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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

CheckV assesses the quality and completeness of metagenome-assembled viral genomes

Abstract Millions of new viral sequences have been identified from metagenomes, but the quality and completeness of these sequences vary considerably. Here we present CheckV, an automated pipeline for identifying closed viral genomes, estimating the completeness of genome fragments and removing flanking host regions from integrated proviruses. CheckV estimates completeness by comparing sequences with a large database of complete viral genomes, including 76,262 identified from a systematic search of publicly available metagenomes, metatranscriptomes and metaviromes. After validation on mock datasets and comparison to existing methods, we applied CheckV to large and diverse collections of metagenome-assembled viral sequences, including IMG/VR and the Global Ocean Virome. This revealed 44,652 high-quality viral genomes (that is, >90% complete), although the vast majority of sequences were small fragments, which highlights the challenge of assembling viral genomes from short-read metagenomes. Additionally, we found that removal of host contamination substantially improved the accurate identification of auxiliary metabolic genes and interpretation of viral-encoded functions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Tunturi virus isolates and metagenome-assembled viral genomes provide insights into the virome of Acidobacteriota in Arctic tundra soils

Arctic soils are climate-critical areas, where microorganisms play crucial roles in nutrient cycling processes. Acidobacteriota are phylogenetically and physiologically diverse bacteria that are abundant and active in Arctic tundra soils. Still, surprisingly little is known about acidobacterial viruses in general and those residing in the Arctic in particular. Here, we applied both culture-dependent and -independent methods to study the virome of Acidobacteriota in Arctic soils. Five virus isolates, Tunturi 1–5, were obtained from Arctic tundra soils, Kilpisjärvi, Finland (69°N), using Tunturiibacter spp. strains originating from the same area as hosts. The new virus isolates have tailed particles with podo- (Tunturi 1, 2, 3), sipho- (Tunturi 4), or myovirus-like (Tunturi 5) morphologies. The dsDNA genomes of the viral isolates are 63–98 kbp long, except Tunturi 5, which is a jumbo phage with a 309-kbp genome. Tunturi 1 and Tunturi 2 share 88% overall nucleotide identity, while the other three are not related to one another. For over half of the open reading frames in Tunturi genomes, no functions could be predicted. To further assess the Acidobacteriota-associated viral diversity in Kilpisjärvi soils, bulk metagenomes from the same soils were explored and a total of 1881 viral operational taxonomic units (vOTUs) were bioinformatically predicted. Almost all vOTUs (98%) were assigned to the class Caudoviricetes. For 125 vOTUs, including five (near-)complete ones, Acidobacteriota hosts were predicted. Acidobacteriota-linked vOTUs were abundant across sites, especially in fens. Terriglobia-associated proviruses were observed in Kilpisjärvi soils, being related to proviruses from distant soils and other biomes. Approximately genus- or higher-level similarities were found between the Tunturi viruses, Kilpisjärvi vOTUs, and other soil vOTUs, suggesting some shared groups of Acidobacteriota viruses across soils. This study provides acidobacterial virus isolates as laboratory models for future research and adds insights into the diversity of viral communities associated with Acidobacteriota in tundra soils. Predicted virus-host links and viral gene functions suggest various interactions between viruses and their host microorganisms. Largely unknown sequences in the isolates and metagenome-assembled viral genomes highlight a need for more extensive sampling of Arctic soils to better understand viral functions and contributions to ecosystem-wide cycling processes in the Arctic.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Structural and functional analyses of SARS-CoV-2 Nsp3 and its specific interactions with the 5’ UTR of the viral genome

