Loop Of VP1 Research Articles

Virion Structure of Black Queen Cell Virus, a Common Honeybee Pathogen.

ABSTRACTViral diseases are a major threat to honeybee (Apis mellifera) populations worldwide and therefore an important factor in reliable crop pollination and food security. Black queen cell virus (BQCV) is the etiological agent of a fatal disease of honeybee queen larvae and pupae. The virus belongs to the genus Triatovirus from the family Dicistroviridae, which is part of the order Picornavirales. Here we present a crystal structure of BQCV determined to a resolution of 3.4 Å. The virion is formed by 60 copies of each of the major capsid proteins VP1, VP2, and VP3; however, there is no density corresponding to a 75-residue-long minor capsid protein VP4 encoded by the BQCV genome. We show that the VP4 subunits are present in the crystallized virions that are infectious. This aspect of the BQCV virion is similar to that of the previously characterized triatoma virus and supports the recent establishment of the separate genus Triatovirus within the family Dicistroviridae. The C terminus of VP1 and CD loops of capsid proteins VP1 and VP3 of BQCV form 34-Å-tall finger-like protrusions at the virion surface. The protrusions are larger than those of related dicistroviruses.IMPORTANCE The western honeybee is the most important pollinator of all, and it is required to sustain the agricultural production and biodiversity of wild flowering plants. However, honeybee populations worldwide are suffering from virus infections that cause colony losses. One of the most common, and least known, honeybee pathogens is black queen cell virus (BQCV), which at high titers causes queen larvae and pupae to turn black and die. Here we present the three-dimensional virion structure of BQCV, determined by X-ray crystallography. The structure of BQCV reveals large protrusions on the virion surface. Capsid protein VP1 of BQCV does not contain a hydrophobic pocket. Therefore, the BQCV virion structure provides evidence that capsid-binding antiviral compounds that can prevent the replication of vertebrate picornaviruses may be ineffective against honeybee virus infections.

Enterovirus D68 Infections Associated with Severe Respiratory Illness in Elderly Patients and Emergence of a Novel Clade in Hong Kong.

Despite the recent emergence of enterovirus D68 (EV-D68), its clinical impact on adult population is less well defined. To better define the epidemiology of EV-D68, 6,800 nasopharyngeal aspirates (NPAs) from 2010–2014 were subject to EV-D68 detection by RT-PCR and sequencing of 5′UTR and partial VP1. EV-D68 was detected in 30 (0.44%) NPAs from 22 children and 8 adults/elderlies. Sixteen patients (including five elderly) (53%) had pneumonia and 13 (43%) patients were complicated by small airway disease exacerbation. Phylogenetic analysis of VP1, 2C and 3D regions showed four distinct lineages of EV-D68, clade A1, A2, B1 and B3, with adults/elderlies exclusively infected by clade A2. The potentially new clade, B3, has emerged in 2014, while strains closely related to recently emerged B1 strains in the United States were also detected as early as 2011 in Hong Kong. The four lineages possessed distinct aa sequence patterns in BC and DE loops. Amino acid residues 97 and 140, within BC and DE-surface loops of VP1 respectively, were under potential positive selection. EV-D68 infections in Hong Kong usually peak in spring/summer, though with a delayed autumn/winter peak in 2011. This report suggests that EV-D68 may cause severe respiratory illness in adults/elderlies with underlying co-morbidities.

Insert engineering and solubility screening improves recovery of virus-like particle subunits displaying hydrophobic epitopes.

The Polyomavirus coat protein, VP1 has been developed as an epitope presentation system able to provoke humoral immunity against a variety of pathogens, such as Influenza and Group A Streptococcus. The ability of the system to carry cytotoxic T cell epitopes on a surface-exposed loop and the impact on protein solubility has not been examined. Four variations of three selected epitopes were cloned into surface-exposed loops of VP1, and expressed in Escherichia coli. VP1 pentamers, also known as capsomeres, were purified via a glutathione-S-transferase tag. Size exclusion chromatography indicated severe aggregation of the recombinant VP1 during enzymatic tag removal resulting from the introduction the hydrophobic epitopes. Inserts were modified to possess double aspartic acid residues at each end of the hydrophobic epitopes and a high-throughput buffer condition screen was implemented with protein aggregation monitored during tag removal by spectrophotometry and dynamic light scattering. These analyses showed that the insertion of charged residues at the extremities of epitopes could improve solubility of capsomeres and revealed multiple windows of opportunity for further condition optimization. A combination of epitope design, pH optimization, and the additive l-arginine permitted the recovery of soluble VP1 pentamers presenting hydrophobic epitopes and their subsequent assembly into virus-like particles.

Crystal Structures of Enterovirus 71 (EV71) Recombinant Virus Particles Provide Insights into Vaccine Design

Hand-foot-and-mouth disease (HFMD) remains a major health concern in the Asia-Pacific regions, and its major causative agents include human enterovirus 71 (EV71) and coxsackievirus A16. A desirable vaccine against HFMD would be multivalent and able to elicit protective responses against multiple HFMD causative agents. Previously, we have demonstrated that a thermostable recombinant EV71 vaccine candidate can be produced by the insertion of a foreign peptide into the BC loop of VP1 without affecting viral replication. Here we present crystal structures of two different naturally occurring empty particles, one from a clinical C4 strain EV71 and the other from its recombinant virus containing an insertion in the VP1 BC loop. Crystal structure analysis demonstrated that the inserted foreign peptide is well exposed on the particle surface without significant structural changes in the capsid. Importantly, such insertions do not seem to affect the virus uncoating process as illustrated by the conformational similarity between an uncoating intermediate of another recombinant virus and that of EV71. Especially, at least 18 residues from the N terminus of VP1 are transiently externalized. Altogether, our study provides insights into vaccine development against HFMD.

Determining the epitope dominance on the capsid of a serotype SAT2 foot-and-mouth disease virus by mutational analyses.

Monoclonal-antibody (MAb)-resistant mutants were used to map antigenic sites on foot-and-mouth disease virus (FMDV), which resulted in the identification of neutralizing epitopes in the flexible βG-βH loop in VP1. For FMDV SAT2 viruses, studies have shown that at least two antigenic sites exist. By use of an infectious SAT2 cDNA clone, 10 structurally exposed and highly variable loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of SAT2/Zimbabwe (ZIM)/7/83 (topotype II) and replaced with the corresponding regions of SAT2/Kruger National Park (KNP)/19/89 (topotype I). Virus neutralization assays using convalescent-phase antisera raised against the parental virus, SAT2/ZIM/7/83, indicated that the mutant virus containing the TQQS-to-ETPV mutation in the N-terminal part of the βG-βH loop of VP1 showed not only a significant increase in the neutralization titer but also an increase in the index of avidity to the convalescent-phase antisera. Furthermore, antigenic profiling of the epitope-replaced and parental viruses with nonneutralizing SAT2-specific MAbs led to the identification of two nonneutralizing antigenic regions. Both regions were mapped to incorporate residues 71 to 72 of VP2 as the major contact point. The binding footprint of one of the antigenic regions encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 48 to 50 of VP1, and the second antigenic region encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 84 to 86 and 109 to 11 of VP1. This is the first time that antigenic regions encompassing residues 71 to 72 of VP2 have been identified on the capsid of a SAT2 FMDV. Monoclonal-antibody-resistant mutants have traditionally been used to map antigenic sites on foot-and-mouth disease virus (FMDV). However, for SAT2-type viruses, which are responsible for most of the FMD outbreaks in Africa and are the most varied of all seven serotypes, only two antigenic sites have been identified. We have followed a unique approach using an infectious SAT2 cDNA genome-length clone. Ten structurally surface-exposed, highly varied loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of the SAT2/ZIM/7/83 virus. These regions were replaced with the corresponding regions of an antigenically disparate virus, SAT2/KNP/19/89. Antigenic profiling of the epitope-replaced and parental viruses with SAT2-specific MAbs led to the identification of two unique antibody-binding footprints on the SAT2 capsid. In this report, evidence for the structural engineering of antigenic sites of a SAT2 capsid to broaden cross-reactivity with antisera is provided.

Evolution of serotype A foot-and-mouth disease virus capsid under neutralizing antibody pressure in vitro

In this study, the Indian foot-and-mouth disease virus (FMDV) vaccine strain (A IND 40/2000) was passaged under homologous bovine convalescent serum (BCS) pressure to gain insight into the evolutionary dynamics of the antigenic sites. A considerable drop in the neutralization titres of the BCS for the isolated variants as compared to the parental population in either virus neutralization or plaque reduction neutralization test was observed. T143K substitution preceding the integrin binding ‘RGD’-motif in the βG-βH loop of VP1 was found to be selected consistently and exclusively under immune pressure. By virtue of its location within an immunodominant site, sequence heterogeneity observed in the field viruses and residues already mapped in the neutralizing monoclonal antibody resistant mutants, position 143 in VP1 was predicted to be a critical residue of an important neutralizing epitope in serotype A FMDV. Using next-generation sequencing approach, the gradual overtaking of the originally dominating major variant by a minor one under a selective environment could be demonstrated. In the control passage regimen, VP2 E131K substitution was fixed within the heparan sulfate binding pocket probably as a result of adaptation to use alternative cellular receptors. But at the same time, these substitutions arising under selection forces other than immune pressure changed the antigenic behaviour of the virus inadvertently.

