-S8c1p213 and its RGG derivative (C) at a multiplicity of infection of 5 PFU per cell. Procedures for the infection in liquid culture and titration of infectivity are described in Materials and Methods. Each value represents the mean of triplicate assays.

FIG. 3

Replication of recombinant FMDV variants in K-562 cells. Cells were infected with the indicated chimeric viruses at a multiplicity of infection of 2 PFU per cell. Procedures for infection of the suspension cultures and titration of infectivity are described in Materials and Methods. Each value represents the mean of duplicate assays.

FIG. 4

Replication in BHK-21 cells of FMDV clones unable to bind heparin. p100c10 and p100RGG denote FMDV C-S8c1p100c10 and C-S8c1p100RGG, respectively; their origins are described in Materials and Methods. The five clones selected for the inability to bind heparin, termed hs-c1 through hs-c5, were selected as detailed in the text (3). Infections of BHK-21 cell monolayers were carried out at a multiplicity of infection of 5 PFU per cell. Procedures for infection in liquid culture and titration of infectivity are described in Materials and Methods. One representative experiment of three is shown for the parental p100c10 and p100RGG viruses.

FIG. 5

Inhibition of FMDV infectivity by peptides A15 and A15 with RGG instead of RGD. The amino acid sequences of peptides A15 and A15-RGG are given in Materials and Methods. BHK-21 cell monolayers (about 5 × 10⁵ cells) were treated with the indicated concentration of peptide A15 or A15-RGG for 45 min at room temperature and further incubated in the presence of FMDV (50 to 150 PFU) for 45 min at 37°C. The monolayers were washed with 0.1 M phosphate buffer (pH 6.0) and overlaid with agar containing medium as described in Materials and Methods. The FMDVs used in different experiments are listed in the right; hs- denotes a clone selected for its inability to bind heparin. Origins of the viruses and procedures for selection of clones and infectivity assays are described in Materials and Methods. Each value is the average of three determinations. Standard deviations are indicated.