Diversity and Distribution of a Novel Genus of Hyperthermophilic Aquificae Viruses Encoding a Proof-Reading Family-A DNA Polymerase
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59 BASIC BIOLOGICAL SCIENCES↗
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Results for “viral genomics”
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RolyPoly (rp) v0.1.0
The Rolypoly pipeline is designed to process raw RNA-seq data and identify potential RNA viral sequences. It is split into several self contained steps: 1. input data filtering and QC, 2. Genome assembly and refinement, 3. Assembly filtering, 4. Mapping to known RNA viral genomes, 5. Searching for RNA viral marker genes. 6. Genome functional and structural annotation. 6. Report preparation and potential downstream analysis The last module, may include taxonomic assignment, host range estimation, and phenotypic prediction. There are many similar software, but they focus on human related viruses, and lack the downstream applications or differ in their sensitivity. The initial user base are non-computational microbial ecologists who wish to better understand the potential RNA viruses in their own generated samples.
Neri, Uri↗
Unveiling the microbial realm with VEBA 2.0: a modular bioinformatics suite for end-to-end genome-resolved prokaryotic, (micro)eukaryotic and viral multi-omics from either short- or long-read sequencing
Abstract The microbiome is a complex community of microorganisms, encompassing prokaryotic (bacterial and archaeal), eukaryotic, and viral entities. This microbial ensemble plays a pivotal role in influencing the health and productivity of diverse ecosystems while shaping the web of life. However, many software suites developed to study microbiomes analyze only the prokaryotic community and provide limited to no support for viruses and microeukaryotes. Previously, we introduced the Viral Eukaryotic Bacterial Archaeal (VEBA) open-source software suite to address this critical gap in microbiome research by extending genome-resolved analysis beyond prokaryotes to encompass the understudied realms of eukaryotes and viruses. Here we present VEBA 2.0 with key updates including a comprehensive clustered microeukaryotic protein database, rapid genome/protein-level clustering, bioprospecting, non-coding/organelle gene modeling, genome-resolved taxonomic/pathway profiling, long-read support, and containerization. We demonstrate VEBA’s versatile application through the analysis of diverse case studies including marine water, Siberian permafrost, and white-tailed deer lung tissues with the latter showcasing how to identify integrated viruses. VEBA represents a crucial advancement in microbiome research, offering a powerful and accessible software suite that bridges the gap between genomics and biotechnological solutions.
59 BASIC BIOLOGICAL SCIENCES↗
Spatial and temporal metagenomics of river compartments reveals viral community dynamics in an urban impacted stream
Although river ecosystems constitute a small fraction of Earth’s total area, they are critical modulators of microbially and virally orchestrated global biogeochemical cycles. However, most studies either use data that is not spatially resolved or is collected at timepoints that do not reflect the short life cycles of microorganisms. To address this gap, we assessed how viral and microbial communities change over a 48-hour period by sampling surface water and pore water compartments of the wastewater-impacted River Erpe in Germany. We sampled every 3 hours resulting in 32 samples for which we obtained metagenomes along with geochemical and metabolite measurements. From our metagenomes, we identified 6,500 viral and 1,033 microbial metagenome assembled genomes (MAGs) and found distinct community membership and abundance associated with each river compartment (e.g., Competibacteraceae in surfacewater and Sulfurimonadaceae in pore water). We show that 17% of our viral MAGs clustered to viruses from other ecosystems like wastewater treatment plants and rivers. Our results also indicated that 70% of the viral community was persistent in surface waters, whereas only 13% were persistent in the pore waters taken from the hyporheic zone. Finally, we predicted linkages between 73 viral genomes and 38 microbial genomes. These putatively linked hosts included members of the Competibacteraceae, which we suggest are potential contributors to river carbon and nitrogen cycling via denitrification and nitrogen fixation. Together, these findings demonstrate that members of the surface water microbiome from this urban river are stable over multiple diurnal cycles. These temporal insights raise important considerations for ecosystem models attempting to constrain dynamics of river biogeochemical cycles.
54 ENVIRONMENTAL SCIENCES↗
Pooled PPIseq: Screening the SARS-CoV-2 and human interface with a scalable multiplexed protein-protein interaction assay platform
Protein-Protein Interactions (PPIs) are a key interface between virus and host, and these interactions are important to both viral reprogramming of the host and to host restriction of viral infection. In particular, viral-host PPI networks can be used to further our understanding of the molecular mechanisms of tissue specificity, host range, and virulence. At higher scales, viral-host PPI screening could also be used to screen for small-molecule antivirals that interfere with essential viral-host interactions, or to explore how the PPI networks between interacting viral and host genomes co-evolve. Current high-throughput PPI assays have screened entire viral-host PPI networks. However, these studies are time consuming, often require specialized equipment, and are difficult to further scale. Here, we develop methods that make larger-scale viral-host PPI screening more accessible. This approach combines the mDHFR split-tag reporter with the iSeq2 interaction-barcoding system to permit massively-multiplexed PPI quantification by simple pooled engineering of barcoded constructs, integration of these constructs into budding yeast, and fitness measurements by pooled cell competitions and barcode-sequencing. We applied this method to screen for PPIs between SARS-CoV-2 proteins and human proteins, screening in triplicate >180,000 ORF-ORF combinations represented by >1,000,000 barcoded lineages. Our results complement previous screens by identifying 74 putative PPIs, including interactions between ORF7A with the taste receptors TAS2R41 and TAS2R7, and between NSP4 with the transmembrane KDELR2 and KDELR3. We show that this PPI screening method is highly scalable, enabling larger studies aimed at generating a broad understanding of how viral effector proteins converge on cellular targets to effect replication.
60 APPLIED LIFE SCIENCES↗
Phage-specific metabolic reprogramming of virocells
Ocean viruses are abundant and infect 20–40% of surface microbes. Infected cells, termed virocells, are thus a predominant microbial state. Yet, virocells and their ecosystem impacts are understudied, thus precluding their incorporation into ecosystem models. Here we investigated how unrelated bacterial viruses (phages) reprogram one host into contrasting virocells with different potential ecosystem footprints. We independently infected the marine Pseudoalteromonas bacterium with siphovirus PSA-HS2 and podovirus PSA-HP1. Time-resolved multi-omics unveiled drastically different metabolic reprogramming and resource requirements by each virocell, which were related to phage–host genomic complementarity and viral fitness. Namely, HS2 was more complementary to the host in nucleotides and amino acids, and fitter during infection than HP1. Functionally, HS2 virocells hardly differed from uninfected cells, with minimal host metabolism impacts. HS2 virocells repressed energy-consuming metabolisms, including motility and translation. Contrastingly, HP1 virocells substantially differed from uninfected cells. They repressed host transcription, responded to infection continuously, and drastically reprogrammed resource acquisition, central carbon and energy metabolisms. Ecologically, this work suggests that one cell, infected versus uninfected, can have immensely different metabolisms that affect the ecosystem differently. Finally, we relate phage–host genome complementarity, virocell metabolic reprogramming, and viral fitness in a conceptual model to guide incorporating viruses into ecosystem models.
Environmental Sciences & Ecology↗
Structures of diverse poxin cGAMP nucleases reveal a widespread role for cGAS-STING evasion in host–pathogen conflict
DNA viruses in the family Poxviridae encode poxin enzymes that degrade the immune second messenger 2'3'-cGAMP to inhibit cGAS-STING immunity in mammalian cells. The closest homologs of poxin exist in the genomes of insect viruses suggesting a key mechanism of cGAS-STING evasion may have evolved outside of mammalian biology. Here we use a biochemical and structural approach to discover a broad family of 369 poxins encoded in diverse viral and animal genomes and define a prominent role for 2'3'-cGAMP cleavage in metazoan host-pathogen conflict. Structures of insect poxins reveal unexpected homology to flavivirus proteases and enable identification of functional self-cleaving poxins in RNA-virus polyproteins. Our data suggest widespread 2'3'-cGAMP signaling in insect antiviral immunity and explain how a family of cGAS-STING evasion enzymes evolved from viral proteases through gain of secondary nuclease activity. Poxin acquisition by poxviruses demonstrates the importance of environmental connections in shaping evolution of mammalian pathogens.
59 BASIC BIOLOGICAL SCIENCES↗
Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis
Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation1. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. However, many viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.
