The updated Structural Database of Allergenic Proteins (SDAP 2.0) provides 3D models for allergens and incorporated bioinformatics tools.

Background: Allergenic proteins can cause IgE-mediated adverse reactions in sensitized individuals. Although the sequences of many allergenic proteins have been identified, bioinformatics data analysis with advanced computational methods and modeling is needed to identify the basis for IgE binding and cross-reactivity. Objective: We aim to present the features and use of the updated Structural Database of Allergenic Proteins 2.0 (SDAP 2.0) webserver, a unique, publicly available resource to compare allergens using specially designed computational tools and new high-quality 3-D models for most known allergens. Methods: Previously developed and novel software tools for identifying cross-reactive allergens using sequence and structure similarity are implemented in SDAP 2.0. A comprehensive set of high-quality 3-D models of most allergens was generated with the state-of-the-art AlphaFold 2 software. A graphics tool enables the interactive visualization of IgE epitopes on experimentally determined and modeled 3-D structures. Results: A user can search for allergens similar to a given input sequence with the FASTA algorithm or the window-based World Health Organization/International Union of Immunological Societies (WHO/IUIS) guidelines on safety concerns of novel food products. Peptides similar to known IgE epitopes can be identified with the property distance tool and conformational epitopes by the Cross-React method. The updated database contains 1657 manually curated sequences including all allergens from the IUIS database, 334 experimentally determined X-ray or NMR structures, and 1565 3-D models. Each allergen/isoallergen is classified according to its protein family. Conclusions: SDAP provides access to the steadily increasing information on allergenic structures and epitopes with integrated bioinformatics tools to identify and analyze their similarities. In addition to serving the research and regulatory community, it provides clinicians with tools to identify potential coallergies in a sensitive patient and can help companies to design hypoallergenic foods and immunotherapies.

Alanine Scanning of the Unstructured Region of Ara h 2 and of a Related Mimotope Reveals Critical Amino Acids for IgE Binding.

SCOPE: The unstructured region of Ara h 2, referred to as epitope 3, contains a repeated motif, DYPSh (h = hydroxyproline) that is important for IgE binding. METHODS AND RESULTS: IgE binding assays to 20mer and shorter peptides of epitope 3, defines a 16mer core sequence containing one copy of the DPYSh motif, DEDSYERDPYShSQDP. This study performs alanine scanning of this and a related 12mer mimotope, LLDPYAhRAWTK. IgE binding, using a pool of 10 sera and with individual sera, is greatly reduced when alanine is substituted for aspartate at position 8 (D8; p < 0.01), tyrosine at position 10 (Y10; p < 0.01), and hydroxyproline at position 12 (h12; p < 0.001). IgE binding to alanine-substituted peptides of a mimotope containing the DPY_h motif confirm the critical importance of Y (p < 0.01) and h (p < 0.01), but not D. Molecular modeling of the core and mimotope suggests an h-dependent conformational basis for the recognition of these sequences by polyclonal IgE. CONCLUSIONS: IgE from pooled sera and individual sera differentially bound amino acids throughout the sequences of Epitope 3 and its mimotope, with Y10 and h12 being most important for all sera. These results are highly significant for designing hypoallergenic forms of Ara h 2.

PCP consensus protein/peptide alphavirus antigens stimulate broad spectrum neutralizing antibodies.

