Barry Baxt

The effect of peptides containing the arginine-glycine-aspartic acid sequence on the adsorption of foot-and-mouth disease virus to tissue culture cells

Sequencing of the VP1 of a large number of subtypes of foot-and-mouth disease virus (FMDV) has revealed the presence of a conserved arginine-glycine-aspartic acid (RGD) sequence located in a highly exposed region. This sequence has been shown to be essential for the interaction of certain extracellular matrix and adhesion proteins with a superfamily of cell-surface receptors called integrins. We have examined the effects of synthetic peptides containing the RGD sequence on the binding of eight different subtypes of FMDV to tissue culture cells. The results showed that such peptides inhibited viral adsorption by 50–80%. The inhibition was dose dependent but not as great as that achieved by using a saturating amount of virus as an inhibitor. Substitution of other amino acids for any of the three main residues lowered the inhibitory properties of the peptides. These results suggest that the RGD sequence in FMDV VP1 appears to be important for the interaction of virus with cellular receptor sites.

Continuous porcine kidney cell line constitutively expressing bovine αVβ6 integrin with increased susceptibility to foot and mouth disease virus

ABSTRACT Foot-and-mouth disease (FMD) is a worldwide problem limiting the trade of animals and their products from affected countries. The rapid isolation, serotyping, and vaccine matching of FMD virus from disease outbreaks is critical for enabling the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Some primary cells have been shown to be highly susceptible to most strains of FMD virus (FMDV) but are difficult and expensive to prepare and maintain. Since the αVβ6 integrin is a principal receptor for FMDV, we transduced a bovine kidney cell line to stably express both the αV and β6 bovine integrin subunits. This stable cell line (LFBK-αVβ6) showed β6 expression and enhanced susceptibility to FMDV infection for ≥100 cell passages. LFBK-αVβ6 cells were highly sensitive for detecting all serotypes of FMDV from experimentally infected animals, including the porcinophilic FMDV strain O/TAW/97. In comparison to other cell types that are currently used for virus isolation, LFBK-αVβ6 cells were more effective at detecting FMDV in clinical samples, supporting their use as a more sensitive tool for virus isolation.

Foot-and-mouth disease virus utilizes an autophagic pathway during viral replication

n Abstractn n Foot-and-mouth disease virus (FMDV) is the type species of the Aphthovirus genus within the Picornaviridae family. Infection of cells with positive-strand RNA viruses results in a rearrangement of intracellular membranes into viral replication complexes. The origin of these membranes remains unknown; however induction of the cellular process of autophagy is beneficial for the replication of poliovirus, suggesting that it might be advantageous for other picornaviruses. By using confocal microscopy we showed in FMDV-infected cells co-localization of non-structural viral proteins 2B, 2C and 3A with LC3 (an autophagosome marker) and viral structural protein VP1 with Atg5 (autophagy-related protein), and LC3 with LAMP-1. Importantly, treatment of FMDV-infected cell with autophagy inducer rapamycin, increased viral yield, and inhibition of autophagosomal pathway by 3-methyladenine or small-interfering RNAs, decreased viral replication. Altogether, these studies strongly suggest that autophagy may play an important role during the replication of FMDV.n n

Early interactions of foot-and-mouth disease virus with cultured cells.

Abstract The adsorption of foot-and-mouth disease virus (FMDV) types A 12 119, O 1B , and C 3Res were studied in baby hamster kidney (BHK-21) cells by measuring the amount of radioactively labeled purified virus which remained attached to cells at various times after infection. At 4°, over half of the maximum adsorption of type A virus occurred within 15 min, and approximately 65% of the radioactivity bound by 45 min was removed by brief treatment with EDTA, indicating the assay measures primarily adsorption and not penetration. Upon shifting the temperature from 4 to 37°, about 60 to 70% of the bound virus eluted by 1 hr in an unmodified form. Only 140 S virus particles and 12 S subunits could be found associated with the infected cell 20 min after shifting the temperature from 4 to 37°. By 50 min, the number of 140 S particles decreased slightly. The number of viral receptors per BHK-21 cell was determined to be 1–2.5 × 10 4 for virus types A 12 and O 1B . Competition experiments between FMDV types A 12 , O 1B , and C 3Res indicated that they can utilize at least some common receptor sites on cells.

Foot-and-mouth disease virion RNA: Studies on the relation between the length of its 3′-poly(A) segment and infectivity

Abstract Intact 37 S RNA was extracted from foot-and-mouth disease virus (FMDV) (type A12 strain 119) and fractionated into oligo(dT)-cellulose unbound and bound fractions. The RNA in each fraction appeared to contain no hidden breaks as determined by sucrose gradient centrifugation under denaturing conditions. Combined pancreatic RNase A and T1 digestion of [3H]adenosine-labeled unbound and bound virion RNA yielded, in both cases, a poly(A) fragment that migrated considerably faster than 4 S RNA during gel electrophoresis. The length of the poly(A) fragment of unbound virion RNA is less than 10 residues of adenosine, whereas the length of the poly(A) fragment of bound virion RNA contains a segment of approximately 40 residues at its 3′ end. The specific infectivity of unbound virion RNA is essentially the same as bound virion RNA.

