Examination of soluble integrin resistant mutants of foot-and-mouth disease virus

Background: Foot-and-mouth disease virus (FMDV) initiates infection via recognition of one of at least four cell-surface integrin molecules a(v)beta(1), a(v)beta(3), a(v)beta(6), or a(v)beta(8) by a highly conserved Arg-Gly-Asp (RGD) amino acid sequence motif located in the G-H loop of VP1. Within the animal host, the a(v)beta(6) interaction is believed to be the most relevant. Sub-neutralizing levels of soluble secreted a(v)beta(6) (ssa(v)beta(6)) was used as a selective pressure during passages in vitro to explore the plasticity of that interaction. Results: Genetically stable soluble integrin resistant (SIR) FMDV mutants derived from A24 Cruzeiro were selected after just 3 passages in cell culture in the presence of sub-neutralizing levels of ssa(v)beta(6). SIR mutants were characterized by: replication on selective cell lines, plaque morphology, relative sensitivity to ssa(v)beta(6) neutralization, relative ability to utilize a(v)beta(6) for infection, as well as sequence and structural changes. All SIR mutants maintained an affinity for a(v)beta(6). Some developed the ability to attach to cells expressing heparan sulfate (HS) proteoglycan, while others appear to have developed affinity for a still unknown third receptor. Two classes of SIR mutants were selected that were highly or moderately resistant to neutralization by ssa(v)beta(6). Highly resistant mutants displayed a G145D substitution (RGD to RDD), while moderately resistant viruses exhibited a L150P/R substitution at the conserved RGD + 4 position. VP1 G-H loop homology models for the A-type SIR mutants illustrated potential structural changes within the integrin-binding motif by these 2 groups of mutations. Treatment of O1 Campos with ssa(v)beta(6) resulted in 3 SIR mutants with a positively charged VP3 mutation allowing for HS binding. Conclusions: These findings illustrate how FMDV particles rapidly gain resistance to soluble receptor prophylactic measures in vitro. Two different serotypes developed distinct capsid mutations to circumvent the presence of sub-neutralizing levels of the soluble cognate receptor, all of which resulted in a modified receptor tropism that expanded the cell types susceptible to FMDV. The identification of some of these adaptive mutations in known FMDV isolates suggests these findings have implications beyond the cell culture system explored in these studies.