Vaccines based on proteins and peptides may be safer and if calculated based on many sequences, more broad-spectrum than those designed based on single strains. Physicochemical Property Consensus (PCPcon) alphavirus (AV) antigens from the B-domain of the E2 envelope protein were designed, synthesized recombinantly and shown to be immunogenic (i.e. sera after inoculation detected the antigen in dotspots and ELISA). Antibodies in sera after inoculation with B-region antigens based on individual AV species (eastern or Venezuelan equine encephalitis (EEEVcon, VEEVcon), or chikungunya (CHIKVcon) bound only their cognate protein, while those designed against multiple species (Mosaikcon and EVCcon) recognized all three serotype specific antigens. The VEEVcon and EEEVcon sera only showed antiviral activity against their related strains (in plaque reduction neutralization assays (PRNT50/80). Peptides designed to surface exposed areas of the E2-A-domain of CHIKVcon were added to CHIKVcon inocula to provide anti-CHIKV antibodies. EVCcon, based on three different alphavirus species, combined with E2-A-domain peptides from AllAVcon, a PCPcon of 24 diverse AV, generated broad spectrum, antiviral antibodies against VEEV, EEEV and CHIKV, AV with less than 35% amino acid identity to each other (>65% diversity). This is a promising start to a molecularly defined vaccine against all AV. Further study with these antigens can illuminate what areas are most important for a robust immune response, resistant to mutations in rapidly evolving viruses. The validated computational methods can also be used to design broad spectrum antigens against many other pathogen families.

Still SDAPing Along: 20 Years of the Structural Database of Allergenic Proteins.

The introduction of plant extracts to mitigate the symptoms of "hay fever", about a century ago, led to discoveries beginning sixty years ago on determining the sequences and eventually structures of allergenic proteins. As more proteins were cloned, there was a need to rapidly identify and categorize those with significant similarity to known allergens. The Structural Database of Allergenic Proteins (SDAP) was created at the beginning of the 21st century as the first cross-referenced website to allow rapid overview of the structures and sequences of allergenic proteins. SDAP provides a way to identify sequence and functional similarities between these proteins, despite the complex nomenclature system based on the Latin names of their different sources. A rapid FASTA search simplifies grouping allergens from the same structural or functional family. SDAP also provides an overview of the rapidly expanding literature on the sequence, structure and epitopes of allergenic proteins and a way to estimate the potential allergenicity of novel proteins based on rules provided by the IUIS. Twenty years and a pandemic later, the list of allergenic proteins and their attributes continues to grow. SDAP is expanding and improving to allow rapid access to all this information.

Regional and temporal coordinated mutation patterns in SARS-CoV-2 spike protein revealed by a clustering and network analysis.

SARS-CoV-2 has steadily mutated during its spread to > 300 million people throughout the world. The WHO has designated strains with certain mutations, "variants of concern" (VOC), as they may have higher infectivity and/or resist neutralization by antibodies in sera of vaccinated individuals and convalescent patients. Methods to detect regionally emerging VOC are needed to guide treatment and vaccine design. Cluster and network analysis was applied to over 1.2 million sequences of the SARS-CoV-2 spike protein from 36 countries in the GISAID database. While some mutations rapidly spread throughout the world, regionally specific groups of variants were identified. Strains circulating in each country contained different sets of high frequency mutations, many of which were known VOCs. Mutations within clusters increased in frequency simultaneously. Low frequency, but highly correlated mutations detected by the method could signal emerging VOCs, especially if they occur at higher frequency in other regions. An automated version of our method to find high frequency mutations in a set of SARS-COV-2 spike sequences is available online at http://curie.utmb.edu/SAR.html .

Design of peptides with high affinity binding to a monoclonal antibody as a basis for immunotherapy.

About half of the US population is sensitized to one or more allergens, as found by a National Health and Nutrition Examination Survey (NHANES). The most common treatment for seasonal allergic responses is the daily use of oral antihistamines, which can control some of the symptoms, but are not effective for nasal congestion, and can be debilitating in many patients. Peptide immunotherapy is a promising new approach to treat allergic airway diseases. The small size of the immunogens cannot lead to an unwanted allergic reaction in sensitized patients, and the production of peptides with sufficient amounts for immunotherapy is time- and cost-effective. However, it is not known what peptides are the most effective for an immunotherapy of allergens. We previously produced a unique monoclonal antibody (mAb) E58, which can inhibit the binding of multiple groups of mAbs and human IgEs from patients affected by the major group 1 allergens of ragweed (Amb a 1) and conifer pollens (Jun a 1, Cup s 1, and Cry j 1). Here, we demonstrated that a combined approach, starting from two linear E58 epitopes of the tree pollen allergen Jun a 1 and the ragweed pollen allergen Amb a 1, and residue modifications suggested by molecular docking calculations and peptide design could identify a large number of high affinity binding peptides. We propose that this combined experimental and computational approach by structural analysis of linear IgE epitopes and peptide design, can lead to potential new candidates for peptide immunotherapy.