ABSTRACT Non-structural protein 3 (Nsp3) is the largest open reading frame encoded in the SARS-CoV-2 genome, essential for the formation of double-membrane vesicles (DMV) wherein viral RNA replication occurs. We conducted an extensive structure-function analysis of Nsp3 and determined the crystal structures of the ubiquitin-like 1 (Ubl1), nucleic acid binding (NAB), β-coronavirus-specific marker (βSM) domains, and a sub-region of the Y domain of this protein. We show that the Ubl1, ADP-ribose phosphatase (ADRP), human SARS Unique (HSUD), NAB, and Y domains of Nsp3 bind the 5’ UTR of the viral genome and that the Ubl1 and Y domains possess affinity for recognition of this region, suggesting high specificity. The Ubl1-Nucleocapsid (N) protein complex binds the 5’ UTR with greater affinity than the individual proteins alone. Our results suggest that multiple domains of Nsp3, particularly Ubl1 and Y, shepherd the 5’ UTR of the viral genome during translocation through the DMV membrane, priming the Ubl1 domain to load the genome onto N protein. IMPORTANCE The largest protein encoded by the SARS-CoV-2 genome is Nsp3. In infected cells, this multi-domain protein forms a pore structure in the virus-induced double-membrane vesicles (DMV). We have incomplete data on Nsp3 molecular structure, and here, we describe crystal structures for multiple domains of Nsp3. It is thought that newly replicated viral RNA transits through the DMV pore; however, we possess incomplete data on which regions of Nsp3 actually interact with RNA. Here, we present data showing that five domains of Nsp3 interact with the 5’ UTR of the SARS-CoV-2 RNA, including the Y domain for which no function has ever been discovered. These data suggest that the pore structure plays an active role in recognizing the terminal end of the genome, transiting and loading the viral RNA onto the cytoplasmic nucleocapsid protein. These data help expand our knowledge of Nsp3 structure and function and the SARS-CoV-2 replication cycle.

Microbiology↗
DOE OSTI2020

Visualizing a viral genome with contrast variation small angle X-ray scattering

Despite the threat to human health posed by some single-stranded RNA viruses, little is understood about their assembly. The goal of this work is to introduce a new tool for watching an RNA genome direct its own packaging and encapsidation by proteins. Contrast variation small-angle X-ray scattering (CV-SAXS) is a powerful tool with the potential to monitor the changing structure of a viral RNA through this assembly process. The proteins, though present, do not contribute to the measured signal. As a first step in assessing the feasibility of viral genome studies, the structure of encapsidated MS2 RNA was exclusively detected with CV-SAXS and compared with a structure derived from asymmetric cryo-EM reconstructions. Additional comparisons with free RNA highlight the significant structural rearrangements induced by capsid proteins and invite the application of time-resolved CV-SAXS to reveal interactions that result in efficient viral assembly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Expanding standards in viromics: in silico evaluation of dsDNA viral genome identification, classification, and auxiliary metabolic gene curation

Viruses influence global patterns of microbial diversity and nutrient cycles. Though viral metagenomics (viromics), specifically targeting dsDNA viruses, has been critical for revealing viral roles across diverse ecosystems, its analyses differ in many ways from those used for microbes. To date, viromics benchmarking has covered read pre-processing, assembly, relative abundance, read mapping thresholds and diversity estimation, but other steps would benefit from benchmarking and standardization. Here we use in silico-generated datasets and an extensive literature survey to evaluate and highlight how dataset composition (i.e., viromes vs bulk metagenomes) and assembly fragmentation impact (i) viral contig identification tool, (ii) virus taxonomic classification, and (iii) identification and curation of auxiliary metabolic genes (AMGs). The in silico benchmarking of five commonly used virus identification tools show that gene-content-based tools consistently performed well for long (≥3 kbp) contigs, while k -mer- and blast-based tools were uniquely able to detect viruses from short (≤3 kbp) contigs. Notably, however, the performance increase of k -mer- and blast-based tools for short contigs was obtained at the cost of increased false positives (sometimes up to ~5% for virome and ~75% bulk samples), particularly when eukaryotic or mobile genetic element sequences were included in the test datasets. Furthermore, for viral classification, variously sized genome fragments were assessed using gene-sharing network analytics to quantify drop-offs in taxonomic assignments, which revealed correct assignations ranging from ~95% (whole genomes) down to ~80% (3 kbp sized genome fragments). A similar trend was also observed for other viral classification tools such as VPF-class, ViPTree and VIRIDIC, suggesting that caution is warranted when classifying short genome fragments and not full genomes. Finally, we highlight how fragmented assemblies can lead to erroneous identification of AMGs and outline a best-practices workflow to curate candidate AMGs in viral genomes assembled from metagenomes. Together, these benchmarking experiments and annotation guidelines should aid researchers seeking to best detect, classify, and characterize the myriad viruses ‘hidden’ in diverse sequence datasets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis for cloverleaf RNA-initiated viral genome replication

