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At least 1,657 records · Page 92
All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

The Crystal Structure of the Ubiquitin-like Domain of Ribosome Assembly Factor Ytm1 and Characterization of Its Interaction with the AAA-ATPase Midasin

The synthesis of eukaryotic ribosomes is a complex, energetically demanding process requiring the aid of numerous non-ribosomal factors, such as the PeBoW complex. The mammalian PeBoW complex, composed of Pes1, Bop1, and WDR12, is essential for the processing of the 32S preribosomal RNA. Previous work in Saccharomyces cerevisiae has shown that release of the homologous proteins in this complex (Nop7, Erb1, and Ytm1, respectively) from preribosomal particles requires Rea1 (midasin or MDN1 in humans), a large dynein-like protein. Midasin contains a C-terminal metal ion-dependent adhesion site (MIDAS) domain that interacts with the N-terminal ubiquitin-like (UBL) domain of Ytm1/WDR12 as well as the UBL domain of Rsa4/Nle1 in a later step in the ribosome maturation pathway. Here we present the crystal structure of the UBL domain of the WDR12 homologue from S. cerevisiae at 1.7 Å resolution and demonstrate that human midasin binds to WDR12 as well as Nle1 through their respective UBL domains. Midasin contains a well conserved extension region upstream of the MIDAS domain required for binding WDR12 and Nle1, and the interaction is dependent upon metal ion coordination because removal of the metal or mutation of residues that coordinate the metal ion diminishes the interaction. Mammalian WDR12 displays prominent nucleolar localization that is dependent upon active ribosomal RNA transcription. Here, based upon these results, we propose that release of the PeBoW complex and subsequent release of Nle1 by midasin is a well conserved step in the ribosome maturation pathway in both yeast and mammalian cells.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

The structure of the Thermococcus gammatolerans McrB N-terminal domain reveals a new mode of substrate recognition and specificity among McrB homologs

McrBC is a two-component, modification-dependent restriction system that cleaves foreign DNA-containing methylated cytosines. Previous crystallographic studies have shown that Escherichia coli McrB uses a base-flipping mechanism to recognize these modified substrates with high affinity. The side chains stabilizing both the flipped base and the distorted duplex are poorly conserved among McrB homologs, suggesting that other mechanisms may exist for binding modified DNA. In this paper we present the structures of the Thermococcus gammatolerans McrB DNA-binding domain (TgