Protein Interaction Mapping Identifies RBBP6 as a Negative Regulator of Ebola Virus Replication.

Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.

Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

Design of universal Ebola virus vaccine candidates via immunofocusing.

The Ebola virus causes hemorrhagic fever in humans and poses a significant threat to global public health. Although two viral vector vaccines have been approved to prevent Ebola virus disease, they are distributed in the limited ring vaccination setting and only indicated for prevention of infection from orthoebolavirus zairense (EBOV)-one of three orthoebolavirus species that have caused previous outbreaks. Ebola virus glycoprotein GP mediates viral infection and serves as the primary target of neutralizing antibodies. Here, we describe a universal Ebola virus vaccine approach using a structure-guided design of candidates with hyperglycosylation that aims to direct antibody responses away from variable regions and toward conserved epitopes of GP. We first determined the hyperglycosylation landscape on Ebola virus GP and used that to generate hyperglycosylated GP variants with two to four additional glycosylation sites to mask the highly variable glycan cap region. We then created vaccine candidates by displaying wild-type or hyperglycosylated GP variants on ferritin nanoparticles (Fer). Immunization with these antigens elicited potent neutralizing antisera against EBOV in mice. Importantly, we observed consistent cross-neutralizing activity against Bundibugyo virus and Sudan virus from hyperglycosylated GP-Fer with two or three additional glycans. In comparison, elicitation of cross-neutralizing antisera was rare in mice immunized with wild-type GP-Fer. These results demonstrate a potential strategy to develop universal Ebola virus vaccines that confer cross-protective immunity against existing and emerging filovirus species.

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, and exert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.

CyVerse: Cyberinfrastructure for open science.

CyVerse, the largest publicly-funded open-source research cyberinfrastructure for life sciences, has played a crucial role in advancing data-driven research since the 2010s. As the technology landscape evolved with the emergence of cloud computing platforms, machine learning and artificial intelligence (AI) applications, CyVerse has enabled access by providing interfaces, Software as a Service (SaaS), and cloud-native Infrastructure as Code (IaC) to leverage new technologies. CyVerse services enable researchers to integrate institutional and private computational resources, custom software, perform analyses, and publish data in accordance with open science principles. Over the past 13 years, CyVerse has registered more than 124,000 verified accounts from 160 countries and was used for over 1,600 peer-reviewed publications. Since 2011, 45,000 students and researchers have been trained to use CyVerse. The platform has been replicated and deployed in three countries outside the US, with additional private deployments on commercial clouds for US government agencies and multinational corporations. In this manuscript, we present a strategic blueprint for creating and managing SaaS cyberinfrastructure and IaC as free and open-source software.

Standards recommendations for the Earth BioGenome Project.

A global international initiative, such as the Earth BioGenome Project (EBP), requires both agreement and coordination on standards to ensure that the collective effort generates rapid progress toward its goals. To this end, the EBP initiated five technical standards committees comprising volunteer members from the global genomics scientific community: Sample Collection and Processing, Sequencing and Assembly, Annotation, Analysis, and IT and Informatics. The current versions of the resulting standards documents are available on the EBP website, with the recognition that opportunities, technologies, and challenges may improve or change in the future, requiring flexibility for the EBP to meet its goals. Here, we describe some highlights from the proposed standards, and areas where additional challenges will need to be met.

Network medicine framework for identifying drug-repurposing opportunities for COVID-19.

The COVID-19 pandemic has highlighted the need to quickly and reliably prioritize clinically approved compounds for their potential effectiveness for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Here, we deployed algorithms relying on artificial intelligence, network diffusion, and network proximity, tasking each of them to rank 6,340 drugs for their expected efficacy against SARS-CoV-2. To test the predictions, we used as ground truth 918 drugs experimentally screened in VeroE6 cells, as well as the list of drugs in clinical trials that capture the medical community's assessment of drugs with potential COVID-19 efficacy. We find that no single predictive algorithm offers consistently reliable outcomes across all datasets and metrics. This outcome prompted us to develop a multimodal technology that fuses the predictions of all algorithms, finding that a consensus among the different predictive methods consistently exceeds the performance of the best individual pipelines. We screened in human cells the top-ranked drugs, obtaining a 62% success rate, in contrast to the 0.8% hit rate of nonguided screenings. Of the six drugs that reduced viral infection, four could be directly repurposed to treat COVID-19, proposing novel treatments for COVID-19. We also found that 76 of the 77 drugs that successfully reduced viral infection do not bind the proteins targeted by SARS-CoV-2, indicating that these network drugs rely on network-based mechanisms that cannot be identified using docking-based strategies. These advances offer a methodological pathway to identify repurposable drugs for future pathogens and neglected diseases underserved by the costs and extended timeline of de novo drug development.

