Structure of the complex of an Fab fragment of a neutralizing antibody with foot-and-mouth disease virus: positioning of a highly mobile antigenic loop

Bluetongue virus (BTV) is an arthropod-borne virus transmitted by Culicoides species to vertebrate hosts.The double-capsid virion is infectious for Culicoides vector and mammalian cells, while the inner core is infectious for only Culicoides-derived cells. The recently determined crystal structure of the BTV core has revealed an accessible RGD motif between amino acids 168 to 170 of the outer core protein VP7, whose structure and position would be consistent with a role in cell entry. To delineate the biological role of the RGD sequence within VP7, we have introduced point mutations in the RGD tripeptide and generated three recombinant baculoviruses, each expressing a mutant derivative of VP7 (VP7-AGD, VP7-ADL, and VP7-AGQ). Each expressed mutant protein was purified, and the oligomeric nature and secondary structure of each was compared with those of the wild-type (wt) VP7 molecule. Each mutant VP7 protein was used to generate empty core-like particles (CLPs) and were shown to be biochemically and morphologically identical to those of wt CLPs. However, when mutant CLPs were used in an in vitro cell binding assay, each showed reduced binding to Culicoides cells compared to wt CLPs. Twelve monoclonal antibodies (MAbs) was generated using purified VP7 or CLPs as a source of antigen and were utilized for epitope mapping with available chimeric VP7 molecules and the RGD mutants. Several MAbs bound to the RGD motif on the core, as shown by immunogold labeling and cryoelectron microscopy. RGD-specific MAb H1.5, but not those directed to other regions of the core, inhibited the binding activity of CLPs to the Culicoides cell surface. Together, these data indicate that the RGD motif present on BTV VP7 is responsible for Culicoides cell binding activity.Orbiviruses (within the family Reoviridae), are vectored to vertebrate species by arthropods (e.g., gnats, mosquitoes and ticks) and are able to replicate in both hosts. Bluetongue virus (BTV) is the prototype virus of the genus and is transmitted by gnats (Culicoides species), causing diseases of economic importance in ruminants in many parts of the world. Vector-virus interactions play a crucial role in vector-borne disease epidemiology. The spread of Culicoides species from BTV-endemic to non-BTV (or related African horsesickness virus, AHSV, and epizootic hemorrhagic disease virus EHDV, of deer) regions of the world in the past highlights the concern that these viruses are a threat to regions of the world that are presently free from them.The initiation of a virus infection involves virus binding to ligands on the cell surface prior to cell entry by a number of mechanisms (depending on the virus). Like many other viruses, BTV appears to utilize a protein molecule(s) of mammalian cells as a receptor (20); however, it is also possible that alternative receptors may be utilized in different tissues and in different species and as accessory molecules.BTV has a genome composed of 10 segments of doublestranded RNA packaged within a double icosahedral capsid. The outer capsi...

Section: Resultsmentioning

confidence: 99%

RGD Tripeptide of Bluetongue Virus VP7 Protein Is Responsible for Core Attachment to Culicoides Cells

Tan

Nason²,

Staeuber

et al. 2001

“…Studies of sitedirected mutagenesis of infectious cDNA copies of the FMDV genome (40, 44, 49), inhibition of infectivity by synthetic peptides (48), and binding of antibodies to substituted peptides (63) have defined those amino acid residues which are involved in cell receptor recognition and antibody binding. In FMDV of serotype C (clone C-S8c1, derived from natural isolate C-Sta Pau Sp/70 [59]), the RGD motif is directly involved in both integrin recognition (30,48) and binding of several neutralizing antibodies (31,56,(61)(62)(63). In spite of being subjected to strong selective pressure by antibodies, the RGD triplet was invariant among natural FMDV isolates, in populations of FMDV C-S8c1 subjected to intense selection by neutralizing antibodies (8), and among 81 monoclonal antibody (MAb) escape mutants of FMDV C-S8c1 (43,46,47).…”

Section: Rna Viruses Mutate At Rates Of 10mentioning

confidence: 99%

Cell Recognition by Foot-and-Mouth Disease Virus That Lacks the RGD Integrin-Binding Motif: Flexibility in Aphthovirus Receptor Usage

Baranowski

Ruiz-Jarabo

Sevilla

et al. 2000

150

179

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 ...

“…Receptor binding sites may be found in cavities that are not readily accessible to antibodies (5,50) or sequestered within structures that are not exposed until after binding of another receptor (31). In other cases, the receptor binding sites appear to be exposed on the surface of the virus and to overlap substantially with antibody binding sites that have been defined (16,23). Parvoviruses have small, 260-Å-diameter, icosahedral, nonenveloped capsids that package a single-stranded DNA genome of about 5 kb.…”

mentioning

confidence: 99%

Structural Comparison of Different Antibodies Interacting with Parvovirus Capsids

Hafenstein

Bowman

Sun

et al. 2009

107

The structures of canine parvovirus (CPV) and feline parvovirus (FPV) complexed with antibody fragments from eight different neutralizing monoclonal antibodies were determined by cryo-electron microscopy (cryoEM) reconstruction to resolutions varying from 8.5 to 18 Å. The crystal structure of one of the Fab molecules and the sequence of the variable domain for each of the Fab molecules have been determined. The structures of Fab fragments not determined crystallographically were predicted by homology modeling according to the amino acid sequence. Fitting of the Fab and virus structures into the cryoEM densities identified the footprints of each antibody on the viral surface. As anticipated from earlier analyses, the Fab binding sites are directed to two epitopes, A and B. The A site is on an exposed part of the surface near an icosahedral threefold axis, whereas the B site is about equidistant from the surrounding five-, three-, and twofold axes. One antibody directed to the A site binds CPV but not FPV. Two of the antibodies directed to the B site neutralize the virus as Fab fragments. The differences in antibody properties have been linked to the amino acids within the antibody footprints, the position of the binding site relative to the icosahedral symmetry elements, and the orientation of the Fab structure relative to the surface of the virus. Most of the exposed surface area was antigenic, although each of the antibodies had a common area of overlap that coincided with the positions of the previously mapped escape mutations.