Conformational Control of Fast Asparagine Deamidation in a Norovirus Capsid Protein.

Accelerated spontaneous deamidation of asparagine 373 and subsequent conversion into an isoaspartate has been shown to attenuate the binding of histo blood group antigens (HBGAs) to the protruding domain (P-domain) of the capsid protein of a prevalent norovirus strain (GII.4). Here, we link an unusual backbone conformation of asparagine 373 to its fast site-specific deamidation. NMR spectroscopy and ion exchange chromatography have been used to monitor the deamidation reaction of P-domains of two closely related GII.4 norovirus strains, specific point mutants, and control peptides. MD simulations over several microseconds have been instrumental to rationalize the experimental findings. While conventional descriptors such as available surface area, root-mean-square fluctuations, or nucleophilic attack distance fail as explanations, the population of a rare syn-backbone conformation distinguishes asparagine 373 from all other asparagine residues. We suggest that stabilization of this unusual conformation enhances the nucleophilicity of the backbone nitrogen of aspartate 374, in turn accelerating the deamidation of asparagine 373. This finding should be relevant to the development of reliable prediction algorithms for sites of rapid asparagine deamidation in proteins.

On the way to plant data commons - a genotyping use case.

Over the last years it has been observed that the progress in data collection in life science has created increasing demand and opportunities for advanced bioinformatics. This includes data management as well as the individual data analysis and often covers the entire data life cycle. A variety of tools have been developed to store, share, or reuse the data produced in the different domains such as genotyping. Especially imputation, as a subfield of genotyping, requires good Research Data Management (RDM) strategies to enable use and re-use of genotypic data. To aim for sustainable software, it is necessary to develop tools and surrounding ecosystems, which are reusable and maintainable. Reusability in the context of streamlined tools can e.g. be achieved by standardizing the input and output of the different tools and adapting to open and broadly used file formats. By using such established file formats, the tools can also be connected with others, improving the overall interoperability of the software. Finally, it is important to build strong communities that maintain the tools by developing and contributing new features and maintenance updates. In this article, concepts for this will be presented for an imputation service.

The chromatin remodeling protein ATRX positively regulates IRF3-dependent type I interferon production and interferon-induced gene expression.

The chromatin remodeling protein alpha thalassemia/mental retardation syndrome X-linked (ATRX) is a component of promyelocytic leukemia nuclear bodies (PML-NBs) and thereby mediates intrinsic immunity against several viruses including human cytomegalovirus (HCMV). As a consequence, viruses have evolved different mechanisms to antagonize ATRX, such as displacement from PML-NBs or degradation. Here, we show that depletion of ATRX results in an overall impaired antiviral state by decreasing transcription and subsequent secretion of type I IFNs, which is followed by reduced expression of interferon-stimulated genes (ISGs). ATRX interacts with the transcription factor interferon regulatory factor 3 (IRF3) and associates with the IFN-ß promoter to facilitate transcription. Furthermore, whole transcriptome sequencing revealed that ATRX is required for efficient IFN-induced expression of a distinct set of ISGs. Mechanistically, we found that ATRX positively modulates chromatin accessibility specifically upon IFN signaling, thereby affecting promoter regions with recognition motifs for AP-1 family transcription factors. In summary, our study uncovers a novel co-activating function of the chromatin remodeling factor ATRX in innate immunity that regulates chromatin accessibility and subsequent transcription of interferons and ISGs. Consequently, ATRX antagonization by viral proteins and ATRX mutations in tumors represent important strategies to broadly compromise both intrinsic and innate immune responses.

DivBrowse-interactive visualization and exploratory data analysis of variant call matrices.

BACKGROUND: The sequencing of whole genomes is becoming increasingly affordable. In this context, large-scale sequencing projects are generating ever larger datasets of species-specific genomic diversity. As a consequence, more and more genomic data need to be made easily accessible and analyzable to the scientific community. FINDINGS: We present DivBrowse, a web application for interactive visualization and exploratory analysis of genomic diversity data stored in Variant Call Format (VCF) files of any size. By seamlessly combining BLAST as an entry point together with interactive data analysis features such as principal component analysis in one graphical user interface, DivBrowse provides a novel and unique set of exploratory data analysis capabilities for genomic biodiversity datasets. The capability to integrate DivBrowse into existing web applications supports interoperability between different web applications. Built-in interactive computation of principal component analysis allows users to perform ad hoc analysis of the population structure based on specific genetic elements such as genes and exons. Data interoperability is supported by the ability to export genomic diversity data in VCF and General Feature Format 3 files. CONCLUSION: DivBrowse offers a novel approach for interactive visualization and analysis of genomic diversity data and optionally also gene annotation data by including features like interactive calculation of variant frequencies and principal component analysis. The use of established standard file formats for data input supports interoperability and seamless deployment of application instances based on the data output of established bioinformatics pipelines.