ORF10-Cullin-2-ZYG11B complex is not required for SARS-CoV-2 infection.

In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2ZYG11B). Since CRL2ZYG11B mediates protein degradation, one possible role for ORF10 is to "hijack" CRL2ZYG11B in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2ZYG11B or functions in other ways, for example, as an inhibitor or substrate of CRL2ZYG11B While we confirm the ORF10-ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2ZYG11B Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2ZYG11B is not relevant for SARS-CoV-2 infection in vitro.

Anti-SARS-CoV-2 activity of targeted kinase inhibitors: Repurposing clinically available drugs for COVID-19 therapy.

Coronavirus disease 2019 (COVID-19) remains a major public health concern, and vaccine unavailability, hesitancy, or failure underscore the need for discovery of efficacious antiviral drug therapies. Numerous approved drugs target protein kinases associated with viral life cycle and symptoms of infection. Repurposing of kinase inhibitors is appealing as they have been vetted for safety and are more accessible for COVID-19 treatment. However, an understanding of drug mechanism is needed to improve our understanding of the factors involved in pathogenesis. We tested the in vitro activity of three kinase inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including inhibitors of AXL kinase, a host cell factor that contributes to successful SARS-CoV-2 infection. Using multiple cell-based assays and approaches, gilteritinib, nintedanib, and imatinib were thoroughly evaluated for activity against SARS-CoV-2 variants. Each drug exhibited antiviral activity, but with stark differences in potency, suggesting differences in host dependency for kinase targets. Importantly, for gilteritinib, the amount of compound needed to achieve 90% infection inhibition, at least in part involving blockade of spike protein-mediated viral entry and at concentrations not inducing phospholipidosis (PLD), approached a clinically achievable concentration. Knockout of AXL, a target of gilteritinib and nintedanib, impaired SARS-CoV-2 variant infectivity, supporting a role for AXL in SARS-CoV-2 infection and supporting further investigation of drug-mediated AXL inhibition as a COVID-19 treatment. This study supports further evaluation of AXL-targeting kinase inhibitors as potential antiviral agents and treatments for COVID-19. Additional mechanistic studies are needed to determine underlying differences in virus response.

A Naturally Occurring Polymorphism in the Base of Sudan Virus Glycoprotein Decreases Glycoprotein Stability in a Species-Dependent Manner.

Sudan virus (SUDV) is one of five filoviruses that compose the genus Ebolavirus that has been responsible for episodic outbreaks in Central Africa. While the SUDV glycoprotein (GP) structure has been solved, GP residues that affect SUDV entry have not been extensively examined; many of the entry characteristics of SUDV GP are inferred from studies with the Zaire Ebola virus (EBOV) GP. Here, we investigate the effect on virus entry of a naturally occurring polymorphism in SUDV GP. Two of the earliest SUDV isolates contain glutamine at residue 95 (Q95) within the base region of GP1, whereas more recent SUDV isolates and GPs from all other ebolaviruses carry lysine at this position (K95). A K95Q change dramatically decreased titers of pseudovirions bearing SUDV GP, whereas the K95Q substitution in EBOV GP had no effect on titer. We evaluated virus entry to identify SUDV GP Q95-specific entry defects. The presence of Q95 in either EBOV or SUDV GP resulted in enhanced sensitivity of GP to proteolytic processing, yet this could not account for the SUDV-specific decrease in GP Q95 infectivity. We found that SUDV GP Q95 pseudovirions were more sensitive to imipramine, a GP-destabilizing antiviral. In contrast, SUDV GP K95 was more stable, requiring elevated temperatures to inhibit virus infection. Thus, the residue present at GP 95 has a critical role in stabilizing the SUDV glycoprotein, whereas this polymorphism has no effect on EBOV GP stability. These results provide novel insights into filovirus species-specific GP structure that affects virus infectivity. IMPORTANCE Filovirus outbreaks are associated with significant morbidity and mortality. Understanding the structural constraints of filoviral GPs that control virus entry into cells is critical for rational development of novel antivirals to block infection. Here, we identify a naturally occurring glutamine (Q) to lysine (K) polymorphism at residue 95 as a critical determinant of Sudan virus GP stability but not Zaire Ebola virus GP stability. We propose that glutamine at residue 95 in Sudan virus GP mediates decreased virus entry, thereby reducing infectivity. Our findings highlight a unique structural characteristic of Sudan virus GP that affects GP-mediated functionality. Further, it provides a cautionary note for the development of future broad-spectrum filovirus antivirals.