Weiss, Trevor↗
Life strategies for Aminicenantia in subseafloor oceanic crust
Abstract After decades studying the microbial “deep biosphere” in subseafloor oceanic crust, the growth and life strategies in this anoxic, low energy habitat remain poorly described. Using both single cell genomics and metagenomics, we reveal the life strategies of two distinct lineages of uncultivated Aminicenantia bacteria from the basaltic subseafloor oceanic crust of the eastern flank of the Juan de Fuca Ridge. Both lineages appear adapted to scavenge organic carbon, as each have genetic potential to catabolize amino acids and fatty acids, aligning with previous Aminicenantia reports. Given the organic carbon limitation in this habitat, seawater recharge and necromass may be important carbon sources for heterotrophic microorganisms inhabiting the ocean crust. Both lineages generate ATP via several mechanisms including substrate-level phosphorylation, anaerobic respiration, and electron bifurcation driving an Rnf ion translocation membrane complex. Genomic comparisons suggest these Aminicenantia transfer electrons extracellularly, perhaps to iron or sulfur oxides consistent with mineralogy of this site. One lineage, called JdFR-78, has small genomes that are basal to the Aminicenantia class and potentially use “primordial” siroheme biosynthetic intermediates for heme synthesis, suggesting this lineage retain characteristics of early evolved life. Lineage JdFR-78 contains CRISPR-Cas defenses to evade viruses, while other lineages contain prophage that may help prevent super-infection or no detectable viral defenses. Overall, genomic evidence points to Aminicenantia being well adapted to oceanic crust environments by taking advantage of simple organic molecules and extracellular electron transport.
Environmental Sciences & Ecology↗
SARS-CoV-2 evolution balances conflicting roles of N protein phosphorylation
All lineages of SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, contain mutations between amino acids 199 and 205 in the nucleocapsid (N) protein that are associated with increased infectivity. The effects of these mutations have been difficult to determine because N protein contributes to both viral replication and viral particle assembly during infection. Here, we used single-cycle infection and virus-like particle assays to show that N protein phosphorylation has opposing effects on viral assembly and genome replication. Ancestral SARS-CoV-2 N protein is densely phosphorylated, leading to higher levels of genome replication but 10-fold lower particle assembly compared to evolved variants with low N protein phosphorylation, such as Delta (N:R203M), Iota (N:S202R), and B.1.2 (N:P199L). A new open reading frame encoding a truncated N protein called N*, which occurs in the B.1.1 lineage and subsequent lineages of the Alpha, Gamma, and Omicron variants, supports high levels of both assembly and replication. Our findings help explain the enhanced fitness of viral variants of concern and a potential avenue for continued viral selection.
Microbiology↗
Lower viral evolutionary pressure under stable versus fluctuating conditions in subzero Arctic brines
Climate change threatens Earth’s ice-based ecosystems which currently offer archives and eco-evolutionary experiments in the extreme. Arctic cryopeg brine (marine-derived, within permafrost) and sea ice brine, similar in subzero temperature and high salinity but different in temporal stability, are inhabited by microbes adapted to these extreme conditions. However, little is known about their viruses (community composition, diversity, interaction with hosts, or evolution) or how they might respond to geologically stable cryopeg versus fluctuating sea ice conditions. We used long- and short-read viromics and metatranscriptomics to study viruses in Arctic cryopeg brine, sea ice brine, and underlying seawater, recovering 11,088 vOTUs (~species-level taxonomic unit), a 4.4-fold increase of known viruses in these brines. More specifically, the long-read-powered viromes doubled the number of longer (≥25 kb) vOTUs generated and recovered more hypervariable regions by >5-fold compared to short-read viromes. Distribution assessment, by comparing to known viruses in public databases, supported that cryopeg brine viruses were of marine origin yet distinct from either sea ice brine or seawater viruses, while 94% of sea ice brine viruses were also present in seawater. A virus-encoded, ecologically important exopolysaccharide biosynthesis gene was identified, and many viruses (~half of metatranscriptome-inferred “active” vOTUs) were predicted as actively infecting the dominant microbial genera Marinobacter and Polaribacter in cryopeg and sea ice brines, respectively. Evolutionarily, microdiversity (intra-species genetic variations) analyses suggested that viruses within the stable cryopeg brine were under significantly lower evolutionary pressures than those in the fluctuating sea ice environment, while many sea ice brine virus-tail genes were under positive selection, indicating virus-host co-evolutionary arms races. Our results confirmed the benefits of long-read-powered viromics in understanding the environmental virosphere through significantly improved genomic recovery, expanding viral discovery and the potential for biological inference. Evidence of viruses actively infecting the dominant microbes in subzero brines and modulating host metabolism underscored the potential impact of viruses on these remote and underexplored extreme ecosystems. Microdiversity results shed light on different strategies viruses use to evolve and adapt when extreme conditions are stable versus fluctuating. Together, these findings verify the value of long-read-powered viromics and provide foundational data on viral evolution and virus-microbe interactions in Earth’s destabilized and rapidly disappearing cryosphere.