Cryo-Electron Microscopy Reconstruction Shows Poliovirus 135S Particles Poised for Membrane Interaction and RNA Release

During infection, binding of mature poliovirus to cell surface receptors induces an irreversible expansion of the capsid, to form an infectious cell-entry intermediate particle that sediments at 135S. In these expanded virions, the major capsid proteins (VP1 to VP3) adopt an altered icosahedral arrangement to open holes in the capsid at 2-fold and quasi-3-fold axes, and internal polypeptides VP4 and the N terminus of VP1, which can bind membranes, become externalized. Cryo-electron microscopy images for 117,330 particles were collected using Leginon and reconstructed using FREALIGN. Improved rigid-body positioning of major capsid proteins established reliably which polypeptide segments become disordered or rearranged. The virus-to-135S transition includes expansion of 4%, rearrangements of the GH loops of VP3 and VP1, and disordering of C-terminal extensions of VP1 and VP2. The N terminus of VP1 rearranges to become externalized near its quasi-3-fold exit, binds to rearranged GH loops of VP3 and VP1, and attaches to the top surface of VP2. These details improve our understanding of subsequent stages of infection, including endocytosis and RNA transfer into the cytoplasm.

SV40 virus-like particles as an effective delivery system and its application to a vaccine carrier

The authors have purified a major capsid protein, VP1 of Simian virus 40 (SV40), using recombinant baculovirus and have established the method of in vitro reassembly of SV40 virus-like particles (SV40-VLPs) from VP1-pentamers. In this reassembly, SV40-VLPs can encapsulate approximately 5 kb exogenous DNA shielded by histone or foreign proteins fused to minor capsid proteins VP2/3 and effectively deliver them into mammalian cells. Insertion of a particular foreign peptide into the surface loops of VP1 provides SV40-VLPs with the ability of cell targeting. Furthermore, SV40-VLPs appear to stimulate innate immunity as a natural adjuvant. Given these characteristics, SV40-VLPs may be a promising vaccine carrier to deliver heterologous antigens for the induction of cytotoxic T lymphocytes without artificial adjuvants. In this review, the authors describe how SV40-VLPs have been developed and engineered, and discuss their potential benefits and challenges as a cytotoxic T lymphocyte-based vaccine platform.

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.

Molecular Epidemiology and Evolution of Human Enterovirus Serotype 68 in Thailand, 2006–2011

BackgroundPublications worldwide have reported on the re-occurrence of human enterovirus 68 (EV68), a rarely detected pathogen usually causing respiratory illness. However, epidemiological data regarding this virus in particular on the Asian continent has so far been limited.Methodology/FindingsWe investigated the epidemiology and genetic variability of EV68 infection among Thai children with respiratory illnesses from 2006–2011 (n = 1810). Semi-nested PCR using primer sets for amplification of the 5′-untranslated region through VP2 was performed for rhino-enterovirus detection. Altogether, 25 cases were confirmed as EV68 infection indicating a prevalence of 1.4% in the entire study population. Interestingly, the majority of samples were children aged >5 years (64%). Also, co-infection with other viruses was found in 28%, while pandemic H1N1 influenza/2009 virus was the most common co-infection. Of EV68-positive patients, 36% required hospitalizations with the common clinical presentations of fever, cough, dyspnea, and wheezing. The present study has shown that EV68 was extremely rare until 2009 (0.9%). An increasing annual prevalence was found in 2010 (1.6%) with the highest detection frequency in 2011 (4.3%). Based on analysis of the VP1 gene, the evolutionary rate of EV68 was estimated at 4.93×10−3 substitutions/site/year. Major bifurcation of the currently circulating EV68 strains occurred 66 years ago (1945.31 with (1925.95–1960.46)95% HPD). Among the current lineages, 3 clusters of EV68 were categorized based on the different molecular signatures in the BC and DE loops of VP1 combined with high posterior probability values. Each cluster has branched off from their common ancestor at least 36 years ago (1975.78 with (1946.13–1984.97)95% HPD).ConclusionDifferences in epidemiological characteristic and seasonal profile of EV68 have been found in this study. Results from Bayesian phylogenetic investigations also revealed that EV68 should be recognized as a genetically diverse virus with a substitution rate identical to that of enterovirus 71 genotype B (4.2×10−3 s/s/y).