An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations.

Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.

The Natural Product Eugenol Is an Inhibitor of the Ebola Virus In Vitro.

PURPOSE: Since the 2014 Ebola virus (EBOV) outbreak in West Africa there has been considerable effort towards developing drugs to treat Ebola virus disease and yet to date there is no FDA approved treatment. This is important as at the time of writing this manuscript there is an ongoing outbreak in the Democratic Republic of the Congo which has killed over 1000. METHODS: We have evaluated a small number of natural products, some of which had shown antiviral activity against other pathogens. This is exemplified with eugenol, which is found in high concentrations in multiple essential oils, and has shown antiviral activity against feline calicivirus, tomato yellow leaf curl virus, Influenza A virus, Herpes Simplex virus type 1 and 2, and four airborne phages. RESULTS: Four compounds possessed EC50 values less than or equal to 11 µM. Of these, eugenol, had an EC50 of 1.3 µM against EBOV and is present in several plants including clove, cinnamon, basil and bay. Eugenol is much smaller and structurally unlike any compound that has been previously identified as an inhibitor of EBOV, therefore it may provide new mechanistic insights. CONCLUSION: This compound is readily accessible in bulk quantities, is inexpensive, and has a long history of human consumption, which endorses the idea for further assessment as an antiviral therapeutic. This work also suggests that a more exhaustive assessment of natural product libraries against EBOV and other viruses is warranted to improve our ability to identify compounds that are so distinct from FDA approved drugs.

Autophagy-Associated Proteins Control Ebola Virus Internalization Into Host Cells.

Ebola virus (EBOV) enters host cells by macropinocytosis, a poorly understood process. Recent studies have suggested that cell factors involved in autophagy, an evolutionally conserved pathway leading to the lysosomal degradation of protein aggregates and organelles during cellular stress, also have roles in macropinocytosis. Here, we demonstrate that autophagy-associated proteins are required for trafficking of EBOV into the cell body. Depleting cells of beclin 1, autophagy-related protein 7, or microtubule-associated protein 1A/B light chain 3B (LC3B) abolished EBOV uptake, owing to a block in vesicle formation at the cell surface. Both LC3B-I and LC3B-II interacted with macropinocytic structures. Our work indicates that, although various forms of LC3B possess an inherent ability to associate with forming macropinosomes, LC3B-II is critical for internalization of macropinocytic vesicles and, therefore, EBOV from the cell surface.

Development of Clinical-Stage Human Monoclonal Antibodies That Treat Advanced Ebola Virus Disease in Nonhuman Primates.

Background: For most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases. Methods: In this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses. Results: Of the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent. Conclusions: This antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.

NanoOK: multi-reference alignment analysis of nanopore sequencing data, quality and error profiles.

MOTIVATION: The Oxford Nanopore MinION sequencer, currently in pre-release testing through the MinION Access Programme (MAP), promises long reads in real-time from an inexpensive, compact, USB device. Tools have been released to extract FASTA/Q from the MinION base calling output and to provide basic yield statistics. However, no single tool yet exists to provide comprehensive alignment-based quality control and error profile analysis--something that is extremely important given the speed with which the platform is evolving. RESULTS: NanoOK generates detailed tabular and graphical output plus an in-depth multi-page PDF report including error profile, quality and yield data. NanoOK is multi-reference, enabling detailed analysis of metagenomic or multiplexed samples. Four popular Nanopore aligners are supported and it is easily extensible to include others. AVAILABILITY AND IMPLEMENTATION: NanoOK is an open-source software, implemented in Java with supporting R scripts. It has been tested on Linux and Mac OS X and can be downloaded from https://github.com/TGAC/NanoOK. A VirtualBox VM containing all dependencies and the DH10B read set used in this article is available from http://opendata.tgac.ac.uk/nanook/. A Docker image is also available from Docker Hub--see program documentation https://documentation.tgac.ac.uk/display/NANOOK. CONTACT: richard.leggett@tgac.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Interferon-γ Inhibits Ebola Virus Infection.