Analysis of Foot-and-Mouth Disease Virus Integrin Receptor Expression in Tissues from Naive and Infected Cattle

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals principally affecting cattle, pigs and sheep. FMD virus (FMDV) uses the alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(6), and alpha(V)beta(8) integrins as receptors in vitro via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the betaG-betaH loop of VP1. Immunofluorescence and confocal microscopy were used to study the expression of two major FMDV receptors, alpha(V)beta(3) and alpha(V)beta(6), within epithelial tissues from FMDV-infected and uninfected cattle in order to understand the role of these receptors in tissue tropism. Integrin alpha(V)beta(6) was expressed by epithelial cells in tissues that are important sites for FMDV replication (i.e. tongue and coronary band). Integrin alpha(V)beta(3) was detected in epithelium of all tissues examined except tongue. In addition, alpha(V)beta(3) expression was associated with blood vessels in all tissues examined. In infected tissues, alpha(V)beta(6) integrin was distributed on the surface of those epithelial cells also expressing FMDV antigen. Although integrin alpha(V)beta(3) has been shown to be a receptor for FMDV, no expression of alpha(V)beta(3) was associated with FMDV-positive keratinocytes in the tongue. In contrast, podal epithelial cells containing FMDV antigen also expressed alpha(V)beta(3) integrin. Thus, at the cellular level the expression of these two integrins correlates with susceptibility to infection and may contribute substantially to viral tropism in FMD pathogenesis.

The adsorption and degradation of foot-and-mouth disease virus by isolated BHK-21 cell plasma membranes.

Abstract Foot-and-mouth disease virus (FMDV) was examined for its ability to adsorb specifically to plasma membranes isolated from BHK-21 cells. The membranes were prepared by the polyethylene glycol-dextran method, and characterized by increases in specific activity of ouabain-sensitive Na + K + -ATPase and 5′-nucleotidase, and by enrichment in 3 H-fucose over unfractionated homogenates. The membranes adsorbed purified radiolabeled FMDV type A 12 119 with kinetics characteristic of intact cells. Plasma membranes prepared from cells pretreated with trypsin were unable to adsorb virus. The adsorption of labeled FMDV was inhibited by unlabeled virus. Treatment of virus with trypsin, which cleaves capsid protein 3, greatly reduced its ability to adsorb to both plasma membranes and intact cells. After adsorption of virus to membranes at 4°, subsequent incubation at 37° under physiological conditions resulted in a rapid elution of bound virus in an unmodified form which reached approximately 80% by 1 hr. Incubation of the membrane-virus complex at 33° under low-salt conditions degraded the virus particles to intact and fragmented viral RNA and 12 S protein subunits. Membrane-induced viral degradation did not occur at 4° but was observed within 5 min after shifting to 33°. Thus, isolated plasma membranes from BHK-21 cells retain receptors for FMDV possessing uncleaved capsid protein 3. In addition, the eclipse and uncoating of FMDV in intact cells probably occurs at the plasma membrane, and in confirmation of previously reported results, the postadsorptive degradation, unlike that of other picornaviruses, occurs in a single step without the production of intermediate subviral particles.

Translation of foot-and-mouth disease virion RNA and processing of the primary cleavage products in a rabbit reticulocyte lysate

Abstract Foot-and-mouth disease (FMD) virion RNA was translated completely in a micrococcal nuclease-treated rabbit reticulocyte cell-free system. The proteins synthesized in vitro were identified (1) on polyacrylamide gels by comparison with virus-specific proteins synthesized in infected cells and authentic virion proteins and (2) on cation exchange columns by comparison of their tryptic peptides to those of virion proteins. The kinetics of appearance of the in vitro synthesized polypeptides was similar to that reported in vivo and the primary products were processed efficiently into virus-specific proteins P56, P43 (VP 0 ), P37, P22, and P12. Processing of primary products P50 and P93 was not as efficient in the absence of exogenous tRNA as in its presence, and only occurred after translation of a region of the genome distal to these polypeptides. The synthesis of an active proteolytic processing enzyme, apparently coded for by FMD virion RNA, was demonstrated.

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

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.

Correction for LaRocco et al., A Continuous Bovine Kidney Cell Line Constitutively Expressing Bovine αVβ6 Integrin Has Increased Susceptibility to Foot-and-Mouth Disease Virus

Volume 51, no. 12, p. [1714–1720][1], 2012. After the initial distribution of the LFBK-αVβ6 cell line, studies were conducted to further characterize the cell line. These studies revealed that the LFBK parental cell line and the transduced LFBK-αVβ6 cell line are of porcine genotype and not of