VP‐1 quasispecies in human infection with polyomavirus BK

Polyomavirus BK is a recognized cause of nephropathy and hemorrhagic cystitis in kidney or allogeneic hematopoietic stem cell transplant recipients. This study explored a role of genetic variations in capsid protein VP-1 gene as a factor in viral pathogenesis. VP-1 was amplified from 7 healthy subjects with viruria, 7 transplant patients with viruria, and 11 patients with viremia or nephropathy. PCR products were cloned and a total of 558 clonal sequences were subjected to phylogenetic analysis using standard methods. VP-1 quasispecies were found in 25/25 and coinfection with different genotypes in 12/25 subjects. Genotype II was found as an unexpected minority species in 5/25 individuals. Recombinant strains of uncertain biologic significance, which frequently contained genotype II and IV sequences were identified in 9/25 subjects. Viremia/nephropathy group was characterized by (a) greater sequence complexity in whole VP-1 versus BC loop and BC loop compared to the HI loop, (b) greater intra-strain genetic diversity in the BC loop compared to whole VP-1 protein and HI loop, (c) more non-synonymous substitutions (dN) in the BC loop compared to whole VP-1 and HI loop, (e) fewer synonymous substitutions (dS) compared to healthy-viruria group, and (f) selection pressure (dN/dS >1.0) exerted on VP-1. In conclusion, this study documents frequent occurrence of quasispecies in a host DNA polymerase dependent virus, which is theoretically expected to show high replication fidelity. Quasispecies occur even in healthy subjects with viruria, but evolutionary selection pressure directed at the viral capsid protein (VP-1) is seen only in patients with viremia or nephropathy.

Molecular Analysis of Virulent Determinants of Enterovirus 71

Enterovirus 71 (EV71) is the most important causative agent of hand, foot and mouth disease (HFMD) in children. In most cases, it is a self-limiting illness. However some EV71 infectious cases can develop severe clinical outcomes, such as encephalitis, meningitis, poliomyelitis like paralysis, and even death. To identify the determinants of virulence, the deduced amino acid sequence of polyprotein and nucleotide sequence of 5′-NTR and 3′-NTR in 25 SC-EV71 strains (strains from severe cases) and 31 MC-EV71 strains (strains from mild cases) were analyzed. Results showed four amino acids on two positions (GlyP710/GlnP710/ArgP710 and GluP729) on the DE and EF loop of VP1, one (LysP930) on the surface of protease 2A and four nucleotides on three positions (GP272, UP488 and AP700/UP700) in the 5'-NTR region are associated with EV71 virulent phenotype. Predicted secondary structure of RNA using the consensus sequence of 5'-NTR by RNAStructure showed the mutation of nucleotide at position 488 in strain BJ08-Z004-3 (position 491 in prototype strain BrCr) can result in the discrepancy of an additional pair of nucleotides and thus change the stability of the second structure of IRES. Fragment base content analysis showed that in the region 696 to 714 bp at the 5'-NTR, where the AP700/UP700 was located, the nucleotide constitution ratios differed significantly between SC-EV71 and MC-EV71 strains. In conclusion, comparative genomic analysis showed that virulence of EV71 strains are mainly determined by the amino acids on two positions of VP1, one position of protease 2A and the nucleotides on three positions in 5'-NTR.

Analysis of SAT1 type foot-and-mouth disease virus capsid proteins: Influence of receptor usage on the properties of virus particles

The three SAT serotype viruses, endemic in Africa, are well known for their difficulty to adapt to cell culture. The viral mechanism involved in foot-and-mouth disease virus (FMDV) tissue tropism and cell-entry is not well understood. A recombinant, small plaque-forming virus (vSAT1tc), derived from a tissue culture-adapted SAT1 virus (SAR/9/81tc), revealed four amino acid substitutions (VP3 Asp192 → Tyr; VP3 Ser217 → Ile; VP1 Ala69 → Gly and VP1 Asn110 → Lys) in the capsid, compared to the SAR/9/81wt isolate collected from infected impala epithelium. One substitution added a positively charged lysine residue to the short βF-βG loop of VP1. Furthermore, vSAT1tc displayed a high affinity for CHO-K1 cells possibly via interaction with negatively charged sulphated polysaccharides while SAT1 impala strain relied strongly on α Vβ6 integrin receptors for cell entry. The cell culture adaptation and small plaque phenotype of vSAT1tc was accompanied by differences in particle aggregation and significant differences in acid stability. Based on limited cross neutralization data, the antigenic features seem to be unchanged. Thus, acquisition of positively charged residues in the virion may be beneficial for adaptation of SAT type field strains to cell culture.