Stable and Inducible Gene Knockdown in Primary Human Fibroblasts: A Versatile Tool to Study the Role of Human Cytomegalovirus Host Cell Factors.

Human fibroblasts represent the most extensively used cell type for the investigation of lytic human cytomegalovirus (HCMV) replication. However, analyzing the function of specific proteins during infection can be challenging since primary cells are difficult to transfect. An alternative approach is the use of lentiviral transduction with vectors for stable or inducible shRNA expression. This approach provides a versatile tool to study the role of host cell factors during HCMV infection. The essential steps to achieve an efficient target protein knockdown are shRNA design, cloning, generation of transgenic lentiviral particles, and, finally, transduction of the cells. However, these steps are highly dependent on the selected vector system. Here we focus on two different vector systems and describe how to successfully generate stable and inducible knockdown fibroblasts. Additionally, we demonstrate different methods to validate the knockdown of the target protein.

The Autophagy-Initiating Protein Kinase ULK1 Phosphorylates Human Cytomegalovirus Tegument Protein pp28 and Regulates Efficient Virus Release.

Autophagy is a catabolic process contributing to intrinsic cellular defense by degrading viral particles or proteins; however, several viruses hijack this pathway for their own benefit. The role of autophagy during human cytomegalovirus (HCMV) replication has not been definitely clarified yet. Utilizing small interfering RNA (siRNA)-based screening, we observed that depletion of many autophagy-related proteins resulted in reduced virus release, suggesting a requirement of autophagy-related factors for efficient HCMV replication. Additionally, we could show that the autophagy-initiating serine/threonine protein kinase ULK1 as well as other constituents of the ULK1 complex were upregulated at early times of infection and stayed upregulated throughout the replication cycle. We demonstrate that indirect interference with ULK1 through inhibition of the upstream regulator AMP-activated protein kinase (AMPK) impaired virus release. Furthermore, this result was verified by direct abrogation of ULK1 kinase activity utilizing the ULK1-specific kinase inhibitors SBI-0206965 and ULK-101. Analysis of viral protein expression in the presence of ULK-101 revealed a connection between the cellular kinase ULK1 and the viral tegument protein pp28 (pUL99), and we identified pp28 as a novel viral substrate of ULK1 by in vitro kinase assays. In the absence of ULK1 kinase activity, large pp28- and pp65-positive structures could be detected in the cytoplasm at late time points of infection. Transmission electron microscopy demonstrated that these structures represent large perinuclear protein accumulations presumably representing aggresomes. Our results indicate that HCMV manipulates ULK1 and further components of the autophagic machinery to ensure the efficient release of viral particles.IMPORTANCE The catabolic program of autophagy represents a powerful immune defense against viruses that is, however, counteracted by antagonizing viral factors. Understanding the exact interplay between autophagy and HCMV infection is of major importance since autophagy-related proteins emerged as promising targets for pharmacologic intervention. Our study provides evidence for a proviral role of several autophagy-related proteins suggesting that HCMV has developed strategies to usurp components of the autophagic machinery for its own benefit. In particular, we observed strong upregulation of the autophagy-initiating protein kinase ULK1 and further components of the ULK1 complex during HCMV replication. In addition, both siRNA-mediated depletion of ULK1 and interference with ULK1 protein kinase activity by two chemically different inhibitors resulted in impaired viral particle release. Thus, we propose that ULK1 kinase activity is required for efficient HCMV replication and thus represents a promising novel target for future antiviral drug development.

The on-premise data sharing infrastructure e!DAL: Foster FAIR data for faster data acquisition.