Ebola virus outbreaks, such as the 2014 Makona epidemic in West Africa, are episodic and deadly. Filovirus antivirals are currently not clinically available. Our findings suggest interferon gamma, an FDA-approved drug, may serve as a novel and effective prophylactic or treatment option. Using mouse-adapted Ebola virus, we found that murine interferon gamma administered 24 hours before or after infection robustly protects lethally-challenged mice and reduces morbidity and serum viral titers. Furthermore, we demonstrated that interferon gamma profoundly inhibits Ebola virus infection of macrophages, an early cellular target of infection. As early as six hours following in vitro infection, Ebola virus RNA levels in interferon gamma-treated macrophages were lower than in infected, untreated cells. Addition of the protein synthesis inhibitor, cycloheximide, to interferon gamma-treated macrophages did not further reduce viral RNA levels, suggesting that interferon gamma blocks life cycle events that require protein synthesis such as virus replication. Microarray studies with interferon gamma-treated human macrophages identified more than 160 interferon-stimulated genes. Ectopic expression of a select group of these genes inhibited Ebola virus infection. These studies provide new potential avenues for antiviral targeting as these genes that have not previously appreciated to inhibit negative strand RNA viruses and specifically Ebola virus infection. As treatment of interferon gamma robustly protects mice from lethal Ebola virus infection, we propose that interferon gamma should be further evaluated for its efficacy as a prophylactic and/or therapeutic strategy against filoviruses. Use of this FDA-approved drug could rapidly be deployed during future outbreaks.

Avian influenza prevalence among hunter-harvested birds in a remote Canadian First Nation community.

INTRODUCTION: Avian influenza virus (AIV) prevalence has been associated with wild game and other bird species. The contamination of these birds may pose a greater risk to those who regularly hunt and consumed infected species. Due to resident concerns communicated by local Band Council, hunter-harvested birds from a remote First Nation community in subArctic Ontario, Canada were assessed for AIV. Hunters, and especially those who live a subsistence lifestyle, are at higher risk of AIV exposure due to their increased contact with wild birds, which represent an important part of their diet. METHODS: Cloacal swabs from 304 harvested game birds representing several species of wild birds commonly hunted and consumed in this First Nation community were analyzed for AIV using real-time reverse transcription polymerase chain reaction. Subtyping was performed using reverse transcription polymerase chain reaction. Sequences were assembled using Lasergene, and the sequences were compared to Genbank. RESULTS: In total, 16 of the 304 cloacal swab samples were positive for AIV. Of the 16 positive samples, 12 were found in mallard ducks, 3 were found in snow geese (wavies), and 1 positive sample was found in partridge. The AIV samples were subtyped, when possible, and found to be positive for the low pathogenic avian influenza virus subtypes H3 and H4. No samples were positive for subtypes of human concern, namely H5 and H7. CONCLUSIONS: This work represents the first AIV monitoring program results of hunter-harvested birds in a remote subsistence First Nation community. Community-level surveillance of AIV in remote subsistence hunting communities may help to identify future risks, while educating those who may have the highest exposure about proper handling of hunted birds. Ultimately, only low pathogenic strains of AIV were found, but monitoring should be continued and expanded to safeguard those with the highest exposure risk to AIV.

CerealsDB 3.0: expansion of resources and data integration.

BACKGROUND: The increase in human populations around the world has put pressure on resources, and as a consequence food security has become an important challenge for the 21st century. Wheat (Triticum aestivum) is one of the most important crops in human and livestock diets, and the development of wheat varieties that produce higher yields, combined with increased resistance to pests and resilience to changes in climate, has meant that wheat breeding has become an important focus of scientific research. In an attempt to facilitate these improvements in wheat, plant breeders have employed molecular tools to help them identify genes for important agronomic traits that can be bred into new varieties. Modern molecular techniques have ensured that the rapid and inexpensive characterisation of SNP markers and their validation with modern genotyping methods has produced a valuable resource that can be used in marker assisted selection. CerealsDB was created as a means of quickly disseminating this information to breeders and researchers around the globe. DESCRIPTION: CerealsDB version 3.0 is an online resource that contains a wide range of genomic datasets for wheat that will assist plant breeders and scientists to select the most appropriate markers for use in marker assisted selection. CerealsDB includes a database which currently contains in excess of a million putative varietal SNPs, of which several hundreds of thousands have been experimentally validated. In addition, CerealsDB also contains new data on functional SNPs predicted to have a major effect on protein function and we have constructed a web service to encourage data integration and high-throughput programmatic access. CONCLUSION: CerealsDB is an open access website that hosts information on SNPs that are considered useful for both plant breeders and research scientists. The recent inclusion of web services designed to federate genomic data resources allows the information on CerealsDB to be more fully integrated with the WheatIS network and other biological databases.