The genomes of positive-strand RNA viruses serve as a template for both protein translation and genome replication. In enteroviruses, a cloverleaf RNA structure at the 5' end of the genome functions as a switch to transition from viral translation to replication by interacting with host poly(C)-binding protein 2 (PCBP2) and the viral 3CD pro protein. We determined the structures of cloverleaf RNA from coxsackievirus and poliovirus. Cloverleaf RNA folds into an H-type four-way junction and is stabilized by a unique adenosine-cytidine-uridine (A•C-U) base triple involving the conserved pyrimidine mismatch region. The two PCBP2 binding sites are spatially proximal and are located on the opposite end from the 3CD pro binding site on cloverleaf. We determined that the A•C-U base triple restricts the flexibility of the cloverleaf stem–loops resulting in partial occlusion of the PCBP2 binding site, and elimination of the A•C-U base triple increases the binding affinity of PCBP2 to the cloverleaf RNA. Based on the cloverleaf structures and biophysical assays, we propose a new mechanistic model by which enteroviruses use the cloverleaf structure as a molecular switch to transition from viral protein translation to genome replication.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

$\mathrm{COBRA}$ improves the completeness and contiguity of viral genomes assembled from metagenomes

Viruses are often studied using metagenome-assembled sequences, but genome incompleteness hampers comprehensive and accurate analyses. Contig Overlap Based Re-Assembly (COBRA) resolves assembly breakpoints based on the de Bruijn graph and joins contigs. Here we benchmarked COBRA using ocean and soil viral datasets. COBRA accurately joined the assembled sequences and achieved notably higher genome accuracy than binning tools. From 231 published freshwater metagenomes, we obtained 7,334 bacteriophage clusters, ~83% of which represent new phage species. Notably, ~70% of these were circular, compared with 34% before COBRA analyses. We expanded sampling of huge phages (≥200 kbp), the largest of which was curated to completion (717 kbp). Improved phage genomes from Rotsee Lake provided context for metatranscriptomic data and indicated the in situ activity of huge phages, whiB-encoding phages and cysC- and cysH-encoding phages. COBRA improves viral genome assembly contiguity and completeness, thus the accuracy and reliability of analyses of gene content, diversity and evolution.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2018

VIRULIGN: fast codon-correct alignment and annotation of viral genomes

Abstract Summary Virus sequence data are an essential resource for reconstructing spatiotemporal dynamics of viral spread as well as to inform treatment and prevention strategies. However, the potential benefit of these applications critically depends on accurate and correctly annotated alignments of genetically heterogeneous data. VIRULIGN was built for fast codon-correct alignments of large datasets, with standardized and formalized genome annotation and various alignment export formats. Availability and implementation VIRULIGN is freely available at https://github.com/rega-cev/virulign as an open source software project. Supplementary information Supplementary data is available at Bioinformatics online.

Libin, Pieter J. K. (ORCID:000000033906758X)↗
DOE OSTI2022

Propagation of viral genomes by replicating ammonia-oxidising archaea during soil nitrification