59 BASIC BIOLOGICAL SCIENCES↗
VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature
Microbes play fundamental roles in shaping natural ecosystem properties and functions, but do so under constraints imposed by their viral predators. However, studying viruses in nature can be challenging due to low biomass and the lack of universal gene markers. Though metagenomic short-read sequencing has greatly improved our virus ecology toolkit—and revealed many critical ecosystem roles for viruses—microdiverse populations and fine-scale genomic traits are missed. Some of these microdiverse populations are abundant and the missed regions may be of interest for identifying selection pressures that underpin evolutionary constraints associated with hosts and environments. Though long-read sequencing promises complete virus genomes on single reads, it currently suffers from high DNA requirements and sequencing errors that limit accurate gene prediction. Here we introduce VirION2, an integrated short- and long-read metagenomic wet-lab and informatics pipeline that updates our previous method (VirION) to further enhance the utility of long-read viral metagenomics. Using a viral mock community, we first optimized laboratory protocols (polymerase choice, DNA shearing size, PCR cycling) to enable 76% longer reads (now median length of 6,965 bp) from 100-fold less input DNA (now 1 nanogram). Using a virome from a natural seawater sample, we compared viromes generated with VirION2 against other library preparation options (unamplified, original VirION, and short-read), and optimized downstream informatics for improved long-read error correction and assembly. VirION2 assemblies combined with short-read based data (‘enhanced’ viromes), provided significant improvements over VirION libraries in the recovery of longer and more complete viral genomes, and our optimized error-correction strategy using long- and short-read data achieved 99.97% accuracy. In the seawater virome, VirION2 assemblies captured 5,161 viral populations (including all of the virus populations observed in the other assemblies), 30% of which were uniquely assembled through inclusion of long-reads, and 22% of the top 10% most abundant virus populations derived from assembly of long-reads. Viral populations unique to VirION2 assemblies had significantly higher microdiversity means, which may explain why short-read virome approaches failed to capture them. These findings suggest the VirION2 sample prep and workflow can help researchers better investigate the virosphere, even from challenging low-biomass samples. Our new protocols are available to the research community on protocols.io as a ‘living document’ to facilitate dissemination of updates to keep pace with the rapid evolution of long-read sequencing technology.
Long-reads↗
The N-terminus of varicella-zoster virus glycoprotein B has a functional role in fusion
Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae , including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native, full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109 KSQD 112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109 AAAA 112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae .
59 BASIC BIOLOGICAL SCIENCES↗
Genetically engineered Foot and Mouth Disease Virus and related proteins, polynucleotides, compositions, methods and systems
Genetically engineered Foot and Mouth Disease Virus (FMDV) and related engineered proteins and polynucleotides, nanolipoprotein particles, compositions, methods and systems are described. The genetically engineered FMDV is modified by the strategic insertion of a protein tag into select regions of the FMDV genome which encode viral proteins that are exposed on the surface of the FMDV viral capsid. The inserted protein tag is displayed as a decoration or attachment on the viral capsid surface.
Rieder, Aida Elizabeth↗
Genetically engineered foot and mouth disease virus and related proteins, polynucleotides, compositions, methods and systems
Genetically engineered Foot and Mouth Disease Virus (FMDV) and related engineered proteins and polynucleotides, nanolipoprotein particles, compositions, methods and systems are described. The genetically engineered FMDV is modified by the strategic insertion of a protein tag into select regions of the FMDV genome which encode viral proteins that are exposed on the surface of the FMDV viral capsid. The inserted protein tag is displayed as a decoration or attachment on the viral capsid surface.