BACKGROUND: The FAIR data principle as a commitment to support long-term research data management is widely accepted in the scientific community. Although the ELIXIR Core Data Resources and other established infrastructures provide comprehensive and long-term stable services and platforms for FAIR data management, a large quantity of research data is still hidden or at risk of getting lost. Currently, high-throughput plant genomics and phenomics technologies are producing research data in abundance, the storage of which is not covered by established core databases. This concerns the data volume, e.g., time series of images or high-resolution hyper-spectral data; the quality of data formatting and annotation, e.g., with regard to structure and annotation specifications of core databases; uncovered data domains; or organizational constraints prohibiting primary data storage outside institional boundaries. RESULTS: To share these potentially dark data in a FAIR way and master these challenges the ELIXIR Germany/de.NBI service Plant Genomic and Phenomics Research Data Repository (PGP) implements a "bring the infrastructure to the data" approach, which allows research data to be kept in place and wrapped in a FAIR-aware software infrastructure. This article presents new features of the e!DAL infrastructure software and the PGP repository as a best practice on how to easily set up FAIR-compliant and intuitive research data services. Furthermore, the integration of the ELIXIR Authentication and Authorization Infrastructure (AAI) and data discovery services are introduced as means to lower technical barriers and to increase the visibility of research data. CONCLUSION: The e!DAL software matured to a powerful and FAIR-compliant infrastructure, while keeping the focus on flexible setup and integration into existing infrastructures and into the daily research process.

BRIDGE - A Visual Analytics Web Tool for Barley Genebank Genomics.

Genebanks harbor a large treasure trove of untapped plant genetic diversity. A growing world population and a changing climate require an increase in the production and development of stress resistant plant cultivars while decreasing the acreage. These requirements for improved plant cultivars can be supported by the broader exploitation of plant genetic resources (PGR) as inputs for genomics-assisted breeding. To support this process we have developed BRIDGE, a data warehouse and exploratory data analysis tool for genebank genomics of barley (Hordeum vulgare L.). Using efficient technologies for data storage, data transfer and web development, we facilitate access to digital genebank resources of barley by prioritizing the interactive and visual analysis of integrated genotypic and phenotypic data. The underlying data resulted from a barley genebank genomics study cataloging sequence and morphological data of 22,626 barley accessions, mainly from the German Federal ex situ genebank. BRIDGE consists of interactively coupled modules to visualize integrated, curated and quality checked data, such as variation data, results of dimensionality reduction and genome wide association studies (GWAS), phenotyping results, passport data as well as the geographic distribution of germplasm samples. The core component is a manager for custom collections of germplasm. A search module to find and select germplasm by passport and phenotypic attributes is included as well as modules to export genotypic data in gzip-compressed variant call format (VCF) files and phenotypic data in MIAPPE-compliant ISA-Tab files. BRIDGE is accessible at the following URL: https://bridge.ipk-gatersleben.de.

Programmatic Access to FAIRified Digital Plant Genetic Resources.

Genetic variance within the genotype of population and its mapping to phenotype variance in a systematic and high throughput manner is of interest for biodiversity and breeding research. Beside the established and efficient high throughput genotype technologies, phenotype capabilities got increased focus in the last decade. This results in an increasing amount of phenotype data from well scaling, automated sensor platform. Thus, data stewardship is a central component to make experimental data from multiple domains interoperable and re-usable. To ensure a standard and comprehensive sharing of scientific and experimental data among domain experts, FAIR data principles are utilized for machine read-ability and scale-ability. In this context, BrAPI consortium, provides a comprehensive and commonly agreed FAIRed guidelines to offer a BrAPI layered scientific data in a RESTful manner. This paper presents the concepts, best practices and implementations to meet these challenges. As one of the worlds leading plant research institutes it is of vital interest for the IPK-Gatersleben to transform legacy data infrastructures into a bio-digital resource center for plant genetics resources (PGR). This paper also demonstrates the benefits of integrated database back-ends, established data stewardship processes, and FAIR data exposition in a machine-readable, highly scalable programmatic interfaces.

isa4j: a scalable Java library for creating ISA-Tab metadata.

Experimental data is only useful to other researchers if it is findable, accessible, interoperable, and reusable (FAIR). The ISA-Tab framework enables scientists to publish metadata about their experiments in a plain text, machine-readable format that aims to confer that interoperability and reusability. A Python software package (isatools) is currently being developed to programmatically produce these metadata files. For Java-based environments, there is no equivalent solution yet. While the isatools package provides a lot of flexibility and a wealth of different features for the Python ecosystem, a package for JVM-based applications might offer the speed and scalability needed for writing very large ISA-Tab files, making the ISA framework available in an even wider range of situations and environments. Here we present a light-weight and scalable Java library (isa4j) for generating metadata files in the ISA-Tab format, which elegantly integrates into existing JVM applications and especially shines at generating very large files. It is modeled after the ISA core specifications and designed in keeping with isatools conventions, making it consistent and intuitive to use for the community. isa4j is implemented in Java (JDK11+) and freely available under the terms of the MIT license from the Central Maven Repository ( https://mvnrepository.com/artifact/de.ipk-gatersleben/isa4j). The source code, detailed documentation, usage examples and performance evaluations can be found at https://github.com/IPK-BIT/isa4j.