Synthetic proteins for COVID-19 diagnostics.

There is an urgent need for inexpensive, rapid and specific antigen-based assays to test for vaccine efficacy and detect infection with SARS-CoV-2 and its variants. We have identified a small, synthetic protein (JS7), representing a region of maximum variability within the receptor binding domain (RBD), which binds antibodies in sera from nine patients with PCR-verified COVID-19 of varying severity. Antibodies binding to either JS7 or the SARS-CoV-2 recombinant RBD, as well as those that disrupt binding between a fragment of the ACE2 receptor and the RBD, are proportional to disease severity and clinical outcome. Binding to JS7 was inhibited by linear peptides from the RBD interface with ACE2. Variants of JS7, such as E484K or N501Y, can be quickly synthesized in pure form in large quantities by automated methods. JS7 and related synthetic antigens can provide a basis for specific diagnostics for SARS-CoV-2 infections.

The importance of the 2S albumins for allergenicity and cross-reactivity of peanuts, tree nuts, and sesame seeds.

Allergies to peanuts, tree nuts, and sesame seeds are among the most important food-related causes of anaphylaxis. Important clinical questions include: Why is there a variable occurrence of coallergy among these foods and Is this immunologically mediated? The clinical and immunologic data summarized here suggest an immunologic basis for these coallergies that is based on similarities among the 2S albumins. Data from component resolved diagnostics have highlighted the relationship between IgE binding to these allergens and the presence of IgE-mediated food allergy. Furthermore, in vitro and in vivo experiments provide strong evidence that the 2S albumins are the most important allergens in peanuts for inducing an allergic effector response. Although the 2S albumins are diverse, they have a common disulfide-linked core with similar physicochemical properties that make them prime candidates to explain much of the observed coallergy among peanuts, tree nuts, and sesame seeds. The well-established frequency of cashew and pistachio nut coallergy (64%-100%) highlights how the structural similarities among their 2S albumins may account for observed clinical cross-reactivity. A complete understanding of the physicochemical properties of the 2S albumins in peanuts, tree nuts, and sesame seeds will enhance our ability to diagnose, treat, and ultimately prevent these allergies.

Producing physicochemical property consensus alphavirus protein antigens for broad spectrum vaccine design.

There is a pressing need for new vaccines against alphaviruses, which can cause fatal encephalitis (Venezuelan equine encephalitis virus (VEEV) and others) and severe arthralgia (e.g. Chikungunya virus, CHIKV). These positive-strand RNA viruses are diverse and evolve rapidly, meaning that the sequence of any vaccine should cover multiple strains that may be quite different from any previous isolate. Here, consensus proteins were produced to represent the common physicochemical properties (PCPs) of the epitope rich, B domain of the E2 envelope protein. PCP-consensus proteins were based on multiple strains of VEEV (VEEVcon) and CHIKV (CHIKVcon) or the conserved PCPs of 24 different alphaviruses (AllAVcon). The AllAVcon was altered to include binding sites for neutralizing antibodies of both VEEV and CHIKV strains (Mosaikcon). All four designed proteins were produced solubly in E. coli and purified. They formed the ß-strand core expected from experimental structures of this region of the wild type E2 proteins as indicated by circular dichroism (CD) spectra. Furthermore, the CHIKVcon protein bound to a structure dependent, CHIKV neutralizing monoclonal antibody. The AllAVcon and Mosaikcon proteins bound to polyclonal antibodies generated during natural infection with either VEEV or CHIKV, indicating they contained epitopes of both serotypes. The Mosaikcon antigen induced antibodies in rabbit sera that recognized both the VEEVcon and CHIKVcon spike proteins. These PCP-consensus antigens are promising starting points for novel, broad-spectrum alphavirus vaccines.

IgE binding to linear epitopes of Ara h 2 in peanut allergic preschool children undergoing oral Immunotherapy.