Rieder, Aida E.↗
Giant virus diversity and host interactions through global metagenomics
Our current knowledge about nucleocytoplasmic large DNA viruses (NCLDVs) is largely derived from viral isolates that are co-cultivated with protists and algae. Here we reconstructed 2,074 NCLDV genomes from sampling sites across the globe by building on the rapidly increasing amount of publicly available metagenome data. This led to an 11-fold increase in phylogenetic diversity and a parallel 10-fold expansion in functional diversity. Analysis of 58,023 major capsid proteins from large and giant viruses using metagenomic data revealed the global distribution patterns and cosmopolitan nature of these viruses. The discovered viral genomes encoded a wide range of proteins with putative roles in photosynthesis and diverse substrate transport processes, indicating that host reprogramming is probably a common strategy in the NCLDVs. Furthermore, inferences of horizontal gene transfer connected viral lineages to diverse eukaryotic hosts. We anticipate that the global diversity of NCLDVs that we describe here will establish giant viruses-which are associated with most major eukaryotic lineages-as important players in ecosystems across Earth's biomes.
59 BASIC BIOLOGICAL SCIENCES↗
Microbial colonization and persistence in deep fractured shales is guided by metabolic exchanges and viral predation
Background: Microbial colonization of subsurface shales following hydraulic fracturing offers the opportunity to study coupled biotic and abiotic factors that impact microbial persistence in engineered deep subsurface ecosystems. Shale formations underly much of the continental USA and display geographically distinct gradients in temperature and salinity. Complementing studies performed in eastern USA shales that contain brine-like fluids, here we coupled metagenomic and metabolomic approaches to develop the first genome-level insights into ecosystem colonization and microbial community interactions in a lower-salinity, but high-temperature western USA shale formation. Results: We collected materials used during the hydraulic fracturing process (i.e., chemicals, drill muds) paired with temporal sampling of water produced from three different hydraulically fractured wells in the STACK (Sooner Trend Anadarko Basin, Canadian and Kingfisher) shale play in OK, USA. Relative to other shale formations, our metagenomic and metabolomic analyses revealed an expanded taxonomic and metabolic diversity of microorganisms that colonize and persist in fractured shales. Importantly, temporal sampling across all three hydraulic fracturing wells traced the degradation of complex polymers from the hydraulic fracturing process to the production and consumption of organic acids that support sulfate- and thiosulfate-reducing bacteria. Furthermore, we identified 5587 viral genomes and linked many of these to the dominant, colonizing microorganisms, demonstrating the key role that viral predation plays in community dynamics within this closed, engineered system. Lastly, top-side audit sampling of different source materials enabled genome-resolved source tracking, revealing the likely sources of many key colonizing and persisting taxa in these ecosystems. Conclusions: These findings highlight the importance of resource utilization and resistance to viral predation as key traits that enable specific microbial taxa to persist across fractured shale ecosystems. We also demonstrate the importance of materials used in the hydraulic fracturing process as both a source of persisting shale microorganisms and organic substrates that likely aid in sustaining the microbial community. Moreover, we showed that different physicochemical conditions (i.e., salinity, temperature) can influence the composition and functional potential of persisting microbial communities in shale ecosystems. Together, these results expand our knowledge of microbial life in deep subsurface shales and have important ramifications for management and treatment of microbial biomass in hydraulically fractured wells.
59 BASIC BIOLOGICAL SCIENCES↗
IMG/VR v4: an expanded database of uncultivated virus genomes within a framework of extensive functional, taxonomic, and ecological metadata
Viruses are widely recognized as critical members of all microbiomes. Metagenomics enables large-scale exploration of the global virosphere, progressively revealing the extensive genomic diversity of viruses on Earth and highlighting the myriad of ways by which viruses impact biological processes. IMG/VR provides access to the largest collection of viral sequences obtained from (meta)genomes, along with functional annotation and rich metadata. A web interface enables users to efficiently browse and search viruses based on genome features and/or sequence similarity. Here, for this work, we present the fourth version of IMG/VR, composed of >15 million virus genomes and genome fragments, a ≈6-fold increase in size compared to the previous version. These clustered into 8.7 million viral operational taxonomic units, including 231 408 with at least one high-quality representative. Viral sequences in IMG/VR are now systematically identified from genomes, metagenomes, and metatranscriptomes using a new detection approach (geNomad), and IMG standard annotation are complemented with genome quality estimation using CheckV, taxonomic classification reflecting the latest taxonomic standards, and microbial host taxonomy prediction. IMG/VR v4 is available at https://img.jgi.doe.gov/vr, and the underlying data are available to download at https://genome.jgi.doe.gov/portal/IMG_VR.
59 BASIC BIOLOGICAL SCIENCES↗