ProtVar: mapping and contextualizing human missense variation.

Genomic variation can impact normal biological function in complex ways and so understanding variant effects requires a broad range of data to be coherently assimilated. Whilst the volume of human variant data and relevant annotations has increased, the corresponding increase in the breadth of participating fields, standards and versioning mean that moving between genomic, coding, protein and structure positions is increasingly complex. In turn this makes investigating variants in diverse formats and assimilating annotations from different resources challenging. ProtVar addresses these issues to facilitate the contextualization and interpretation of human missense variation with unparalleled flexibility and ease of accessibility for use by the broadest range of researchers. By precalculating all possible variants in the human proteome it offers near instantaneous mapping between all relevant data types. It also combines data and analyses from a plethora of resources to bring together genomic, protein sequence and function annotations as well as structural insights and predictions to better understand the likely effect of missense variation in humans. It is offered as an intuitive web server https://www.ebi.ac.uk/protvar where data can be explored and downloaded, and can be accessed programmatically via an API.

A foundational atlas of autism protein interactions reveals molecular convergence.

Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.

Antigenic mapping of the hemagglutinin of the H9 subtype influenza A viruses using sera from Japanese quail (Coturnix c. japonica).

IMPORTANCE: Determining the relevant amino acids involved in antigenic drift on the surface protein hemagglutinin (HA) is critical to understand influenza virus evolution and efficient assessment of vaccine strains relative to current circulating strains. We used antigenic cartography to generate an antigenic map of the H9 hemagglutinin (HA) using sera produced in one of the most relevant minor poultry species, Japanese quail. Key antigenic positions were identified and tested to confirm their impact on the antigenic profile. This work provides a better understanding of the antigenic diversity of the H9 HA as it relates to reactivity to quail sera and will facilitate a rational approach for selecting more efficacious vaccines against poultry-origin H9 influenza viruses in minor poultry species.

Characterization of A/H7 influenza virus global antigenic diversity and key determinants in the hemagglutinin globular head mediating A/H7N9 antigenic evolution.

IMPORTANCE: A/H7 avian influenza viruses cause outbreaks in poultry globally, resulting in outbreaks with significant socio-economical impact and zoonotic risks. Occasionally, poultry vaccination programs have been implemented to reduce the burden of these viruses, which might result in an increased immune pressure accelerating antigenic evolution. In fact, evidence for antigenic diversification of A/H7 influenza viruses exists, posing challenges to pandemic preparedness and the design of vaccination strategies efficacious against drifted variants. Here, we performed a comprehensive analysis of the global antigenic diversity of A/H7 influenza viruses and identified the main substitutions in the hemagglutinin responsible for antigenic evolution in A/H7N9 viruses isolated between 2013 and 2019. The A/H7 antigenic map and knowledge of the molecular determinants of their antigenic evolution add value to A/H7 influenza virus surveillance programs, the design of vaccines and vaccination strategies, and pandemic preparedness.

Towards a structurally resolved human protein interaction network.

Cellular functions are governed by molecular machines that assemble through protein-protein interactions. Their atomic details are critical to studying their molecular mechanisms. However, fewer than 5% of hundreds of thousands of human protein interactions have been structurally characterized. Here we test the potential and limitations of recent progress in deep-learning methods using AlphaFold2 to predict structures for 65,484 human protein interactions. We show that experiments can orthogonally confirm higher-confidence models. We identify 3,137 high-confidence models, of which 1,371 have no homology to a known structure. We identify interface residues harboring disease mutations, suggesting potential mechanisms for pathogenic variants. Groups of interface phosphorylation sites show patterns of co-regulation across conditions, suggestive of coordinated tuning of multiple protein interactions as signaling responses. Finally, we provide examples of how the predicted binary complexes can be used to build larger assemblies helping to expand our understanding of human cell biology.

Plasticity of the Influenza Virus H5 HA Protein.

Since the emergence of highly pathogenic avian influenza viruses of the H5 subtype, the major viral antigen, hemagglutinin (HA), has undergone constant evolution, resulting in numerous genetic and antigenic (sub)clades. To explore the consequences of amino acid changes at sites that may affect the antigenicity of H5 viruses, we simultaneously mutated 17 amino acid positions of an H5 HA by using a synthetic gene library that, theoretically, encodes all combinations of the 20 amino acids at the 17 positions. All 251 mutant viruses sequenced possessed ≥13 amino acid substitutions in HA, demonstrating that the targeted sites can accommodate a substantial number of mutations. Selection with ferret sera raised against H5 viruses of different clades resulted in the isolation of 39 genotypes. Further analysis of seven variants demonstrated that they were antigenically different from the parental virus and replicated efficiently in mammalian cells. Our data demonstrate the substantial plasticity of the influenza virus H5 HA protein, which may lead to novel antigenic variants.IMPORTANCE The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants.