BACKGROUND: For patients with peanut allergy, there are currently no methods to predict who will develop sustained unresponsiveness (SU) after oral immunotherapy (OIT). OBJECTIVE: Assess IgE binding to peanut (PN), Ara h 2, and specific linear epitopes of Ara h 2 as predictors of the important clinical parameters: eliciting dose threshold and attainment of SU following OIT. METHODS: Samples and clinical data were collected from children undergoing OIT. PN- and Ara h 2-sIgE were quantified by ImmunoCAP® . IgE binding to linear peptides of Ara h 2 and Ara h 6 was measured with peptide microarrays. RESULTS: Values of PN-sIgE correlated with eliciting dose (P = .001) and with a higher likelihood of achieving SU (P < .0001), but these relationships were lost at higher values for PN-sIgE (≥14 kIU for eliciting dose and ≥35 kIU/L for SU). In subjects with PN-sIgE ≥ 14 kIU/L, binding of IgE to epitopes 5 and 6 of Ara h 2 was associated with a lower eliciting dose at baseline challenge (P < .001; Pc  < .02). In subjects with PN-sIgE ≥ 35 kIU/L, a combined model of IgE binding to epitopes 1, 5 and 6 with PN-sIgE was highly predictive of attainment of SU (AUC of 0.86; P = .0067). CONCLUSION: In young patients with peanut allergy, measurement of PN-sIgE and IgE binding to specific linear epitopes of Ara h 2 in baseline samples may allow stratification of patients regarding sensitivity to challenge and outcome of OIT.

Antibody-Mediated Protective Mechanisms Induced by a Trivalent Parainfluenza Virus-Vectored Ebolavirus Vaccine.

Ebolaviruses Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) cause human disease with high case fatality rates. Experimental monovalent vaccines, which all utilize the sole envelope glycoprotein (GP), do not protect against heterologous ebolaviruses. Human parainfluenza virus type 3-vectored vaccines offer benefits, including needle-free administration and induction of mucosal responses in the respiratory tract. Multiple approaches were taken to induce broad protection against the three ebolaviruses. While GP consensus-based antigens failed to elicit neutralizing antibodies, polyvalent vaccine immunization induced neutralizing responses to all three ebolaviruses and protected animals from death and disease caused by EBOV, SUDV, and BDBV. As immunization with a cocktail of antigenically related antigens can skew the responses and change the epitope hierarchy, we performed comparative analysis of antibody repertoire and Fc-mediated protective mechanisms in animals immunized with monovalent versus polyvalent vaccines. Compared to sera from guinea pigs receiving the monovalent vaccines, sera from guinea pigs receiving the trivalent vaccine bound and neutralized EBOV and SUDV at equivalent levels and BDBV at only a slightly reduced level. Peptide microarrays revealed a preponderance of binding to amino acids 389 to 403, 397 to 415, and 477 to 493, representing three linear epitopes in the mucin-like domain known to induce a protective antibody response. Competition binding assays with monoclonal antibodies isolated from human ebolavirus infection survivors demonstrated that the immune sera block the binding of antibodies specific for the GP glycan cap, the GP1-GP2 interface, the mucin-like domain, and the membrane-proximal external region. Thus, administration of a cocktail of three ebolavirus vaccines induces a desirable broad antibody response, without skewing of the response toward preferential recognition of a single virus.IMPORTANCE The symptoms of the disease caused by the ebolaviruses Ebola, Bundibugyo, and Sudan are similar, and their areas of endemicity overlap. However, because of the limited antigenic relatedness of the ebolavirus glycoprotein (GP) used in all candidate vaccines against these viruses, they protect only against homologous and not against heterologous ebolaviruses. Therefore, a broadly specific pan-ebolavirus vaccine is required, and this might be achieved by administration of a cocktail of vaccines. The effects of cocktail administration of ebolavirus vaccines on the antibody repertoire remain unknown. Here, an in-depth analysis of the antibody responses to administration of a cocktail of human parainfluenza virus type 3-vectored vaccines against individual ebolaviruses was performed, which included analysis of binding to GP, neutralization of individual ebolaviruses, epitope specificity, Fc-mediated functions, and protection against the three ebolaviruses. The results demonstrated potent and balanced responses against individual ebolaviruses and no significant reduction of the responses compared to that induced by individual vaccines.