Ammonia-oxidising archaea (AOA) are a ubiquitous component of microbial communities and dominate the first stage of nitrification in some soils. While we are beginning to understand soil virus dynamics, we have no knowledge of the composition or activity of those infecting nitrifiers or their potential to influence processes. This study aimed to characterise viruses having infected autotrophic AOA in two nitrifying soils of contrasting pH by following transfer of assimilated CO 2 -derived 13 C from host to virus via DNA stable-isotope probing and metagenomic analysis. Incorporation of 13 C into low GC mol% AOA and virus genomes increased DNA buoyant density in CsCl gradients but resulted in co-migration with dominant non-enriched high GC mol% genomes, reducing sequencing depth and contig assembly. We therefore developed a hybrid approach where AOA and virus genomes were assembled from low buoyant density DNA with subsequent mapping of 13 C isotopically enriched high buoyant density DNA reads to identify activity of AOA. Metagenome-assembled genomes were different between the two soils and represented a broad diversity of active populations. Sixty-four AOA-infecting viral operational taxonomic units (vOTUs) were identified with no clear relatedness to previously characterised prokaryote viruses. These vOTUs were also distinct between soils, with 42% enriched in 13 C derived from hosts. The majority were predicted as capable of lysogeny and auxiliary metabolic genes included an AOA-specific multicopper oxidase suggesting infection may augment copper uptake essential for central metabolic functioning. These findings indicate virus infection of AOA may be a frequent process during nitrification with potential to influence host physiology and activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The Number and Pattern of Viral Genomic Reassortments are not Necessarily Identifiable from Segment Trees

Reassortment is an evolutionary process common in viruses with segmented genomes. These viruses can swap whole genomic segments during cellular co-infection, giving rise to novel progeny formed from the mixture of parental segments. Since large-scale genome rearrangements have the potential to generate new phenotypes, reassortment is important to both evolutionary biology and public health research. However, statistical inference of the pattern of reassortment events from phylogenetic data is exceptionally difficult, potentially involving inference of general graphs in which individual segment trees are embedded. In this paper, we argue that, in general, the number and pattern of reassortment events are not identifiable from segment trees alone, even with theoretically ideal data. We call this fact the fundamental problem of reassortment, which we illustrate using the concept of the “first-infection tree,” a potentially counterfactual genealogy that would have been observed in the segment trees had no reassortment occurred. Further, we illustrate four additional problems that can arise logically in the inference of reassortment events and show, using simulated data, that these problems are not rare and can potentially distort our observation of reassortment even in small data sets. Finally, we discuss how existing methods can be augmented or adapted to account for not only the fundamental problem of reassortment, but also the four additional situations that can complicate the inference of reassortment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Modeling the Influenza A NP-vRNA-Polymerase Complex in Atomic Detail

Seasonal flu is an acute respiratory disease that exacts a massive toll on human populations, healthcare systems and economies. The disease is caused by an enveloped Influenza virus containing eight ribonucleoprotein (RNP) complexes. Each RNP incorporates multiple copies of nucleoprotein (NP), a fragment of the viral genome (vRNA), and a viral RNA-dependent RNA polymerase (POL), and is responsible for packaging the viral genome and performing critical functions including replication and transcription. A complete model of an Influenza RNP in atomic detail can elucidate the structural basis for viral genome functions, and identify potential targets for viral therapeutics. In this work we construct a model of a complete Influenza A RNP complex in atomic detail using multiple sources of structural and sequence information and a series of homology-modeling techniques, including a motif-matching fragment assembly method. Our final model provides a rationale for experimentally-observed changes to viral polymerase activity in numerous mutational assays. Further, our model reveals specific interactions between the three primary structural components of the RNP, including potential targets for blocking POL-binding to the NP-vRNA complex. The methods developed in this work open the possibility of elucidating other functionally-relevant atomic-scale interactions in additional RNP structures and other biomolecular complexes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus

During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5’-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Pressurized DNA state inside herpes capsids—A novel antiviral target

Drug resistance in viruses represents one of the major challenges of healthcare. As part of an effort to provide a treatment that avoids the possibility of drug resistance, we discovered a novel mechanism of action (MOA) and specific compounds to treat all nine human herpesviruses and animal herpesviruses. The novel MOA targets the pressurized genome state in a viral capsid, “turns off” capsid pressure, and blocks viral genome ejection into a cell nucleus, preventing viral replication. This work serves as a proof-of-concept to demonstrate the feasibility of a new antiviral target—suppressing pressure-driven viral genome ejection —that is likely impervious to developing drug resistance. This pivotal finding presents a platform for discovery of a new class of broad-spectrum treatments for herpesviruses and other viral infections with genome-pressure-dependent replication. A biophysical approach to antiviral treatment such as this is also a vital strategy to prevent the spread of emerging viruses where vaccine development is challenged by high mutation rates or other evasion mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