Energy Landscape for the Membrane Fusion Pathway in Influenza A Hemagglutinin From Discrete Path Sampling.

The conformational change associated with membrane fusion for Influenza A Hemagglutinin is investigated with a model based upon pre- and post-fusion structures of the HA2 component. We employ computational methods based on the potential energy landscape framework to obtain an initial path connecting these two end points, which provides the starting point for refinement of a kinetic transition network. Here we employ discrete path sampling, which provides access to the experimental time and length scales via geometry optimization techniques to identify local minima and the transition states that connect them. We then analyse the distinct phases of the predicted pathway in terms of structure and energetics, and compare with available experimental data and previous simulations. Our results provide the foundations for future work, which will address the effect of mutations, changes in pH, and incorporation of additional components, especially the HA1 chain and the fusion peptide.

Epistatic interactions can moderate the antigenic effect of substitutions in haemagglutinin of influenza H3N2 virus.

We previously showed that single amino acid substitutions at seven positions in haemagglutinin determined major antigenic change of influenza H3N2 virus. Here, the impact of two such substitutions was tested in 11 representative H3 haemagglutinins to investigate context-dependence effects. The antigenic effect of substitutions introduced at haemagglutinin position 145 was fully independent of the amino acid context of the representative haemagglutinins. Antigenic change caused by substitutions introduced at haemagglutinin position 155 was variable and context-dependent. Our results suggest that epistatic interactions with contextual amino acids in the haemagglutinin can moderate the magnitude of antigenic change.

Antigenic Drift of the Influenza A(H1N1)pdm09 Virus Neuraminidase Results in Reduced Effectiveness of A/California/7/2009 (H1N1pdm09)-Specific Antibodies.

The effectiveness of influenza vaccines against circulating A(H1N1)pdm09 viruses was modest for several seasons despite the absence of antigenic drift of hemagglutinin (HA), the primary vaccine component. Since antibodies against HA and neuraminidase (NA) contribute independently to protection against disease, antigenic changes in NA may allow A(H1N1)pdm09 viruses to escape from vaccine-induced immunity. In this study, analysis of the specificities of human NA-specific monoclonal antibodies identified antigenic sites that have changed over time. The impact of these differences on in vitro inhibition of enzyme activity was not evident for polyclonal antisera until viruses emerged in 2013 without a predicted glycosylation site at amino acid 386 in NA. Phylogenetic and antigenic cartography demonstrated significant antigenic changes that in most cases aligned with genetic differences. Typical of NA drift, the antigenic difference is observed in one direction, with antibodies against conserved antigenic domains in A/California/7/2009 (CA/09) continuing to inhibit NA of recent A(H1N1)pdm09 viruses reasonably well. However, ferret CA/09-specific antiserum that inhibited the NA of A/Michigan/45/2015 (MI/15) very well in vitro, protected mice against lethal MI/15 infection poorly. These data show that antiserum against the homologous antigen is most effective and suggest the antigenic properties of NA should not be overlooked when selecting viruses for vaccine production.IMPORTANCE The effectiveness of seasonal influenza vaccines against circulating A(H1N1)pdm09 viruses has been modest in recent years, despite the absence of antigenic drift of HA, the primary vaccine component. Human monoclonal antibodies identified antigenic sites in NA that changed early after the new pandemic virus emerged. The reactivity of ferret antisera demonstrated antigenic drift of A(H1N1)pdm09 NA from 2013 onward. Passive transfer of serum raised against A/California/7/2009 was less effective than ferret serum against the homologous virus in protecting mice against a virus with the NA of more recent virus, A/Michigan/45/2015. Given the long-standing observation that NA-inhibiting antibodies are associated with resistance against disease in humans, these data demonstrate the importance of evaluating NA drift and suggest that vaccine effectiveness might be improved by selecting viruses for vaccine production that have NAs antigenically similar to those of circulating influenza viruses.

Plasticity of Amino Acid Residue 145 Near the Receptor Binding Site of H3 Swine Influenza A Viruses and Its Impact on Receptor Binding and Antibody Recognition.

The hemagglutinin (HA), a glycoprotein on the surface of influenza A virus (IAV), initiates the virus life cycle by binding to terminal sialic acid (SA) residues on host cells. The HA gradually accumulates amino acid substitutions that allow IAV to escape immunity through a mechanism known as antigenic drift. We recently confirmed that a small set of amino acid residues are largely responsible for driving antigenic drift in swine-origin H3 IAV. All identified residues are located adjacent to the HA receptor binding site (RBS), suggesting that substitutions associated with antigenic drift may also influence receptor binding. Among those substitutions, residue 145 was shown to be a major determinant of antigenic evolution. To determine whether there are functional constraints to substitutions near the RBS and their impact on receptor binding and antigenic properties, we carried out site-directed mutagenesis experiments at the single-amino-acid level. We generated a panel of viruses carrying substitutions at residue 145 representing all 20 amino acids. Despite limited amino acid usage in nature, most substitutions at residue 145 were well tolerated without having a major impact on virus replication in vitro All substitution mutants retained receptor binding specificity, but the substitutions frequently led to decreased receptor binding. Glycan microarray analysis showed that substitutions at residue 145 modulate binding to a broad range of glycans. Furthermore, antigenic characterization identified specific substitutions at residue 145 that altered antibody recognition. This work provides a better understanding of the functional effects of amino acid substitutions near the RBS and the interplay between receptor binding and antigenic drift.IMPORTANCE The complex and continuous antigenic evolution of IAVs remains a major hurdle for vaccine selection and effective vaccination. On the hemagglutinin (HA) of the H3N2 IAVs, the amino acid substitution N 145 K causes significant antigenic changes. We show that amino acid 145 displays remarkable amino acid plasticity in vitro, tolerating multiple amino acid substitutions, many of which have not yet been observed in nature. Mutant viruses carrying substitutions at residue 145 showed no major impairment in virus replication in the presence of lower receptor binding avidity. However, their antigenic characterization confirmed the impact of the 145 K substitution in antibody immunodominance. We provide a better understanding of the functional effects of amino acid substitutions implicated in antigenic drift and its consequences for receptor binding and antigenicity. The mutation analyses presented in this report represent a significant data set to aid and test the ability of computational approaches to predict binding of glycans and in antigenic cartography analyses.

Characterization of influenza A(H1N1)pdm09 viruses isolated from Nepalese and Indian outbreak patients in early 2015.

We characterized influenza A(H1N1)pdm09 isolates from large-scale outbreaks that occurred in Nepal and India in early 2015. Although no specific viral features, which may have caused the outbreaks, were identified, an S84N substitution in hemagglutinin was frequently observed. Chronological phylogenetic analysis revealed that these Nepalese and Indian viruses possessing the S84N substitution constitute potential ancestors of the novel genetic subclade 6B.1 virus that spread globally in the following (2015/16) influenza season. Thus, active surveillance of circulating influenza viruses in the Southern Asia region, including Nepal and India, would be beneficial for detecting novel variant viruses prior to their worldwide spread.

Influenza Research Database: An integrated bioinformatics resource for influenza virus research.

The Influenza Research Database (IRD) is a U.S. National Institute of Allergy and Infectious Diseases (NIAID)-sponsored Bioinformatics Resource Center dedicated to providing bioinformatics support for influenza virus research. IRD facilitates the research and development of vaccines, diagnostics and therapeutics against influenza virus by providing a comprehensive collection of influenza-related data integrated from various sources, a growing suite of analysis and visualization tools for data mining and hypothesis generation, personal workbench spaces for data storage and sharing, and active user community support. Here, we describe the recent improvements in IRD including the use of cloud and high performance computing resources, analysis and visualization of user-provided sequence data with associated metadata, predictions of novel variant proteins, annotations of phenotype-associated sequence markers and their predicted phenotypic effects, hemagglutinin (HA) clade classifications, an automated tool for HA subtype numbering conversion, linkouts to disease event data and the addition of host factor and antiviral drug components. All data and tools are freely available without restriction from the IRD website at https://www.fludb.org.

Selection of antigenically advanced variants of seasonal influenza viruses.

Influenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing random mutations in the globular head of the haemagglutinin protein (which includes the antigenic sites) by incubating them with human and/or ferret convalescent sera to human H1N1 and H3N2 viruses. We also selected antigenic escape variants from human viruses treated with convalescent sera and from mice that had been previously immunized against human influenza viruses. Our pilot studies with past influenza viruses identified escape mutants that were antigenically similar to variants that emerged in nature, establishing the feasibility of our approach. Our studies with contemporary human influenza viruses identified escape mutants before they caused an epidemic in 2014-2015. This approach may aid in the prediction of potential antigenic escape variants and the selection of future vaccine candidates before they become widespread in nature.

The global antigenic diversity of swine influenza A viruses.

Swine influenza presents a substantial disease burden for pig populations worldwide and poses a potential pandemic threat to humans. There is considerable diversity in both H1 and H3 influenza viruses circulating in swine due to the frequent introductions of viruses from humans and birds coupled with geographic segregation of global swine populations. Much of this diversity is characterized genetically but the antigenic diversity of these viruses is poorly understood. Critically, the antigenic diversity shapes the risk profile of swine influenza viruses in terms of their epizootic and pandemic potential. Here, using the most comprehensive set of swine influenza virus antigenic data compiled to date, we quantify the antigenic diversity of swine influenza viruses on a multi-continental scale. The substantial antigenic diversity of recently circulating viruses in different parts of the world adds complexity to the risk profiles for the movement of swine and the potential for swine-derived infections in humans.

Amino Acid Substitutions That Affect Receptor Binding and Stability of the Hemagglutinin of Influenza A/H7N9 Virus.

Receptor-binding preference and stability of hemagglutinin have been implicated as crucial determinants of airborne transmission of influenza viruses. Here, amino acid substitutions previously identified to affect these traits were tested in the context of an A/H7N9 virus. Some combinations of substitutions, most notably G219S and K58I, resulted in relatively high affinity for α2,6-linked sialic acid receptor and acid and temperature stability. Thus, the hemagglutinin of the A/H7N9 virus may adopt traits associated with airborne transmission.

Identification of amino acid substitutions supporting antigenic change of influenza A(H1N1)pdm09 viruses.

UNLABELLED: The majority of currently circulating influenza A(H1N1) viruses are antigenically similar to the virus that caused the 2009 influenza pandemic. However, antigenic variants are expected to emerge as population immunity increases. Amino acid substitutions in the hemagglutinin protein can result in escape from neutralizing antibodies, affect viral fitness, and change receptor preference. In this study, we constructed mutants with substitutions in the hemagglutinin of A/Netherlands/602/09 in an attenuated backbone to explore amino acid changes that may contribute to emergence of antigenic variants in the human population. Our analysis revealed that single substitutions affecting the loop that consists of amino acid positions 151 to 159 located adjacent to the receptor binding site caused escape from ferret and human antibodies elicited after primary A(H1N1)pdm09 virus infection. The majority of these substitutions resulted in similar or increased replication efficiency in vitro compared to that of the virus carrying the wild-type hemagglutinin and did not result in a change of receptor preference. However, none of the substitutions was sufficient for escape from the antibodies in sera from individuals that experienced both seasonal and pandemic A(H1N1) virus infections. These results suggest that antibodies directed against epitopes on seasonal A(H1N1) viruses contribute to neutralization of A(H1N1)pdm09 antigenic variants, thereby limiting the number of possible substitutions that could lead to escape from population immunity. IMPORTANCE: Influenza A viruses can cause significant morbidity and mortality in humans. Amino acid substitutions in the hemagglutinin protein can result in escape from antibody-mediated neutralization. This allows the virus to reinfect individuals that have acquired immunity to previously circulating strains through infection or vaccination. To date, the vast majority of A(H1N1)pdm09 strains remain antigenically similar to the virus that caused the 2009 influenza pandemic. However, antigenic variants are expected to emerge as a result of increasing population immunity. We show that single amino acid substitutions near the receptor binding site were sufficient to escape from antibodies specific for A(H1N1)pdm09 viruses but not from antibodies elicited in response to infections with seasonal A(H1N1) and A(H1N1)pdm09 viruses. This study identified substitutions in A(H1N1)pdm09 viruses that support escape from population immunity but also suggested that the number of potential escape variants is limited by previous exposure to seasonal A(H1N1) viruses.

A recommended numbering scheme for influenza A HA subtypes.

Comparisons of residues between sub-types of influenza virus is increasingly used to assess the zoonotic potential of a circulating strain and for comparative studies across subtypes. An analysis of N-terminal cleavage sites for thirteen subtypes of influenza A hemagglutinin (HA) sequences, has previously been described by Nobusawa and colleagues. We have expanded this analysis for the eighteen known subtypes of influenza. Due to differences in the length of HA, we have included strains from multiple clades of H1 and H5, as well as strains of H5 and H7 subtypes with both high and low pathogenicity. Analysis of known structures of influenza A HA enables us to define amino acids which are structurally and functionally equivalent across all HA subtypes using a numbering system based on the mature HA sequence. We provide a list of equivalences for amino acids which are known to affect the phenotype of the virus.

Polymorphisms at amino acid residues 141 and 154 influence conformational variation in ovine PrP.

Polymorphisms in ovine PrP at amino acid residues 141 and 154 are associated with susceptibility to ovine prion disease: Leu141Arg154 with classical scrapie and Phe141Arg154 and Leu141His154 with atypical scrapie. Classical scrapie is naturally transmissible between sheep, whereas this may not be the case with atypical scrapie. Critical amino acid residues will determine the range or stability of structural changes within the ovine prion protein or its functional interaction with potential cofactors, during conversion of PrPC to PrPSc in these different forms of scrapie disease. Here we computationally identified that regions of ovine PrP, including those near amino acid residues 141 and 154, displayed more conservation than expected based on local structural environment. Molecular dynamics simulations showed these conserved regions of ovine PrP displayed genotypic differences in conformational repertoire and amino acid side-chain interactions. Significantly, Leu141Arg154 PrP adopted an extended beta sheet arrangement in the N-terminal palindromic region more frequently than the Phe141Arg154 and Leu141His154 variants. We supported these computational observations experimentally using circular dichroism spectroscopy and immunobiochemical studies on ovine recombinant PrP. Collectively, our observations show amino acid residues 141 and 154 influence secondary structure and conformational change in ovine PrP that may correlate with different forms of scrapie.

Circulating avian influenza viruses closely related to the 1918 virus have pandemic potential.

Wild birds harbor a large gene pool of influenza A viruses that have the potential to cause influenza pandemics. Foreseeing and understanding this potential is important for effective surveillance. Our phylogenetic and geographic analyses revealed the global prevalence of avian influenza virus genes whose proteins differ only a few amino acids from the 1918 pandemic influenza virus, suggesting that 1918-like pandemic viruses may emerge in the future. To assess this risk, we generated and characterized a virus composed of avian influenza viral segments with high homology to the 1918 virus. This virus exhibited pathogenicity in mice and ferrets higher than that in an authentic avian influenza virus. Further, acquisition of seven amino acid substitutions in the viral polymerases and the hemagglutinin surface glycoprotein conferred respiratory droplet transmission to the 1918-like avian virus in ferrets, demonstrating that contemporary avian influenza viruses with 1918 virus-like proteins may have pandemic potential.

Antigenic variation of clade 2.1 H5N1 virus is determined by a few amino acid substitutions immediately adjacent to the receptor binding site.

UNLABELLED: Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are genetically highly variable and have diversified into multiple phylogenetic clades over the past decade. Antigenic drift is a well-studied phenomenon for seasonal human influenza viruses, but much less is known about the antigenic evolution of HPAI H5N1 viruses that circulate in poultry. In this study, we focused on HPAI H5N1 viruses that are enzootic to Indonesia. We selected representative viruses from genetically distinct lineages that are currently circulating and determined their antigenic properties by hemagglutination inhibition assays. At least six antigenic variants have circulated between 2003, when H5N1 clade 2.1 viruses were first detected in Indonesia, and 2011. During this period, multiple antigenic variants cocirculated in the same geographic regions. Mutant viruses were constructed by site-directed mutagenesis to represent each of the circulating antigenic variants, revealing that antigenic differences between clade 2.1 viruses were due to only one or very few amino acid substitutions immediately adjacent to the receptor binding site. Antigenic variants of H5N1 virus evaded recognition by both ferret and chicken antibodies. The molecular basis for antigenic change in clade 2.1 viruses closely resembled that of seasonal human influenza viruses, indicating that the hemagglutinin of influenza viruses from different hosts and subtypes may be similarly restricted to evade antibody recognition. IMPORTANCE: Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for severe outbreaks in both commercial and backyard poultry, causing considerable economic losses and regular zoonotic transmissions to humans. Vaccination is used increasingly to reduce the burden of HPAI H5N1 virus in poultry. Influenza viruses can escape from recognition by antibodies induced upon vaccination or infection through genetic changes in the hemagglutinin protein. The evolutionary patterns and molecular basis of antigenic change in HPAI H5N1 viruses are poorly understood, hampering formulation of optimal vaccination strategies. We have shown here that HPAI H5N1 viruses in Indonesia diversified into multiple antigenic variants, that antigenic differences were due to one or a very few substitutions near the receptor binding site, and that the molecular basis for antigenic change was remarkably similar to that for seasonal human influenza viruses. These findings have consequences for future vaccination and surveillance considerations and contribute to the understanding of the antigenic evolution of influenza viruses.