Distinguishing allergens from non-allergenic homologues using Physical-Chemical Property (PCP) motifs.

Quantitative guidelines to distinguish allergenic proteins from related, but non-allergenic ones are urgently needed for regulatory agencies, biotech companies and physicians. In a previous study, we found that allergenic proteins populate a relatively small number of protein families, as characterized by the Pfam database. However, these families also contain non-allergenic proteins, meaning that allergenic determinants must lie within more discrete regions of the sequence. Thus, new methods are needed to discriminate allergenic proteins within those families. Physical-Chemical Properties (PCP)-motifs specific for allergens within a Pfam class were determined for 17 highly populated protein domains. A novel scoring method based on PCP-motifs that characterize known allergenic proteins within these families was developed, and validated for those domains. The motif scores distinguished sequences of allergens from a large selection of 80,000 randomly selected non-allergenic sequences. The motif scores for the birch pollen allergen (Bet v 1) family, which also contains related fruit and nut allergens, correlated better than global sequence similarities with clinically observed cross-reactivities among those allergens. Further, we demonstrated that the average scores of allergen specific motifs for allergenic profilins are significantly different from the scores of non-allergenic profilins. Several of the selective motifs coincide with experimentally determined IgE epitopes of allergenic profilins. The motifs also discriminated allergenic pectate lyases, including Jun a 1 from mountain cedar pollen, from similar proteins in the human microbiome, which can be assumed to be non-allergens. The latter lacked key motifs characteristic of the known allergens, some of which correlate with known IgE binding sites.

Functional classification of protein toxins as a basis for bioinformatic screening.

Proteins are fundamental to life and exhibit a wide diversity of activities, some of which are toxic. Therefore, assessing whether a specific protein is safe for consumption in foods and feeds is critical. Simple BLAST searches may reveal homology to a known toxin, when in fact the protein may pose no real danger. Another challenge to answer this question is the lack of curated databases with a representative set of experimentally validated toxins. Here we have systematically analyzed over 10,000 manually curated toxin sequences using sequence clustering, network analysis, and protein domain classification. We also developed a functional sequence signature method to distinguish toxic from non-toxic proteins. The current database, combined with motif analysis, can be used by researchers and regulators in a hazard screening capacity to assess the potential of a protein to be toxic at early stages of development. Identifying key signatures of toxicity can also aid in redesigning proteins, so as to maintain their desirable functions while reducing the risk of potential health hazards.

Cross-React: a new structural bioinformatics method for predicting allergen cross-reactivity.

The phenomenon of cross-reactivity between allergenic proteins plays an important role to understand how the immune system recognizes different antigen proteins. Allergen proteins are known to cross-react if their sequence comparison shows a high sequence identity which also implies that the proteins have a similar 3D fold. In such cases, linear sequence alignment methods are frequently used to predict cross-reactivity between allergenic proteins. However, the prediction of cross-reactivity between distantly related allergens continues to be a challenging task. To overcome this problem, we developed a new structure-based computational method, Cross-React, to predict cross-reactivity between allergenic proteins available in the Structural Database of Allergens (SDAP). Our method is based on the hypothesis that we can find surface patches on 3D structures of potential allergens with amino acid compositions similar to an epitope in a known allergen. We applied the Cross-React method to a diverse set of seven allergens, and successfully identified several cross-reactive allergens with high to moderate sequence identity which have also been experimentally shown to cross-react. Based on these findings, we suggest that Cross-React can be used as a predictive tool to assess protein allergenicity and cross-reactivity. Availability and Implementation: : Cross-React is available at: http://curie.utmb.edu/Cross-React.html. Contact: ssnegi@utmb.edu.

A Conserved Structural Signature of the Homeobox Coding DNA in HOX genes.

