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

Paul Lawrence,Michael Larocco,Elizabeth Rieder,Barry Baxt

doi:10.1186/1743-422x-10-2

Paul Lawrence, Michael Larocco + Show 2 more

Open Access

https://doi.org/10.1186/1743-422x-10-2

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Abstract

BackgroundFoot-and-mouth disease virus (FMDV) initiates infection via recognition of one of at least four cell-surface integrin molecules αvβ1, αvβ3, αvβ6, or αvβ8 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 αvβ6 interaction is believed to be the most relevant. Sub-neutralizing levels of soluble secreted αvβ6 (ssαvβ6) was used as a selective pressure during passages in vitro to explore the plasticity of that interaction.ResultsGenetically 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 ssαvβ6. SIR mutants were characterized by: replication on selective cell lines, plaque morphology, relative sensitivity to ssαvβ6 neutralization, relative ability to utilize αvβ6 for infection, as well as sequence and structural changes. All SIR mutants maintained an affinity for αvβ6. 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 ssαvβ6. 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 ssαvβ6 resulted in 3 SIR mutants with a positively charged VP3 mutation allowing for HS binding.ConclusionsThese 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.

Highlights

Foot-and-mouth disease virus (FMDV) is responsible for the most economically important viral disease of cattle and other cloven-hoofed animals [1,2,3,4,5]
The bovine kidney LFBK cell line was selected for these studies on the basis that it is susceptible to infection by all 7 FMDV serotypes, similar to primary bovine kidney cell culture and [29]
RNA Helicase A (RHA), known to be expressed in all 3 cell lines, was detected. These findings allowed for the potential that both αvβ3 and αvβ6 could be found on the surface of LFBK cells, which is consistent with repeated experiments showing the susceptibility of this cell line to non-heparan sulfate (HS)-adapted field isolates of FMDV

Summary

Introduction

Foot-and-mouth disease virus (FMDV) is responsible for the most economically important viral disease of cattle and other cloven-hoofed animals [1,2,3,4,5]. FMDV, the prototypic member of the Aphthovirus genus of Picornaviridae, utilizes in vitro four integrin heterodimers (αvβ, αvβ, αvβ, and αvβ8) for attachment to host cells and entry via clathrincoated pits (CCPs) [6,7,8,9,10,11,12,13,14]. Capsid protein contains a highly conserved Arg-Gly-Asp (RGD) motif, a recognition sequence for the αv-integrin family of cell surface receptors [6,15,16,17]. Foot-and-mouth disease virus (FMDV) initiates infection via recognition of one of at least four cell-surface integrin molecules αvβ, αvβ, αvβ, or αvβ by a highly conserved Arg-Gly-Asp (RGD) amino acid sequence motif located in the G-H loop of VP1. Sub-neutralizing levels of soluble secreted αvβ (ssαvβ6) was used as a selective pressure during passages in vitro to explore the plasticity of that interaction

Results

Discussion

Conclusion