BrAPI-an application programming interface for plant breeding applications.

MOTIVATION: Modern genomic breeding methods rely heavily on very large amounts of phenotyping and genotyping data, presenting new challenges in effective data management and integration. Recently, the size and complexity of datasets have increased significantly, with the result that data are often stored on multiple systems. As analyses of interest increasingly require aggregation of datasets from diverse sources, data exchange between disparate systems becomes a challenge. RESULTS: To facilitate interoperability among breeding applications, we present the public plant Breeding Application Programming Interface (BrAPI). BrAPI is a standardized web service API specification. The development of BrAPI is a collaborative, community-based initiative involving a growing global community of over a hundred participants representing several dozen institutions and companies. Development of such a standard is recognized as critical to a number of important large breeding system initiatives as a foundational technology. The focus of the first version of the API is on providing services for connecting systems and retrieving basic breeding data including germplasm, study, observation, and marker data. A number of BrAPI-enabled applications, termed BrAPPs, have been written, that take advantage of the emerging support of BrAPI by many databases. AVAILABILITY AND IMPLEMENTATION: More information on BrAPI, including links to the specification, test suites, BrAPPs, and sample implementations is available at https://brapi.org/. The BrAPI specification and the developer tools are provided as free and open source.

Genebank genomics highlights the diversity of a global barley collection.

Genebanks hold comprehensive collections of cultivars, landraces and crop wild relatives of all major food crops, but their detailed characterization has so far been limited to sparse core sets. The analysis of genome-wide genotyping-by-sequencing data for almost all barley accessions of the German ex situ genebank provides insights into the global population structure of domesticated barley and points out redundancies and coverage gaps in one of the world's major genebanks. Our large sample size and dense marker data afford great power for genome-wide association scans. We detect known and novel loci underlying morphological traits differentiating barley genepools, find evidence for convergent selection for barbless awns in barley and rice and show that a major-effect resistance locus conferring resistance to bymovirus infection has been favored by traditional farmers. This study outlines future directions for genomics-assisted genebank management and the utilization of germplasm collections for linking natural variation to human selection during crop evolution.

Dynamic regulatory interaction between cytomegalovirus major tegument protein pp65 and protein kinase pUL97 in intracellular compartments, dense bodies and virions.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen of considerable clinical importance. Understanding the processes that are important for viral replication is essential for the development of therapeutic strategies against HCMV infection. The HCMV-encoded protein kinase pUL97 is an important multifunctional regulator of viral replication. Several viral and cellular proteins are phosphorylated by pUL97. The phosphoprotein pp65 is one important substrate of pUL97. It is the most abundant tegument protein of HCMV virions, mediating the upload of other virion constituents and contributing to particle integrity. Further to that, it interferes with host innate immune defences, thereby enabling efficient viral replication. By applying different approaches, we characterized the pp65-pUL97 interaction in various compartments. Specifically, the pUL97 interaction domain of pp65 was defined (282-415). A putative cyclin bridge that enhances pUL97-pp65 interaction was identified. The impact of pUL97 mutation on virion and dense body morphogenesis was addressed using pUL97 mutant viruses. Alterations in the proteome of viral particles were seen, especially with mutant viruses expressing cytoplasmic variants of pUL97. On the basis of these data we postulate a so far poorly recognized functional relationship between pp65 and pUL97, and present a refined model of pp65-pUL97 interaction.

HopZ4 from Pseudomonas syringae, a member of the HopZ type III effector family from the YopJ superfamily, inhibits the proteasome in plants.

The YopJ family of type III effector proteins (T3E) is one of the largest and most widely distributed families of effector proteins, whose members are highly diversified in virulence functions. In the present study, HopZ4, a member of the YopJ family of T3E from the cucumber pathogen Pseudomonas syringae pv. lachrymans is described. HopZ4 shares high sequence similarity with the Xanthomonas T3E XopJ, and a functional analysis suggests a conserved virulence function between these two T3E. As has previously been shown for XopJ, HopZ4 interacts with the proteasomal subunit RPT6 in yeast and in planta to inhibit proteasome activity during infection. The inhibitory effect on the proteasome is dependent on localization of HopZ4 to the plasma membrane as well as on an intact catalytic triad of the effector protein. Furthermore, HopZ4 is able to complement loss of XopJ in Xanthomonas spp., as it prevents precocious host cell death during a compatible Xanthomonas-pepper interaction. The data presented here suggest that different bacterial species employ inhibition of the proteasome as a virulence strategy by making use of conserved T3E from the YopJ family of bacterial effector proteins.