The homeobox encodes a DNA-binding domain found in transcription factors regulating key developmental processes. The most notable examples of homeobox containing genes are the Hox genes, arranged on chromosomes in the same order as their expression domains along the body axis. The mechanisms responsible for the synchronous regulation of Hox genes and the molecular function of their colinearity remain unknown. Here we report the discovery of a conserved structural signature of the 180-base pair DNA fragment comprising the homeobox. We demonstrate that the homeobox DNA has a characteristic 3-base-pair periodicity in the hydroxyl radical cleavage pattern. This periodic pattern is significant in most of the 39 mammalian Hox genes and in other homeobox-containing transcription factors. The signature is present in segmented bilaterian animals as evolutionarily distant as humans and flies. It remains conserved despite the fact that it would be disrupted by synonymous mutations, which raises the possibility of evolutionary selective pressure acting on the structure of the coding DNA. The homeobox coding DNA may therefore have a secondary function, possibly as a regulatory element. The existence of such element may have important consequences for understanding how these genes are regulated.

Conformational IgE epitopes of peanut allergens Ara h 2 and Ara h 6.

BACKGROUND: Cross-linking of IgE antibody by specific epitopes on the surface of mast cells is a prerequisite for triggering symptoms of peanut allergy. IgE epitopes are frequently categorized as linear or conformational epitopes. Although linear IgE-binding epitopes of peanut allergens have been defined, little is known about conformational IgE-binding epitopes. OBJECTIVE: To identify clinically relevant conformational IgE epitopes of the two most important peanut allergens, Ara h 2 and Ara h 6, using phage peptide library. METHODS: A phage 12mer peptide library was screened with allergen-specific IgE from 4 peanut-allergic patients. Binding of the mimotopes to IgE from a total of 29 peanut-allergic subjects was measured by ELISA. The mimotope sequences were mapped on the surface areas of Ara h 2 and Ara h 6 using EpiSearch. RESULTS: Forty-one individual mimotopes were identified that specifically bind anti- Ara h 2/Ara h 6 IgE as well as rabbit anti-Ara h 2 and anti-Ara h 6 IgG. Sequence alignment showed that none of the mimotope sequences match a linear segment of the Ara h 2 or Ara h 6 sequences. EpiSearch analysis showed that all the mimotopes mapped to surface patches of Ara h 2 and Ara h 6. Eight of the mimotopes were recognized by more than 90% of the patients, suggesting immunodominance. Each patient had distinct IgE recognition patterns but the recognition frequency was not correlated to the concentration of peanut specific IgE or to clinical history. CONCLUSIONS: The mimotopes identified in this study represent conformational epitopes. Identification of similar surface patches on Ara h 2 and Ara h 6 further underscores the similarities between these two potent allergens.

Prediction of homoprotein and heteroprotein complexes by protein docking and template-based modeling: A CASP-CAPRI experiment.

We present the results for CAPRI Round 30, the first joint CASP-CAPRI experiment, which brought together experts from the protein structure prediction and protein-protein docking communities. The Round comprised 25 targets from amongst those submitted for the CASP11 prediction experiment of 2014. The targets included mostly homodimers, a few homotetramers, and two heterodimers, and comprised protein chains that could readily be modeled using templates from the Protein Data Bank. On average 24 CAPRI groups and 7 CASP groups submitted docking predictions for each target, and 12 CAPRI groups per target participated in the CAPRI scoring experiment. In total more than 9500 models were assessed against the 3D structures of the corresponding target complexes. Results show that the prediction of homodimer assemblies by homology modeling techniques and docking calculations is quite successful for targets featuring large enough subunit interfaces to represent stable associations. Targets with ambiguous or inaccurate oligomeric state assignments, often featuring crystal contact-sized interfaces, represented a confounding factor. For those, a much poorer prediction performance was achieved, while nonetheless often providing helpful clues on the correct oligomeric state of the protein. The prediction performance was very poor for genuine tetrameric targets, where the inaccuracy of the homology-built subunit models and the smaller pair-wise interfaces severely limited the ability to derive the correct assembly mode. Our analysis also shows that docking procedures tend to perform better than standard homology modeling techniques and that highly accurate models of the protein components are not always required to identify their association modes with acceptable accuracy. Proteins 2016; 84(Suppl 1):323-348. © 2016 Wiley Periodicals, Inc.