Characterisation and epitope mapping of neutralising monoclonal antibodies to A24 Cruzeiro strain of FMDV

Characterisation of seven neutralising monoclonal antibodies (mAbs) produced against foot-and-mouth disease virus A24 Cruzeiro revealed three reactivity groups. Gr-I recognised linear epitopes where as Gr-II was conformation-dependent and trypsin-insensitive. The Gr-III was also conformation-dependent, but trypsin-sensitive. Mar (mAb neutralisation resistant)-mutants could only be produced against Gr-I and Gr-III mAbs. Capsid sequence comparison of Gr-I mar-mutants with parent virus revealed changes in the G–H loop of VP1 at positions 141, 143 and 147. Similarly, a Gr-III mar-mutant showed a change from a highly conserved glycine to a tryptophan at position 148 of VP1 along with three additional changes at the N-terminus of VP1, VP2 and VP4. This residue at 148 of VP1 is located at +2 position after “RGD” and is equivalent to the position identified by the mAb recognising site 5 in serotype O viruses. This site is probably formed because of the interaction of the G–H loop with other residues in different structural proteins.

Genetic characterization of the cell-adapted PanAsia strain of foot-and-mouth disease virus O/Fujian/CHA/5/99 isolated from swine

BackgroundAccording to Office International Des Epizooties (OIE) Bulletin, the PanAsia strain of Foot-and-Mouth Disease Virus (FMDV) was invaded into the People's Republic of China in May 1999. It was confirmed that the outbreaks occurred in Tibet, Hainan and Fujian provinces. In total, 1280 susceptible animals (68 cattle, 1212 swine) were destroyed for the epidemic control.To investigate the distinct biological properties, we performed plaque assay, estimated the pathogenicity in suckling mice and determined the complete genomic sequence of FMDV swine-isolated O/Fujian/CHA/5/99 strain. In addition, a molecular modeling was carried out with the external capsid proteins.ResultsThe pathogenicity study showed that O/Fujian/CHA/5/99 had high virulence with respect to infection in 3-day-old suckling-mice (LD50 = 10-8.3), compared to O/Tibet/CHA/1/99 (LD50 = 10-7.0) which isolated from bovine. The plaque assay was distinguishable between O/Fujian/CHA/5/99 and O/Tibet/CHA/1/99 by their plaque phenotypes. O/Fujian/CHA/5/99 formed large plaque while O/Tibet/CHA/1/99 formed small plaque.The 8,172 nucleotides (nt) of O/Fujian/CHA/5/99 was sequenced, and a phylogenetic tree was generated from the complete nucleotide sequences of VP1 compared with other FMDV reference strains. The identity data showed that O/Fujian/CHA/5/99 is closely related to O/AS/SKR/2002 (94.1% similarity). Based on multiple sequence alignments, comparison of sequences showed that the characteristic nucleotide/amino acid mutations were found in the whole genome of O/Fujian/CHA/5/99.ConclusionOur finding suggested that C275T substitution in IRES of O/Fujian/CHA/5/99 may induce the stability of domain 3 for the whole element function. The structure prediction indicated that most of 14 amino acid substitutions are fixed in the capsid of O/Fujian/CHA/5/99 around B-C loop and E-F loop of VP2 (antigenic site 2), and G-H loop of VP1 (antigenic site 1), respectively. These results implicated that these substitutions close to heparin binding sites (E136G in VP2, A174 S in VP3) and at antigenic site 1 (T142A, A152T and Q153P in VP1) may influence plaque size and the pathogenicity to suckling mice.The potential of genetic characterization would be useful for microevolution and viral pathogenesis of FMDV in the further study.

Mapping of amino acid residues responsible for adhesion of cell culture-adapted foot-and-mouth disease SAT type viruses

Foot-and-mouth disease virus (FMDV) infects host cells by adhering to the α V subgroup of the integrin family of cellular receptors in a Arg–Gly–Asp (RGD) dependent manner. FMD viruses, propagated in non-host cell cultures are reported to acquire the ability to enter cells via alternative cell surface molecules. Sequencing analysis of SAT1 and SAT2 cell culture-adapted variants showed acquisition of positively charged amino acid residues within surface-exposed loops of the outer capsid structural proteins. The fixation of positively charged residues at position 110–112 in the βF–βG loop of VP1 of SAT1 isolates is thought to correlate with the acquisition of the ability to utilise alternative glycosaminoglycan (GAG) molecules for cell entry. Similarly, two SAT2 viruses that adapted readily to BHK-21 cells accumulated positively charged residues at positions 83 and 85 of the βD–βE loop of VP1. Both regions surround the fivefold axis of the virion. Recombinant viruses containing positively charged residues at position 110 and 112 of VP1 were able to infect CHO-K1 cells (that expresses GAG) and demonstrated increased infectivity in BHK-21 cells. Therefore, recombinant SAT viruses engineered to express substitutions that induce GAG-binding could be exploited in the rational design of vaccine seed stocks with improved growth properties in cell cultures.

Crystal structure of equine rhinitis A virus in complex with its sialic acid receptor

Equine rhinitis A virus (ERAV) shares many features with foot-and-mouth disease virus (FMDV) and both are classified within the genus Aphthovirus of the family Picornaviridae. ERAV is used as a surrogate for FMDV research as it does not require high-level biosecurity. In contrast to FMDV, which uses integrins as cellular receptors, the receptor for ERAV has been reported to involve the sugar moiety sialic acid. This study confirmed the importance of sialic acid for cell entry by ERAV and reports the crystal structure of ERAV particles complexed with the receptor analogue 3'-sialyllactose. The receptor is attached to the rim of a capsid pit adjacent to the major immunogenic site and distinct from the sialic acid binding site used by a related picornavirus, the cardiovirus Theiler's murine encephalitis virus. The structure of the major antigenic determinant of the virus, previously identified from antibody escape mutations, is also described as the EF loop of VP1, which forms a hairpin stretching across the capsid surface close to the icosahedral fivefold axis, neighbouring the receptor-binding site, and spanning two protomeric units.

Structure and dynamics of the GH loop of the foot-and-mouth disease virus capsid

The GH loop of VP1 of the foot-and-mouth disease virus capsid is important because it is a major antigenic site and an integrin recognition site. The GH loop is disordered in all X-ray structures of the capsid except for serotype O under reduced conditions in which the loop lies on the capsid surface. Although the structure of the capsid–integrin complex has not yet been determined, the GH loop is known to protrude from the capsid surface when the capsid is bound with an antigen-binding fragment (Fab). To clarify the structure and dynamics of the GH loop under natural unreduced conditions before binding to integrins or Fab fragments, we performed molecular dynamics simulation of 16.3 ns long under rotational symmetry boundary conditions for the capsid of serotype O using the X-ray structure of the reduced capsid for the initial coordinates. When the disulfide bond at the base of the GH loop was formed by the molecular mutation method, the loop protruded into the surrounding water, as reported for Fab–capsid complexes, and fluctuated like a tentacle. After equilibration, the GH loop overlapped the surface of the capsid but continued to fluctuate, being directed toward a 2-fold axis. The conformational change of the GH loop after formation of the disulfide bond was explained by a model of elastic tube. The side chains of arginine and aspartic acid of the integrin recognition residues (RGD tripeptide) extended in opposite directions, and the residues on the C-terminal side of the RGD tripeptide formed a hydrophobic cluster in close proximity of the arginine residue of the tripeptide.

Analysis of Foot-and-Mouth Disease Virus Integrin Receptor Expression in Tissues from Naïve 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.

Theiler's murine encephalomyelitis virus attachment to the gastrointestinal tract is associated with sialic acid binding

DA and GDVII are strains of Theiler’s murine encephalomyelitis virus (TMEV). DA virus mutant DApB encodes VP2 puff B of GDVII, whereas DApBL2M contains VP1 loop II of GDVII with a point mutation in VP2 puff B. Neuraminidase treatment of cells inhibited infection by DA and DApB, but not GDVII or DApBL2M viruses; sialic acid (SA) binding correlated with virus persistence. In virus binding assays to intestine sections, all four TMEVs bound goblet cells and the mucus of the epithelium that was SA dependent. Therefore, differences in SA composition on different cell types can affect tropism and infection.