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-1433Gly 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 ␣ v ␤ 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. RNA viruses mutate at rates of 10Ϫ3 to 10 Ϫ5 misincorporations per nucleotide copied; as a consequence, they evolve as complex mutant distributions termed viral quasispecies (17,19,34,35,51,52,54). Evolution of RNA viral quasispecies does not occur by the steady accumulation of mutations as replication proceeds but rather proceeds as the outcome of population disequilibrium in response to population size variations and environmental modifications. This is reflected in frequent fitness variations of RNA viruses as they replicate in cell culture or in vivo (3,12,18,27,29,33,42,64; reviewed in reference 16). Perturbation of equilibrium may lead to the rapid dominance of subsets of variants which were previously present at low frequency in the mutant spectrum. Expression at the cell surface of particular molecules which can act as receptors or coreceptors for the virus may have a major influence on the mutant distributions in viral quasispecies.Foot-and-mouth disease virus (FMDV) has been used in our laboratory as a model system to study viral quasispecies evolution, including the molecular basis of fitness variations (21, 22) and changes in host cell tropism (3,20). FMDV is an important animal pathogen that belongs to the aphthovirus genus of the Picornaviridae family (5, 55) and infects cattle and other cloven-hooved animals (artiodactyls) (2, 9). Integrin ␣ v ␤ 3 was the first molecule identified as a primary receptor for FMDV (4,6,24,38). Recent evidence suggests that integrin ␣ v ␤ 3 is the functional receptor for FMDV infections of cattle (50). The integrin ...

Evaluation of Genetically Engineered Derivatives of a Chinese Strain of Foot-and-Mouth Disease Virus Reveals a Novel Cell-Binding Site Which Functions in Cell Culture and in Animals

“…Evaluation of receptor binding by using synthetic peptides. The nature of the binding of VpB1-KGE to cells was further evaluated by utilizing a peptide inhibition of plaque formation protocol similar to that reported by Baranowski et al (2). The results of these studies, shown in Fig.…”

“…Specifically, passage of virus in cell culture selects viruses with an expanded host range in cell culture and with altered amino acid residues on their capsid surfaces. In the case of the Chinese virus, the changes were clustered around the fivefold axis of the virion, in a region previously shown to be altered by very high level passage of a type C virus in cell culture (2,3,18,39).…”

“…In addition, cell culture-adapted virus can use heparan sulfate (HS), a cell surface glycosaminoglycan (GAG), as a receptor in order to enter cells (26,48). Furthermore, Domingo and coworkers have reported data showing that derivatives of a type C virus passaged 100 times in cell culture appear to use multiple receptors, which could include molecules that are neither integrins nor HS (2,3,39). …”

A second RGD motif in the 1D capsid protein of a SAT1 type foot-and-mouth disease virus field isolate is not essential for attachment to target cells

“…However, it has been shown that FMDV type O virus strains that contained a KGE sequence in place of the naturally occurring integrin-binding RGD are able to replicate well in cell culture (Zhao et al, 2003), whilst type C viruses have been reported to be capable of initiating infection in the absence of the highly conserved RGD motif (Martinez et al, 1997 andBaranowski et al, 2000). In this study, evidence was obtained indicating RNA replication and virus assembly of the genetically engineered vNAMKGE/SAT2 virus, despite its non-infectious phenotype.…”

“…Tissue culture-adapted viruses can use heparan sulfate, a cell surface glycosaminoglycan, as a receptor to enter cells (Jackson et al, 1996 andSa-Carvalho et al, 1997). More recently, it has become apparent that FMDV may also use receptors of unknown identity that are neither integrin nor heparan sulfate (Martinez et al, 1997, Baranowski et al, 2000and Zhao et al, 2003, thus indicating the existence of other receptor molecules and alternative mechanisms for FMDV entry into cells.…”