Western equine encephalitis virus: evolutionary analysis of a declining alphavirus based on complete genome sequences.

UNLABELLED: Western equine encephalitis virus (WEEV) is an arbovirus from the genus Alphavirus, family Togaviridae, which circulates in North America between birds and mosquitoes, occasionally causing disease in humans and equids. In recent decades, human infection has decreased dramatically; the last documented human case in North America occurred in 1994, and the virus has not been detected in mosquito pools since 2008. Because limited information exists regarding the evolution of WEEV, we analyzed the genomic sequences of 33 low-passage-number strains with diverse geographic and temporal distributions and performed comprehensive phylogenetic analyses. Our results indicated that WEEV is a highly conserved alphavirus with only approximately 5% divergence in its most variable genes. We confirmed the presence of the previously determined group A and B lineages and further resolved group B into three sublineages. We also observed an increase in relative genetic diversity during the mid-20th century, which correlates with the emergence and cocirculation of several group B sublineages. The estimated WEEV population size dropped in the 1990s, with only the group B3 lineage being sampled in the past 20 years. Structural mapping showed that the majority of substitutions in the envelope glycoproteins occurred at the E2-E2 interface. We hypothesize that an event occurred in the mid-20th century that resulted in the increased genetic diversity of WEEV in North America, followed by genetic constriction due to either competitive displacement by the B3 sublineage or stochastic events resulting from a population decline. IMPORTANCE: Western equine encephalitis virus (WEEV) has caused several epidemics that resulted in the deaths of thousands of humans and hundreds of thousands of equids during the past century. During recent decades, human infection decreased drastically and the virus has not been found in mosquito pools since 2008. Because limited information exists regarding the evolution of WEEV, we analyzed 33 complete genome sequences and conducted comprehensive phylogenetic analyses. We confirmed the presence of two major lineages, one of which diverged into three sublineages. Currently, only one of those sublineages is found circulating in nature. Understanding the evolution of WEEV over the past century provides a unique opportunity to observe an arbovirus that is in decline and to better understand what factors can cause said decline.

Membrane fusion triggering: three modules with different structure and function in the upper half of the measles virus attachment protein stalk.

The measles virus (MV) fusion apparatus consists of a fusion protein and an attachment protein named hemagglutinin (H). After receptor-binding through its cuboidal head, the H-protein transmits the fusion-triggering signal through its stalk to the fusion protein. However, the structural basis of signal transmission is unclear because only structures of H-heads without their stalk have been solved. On the other hand, the entire ectodomain structure of the hemagglutinin-neuraminidase protein of another Paramyxovirus revealed a four-helix bundle stalk. To probe the structure of the 95-residue MV H-stalk we individually substituted head-proximal residues (positions 103-153) with cysteine, and biochemically and functionally characterized the resultant proteins. Our results indicate that most residues in the central segment (positions 103-117) can be cross-linked by engineered disulfide bonds, and thus may be engaged in a tetrameric structure. While covalent tetramerization disrupts fusion triggering function, disulfide bond reduction restores it in most positions except Asp-113. The next stalk segment (residues 123-138) also has high propensity to form covalent tetramers, but since these cross-links have little or no effect on function, it can conduct the fusion-triggering signal while remaining in a stabilized tetrameric configuration. This segment may act as a spacer, maintaining H-heads at an optimal height. Finally, the head-proximal segment (residues 139-154) has very limited propensity to trap tetramers, suggesting bifurcation into two flexible linkers clamped by inter-subunit covalent links formed by natural Cys-139 and Cys-154. We discuss the modular structure of the MV H-stalk in the context of membrane fusion triggering and cell entry by Paramyxoviruses.

Base of the measles virus fusion trimer head receives the signal that triggers membrane fusion.

The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses.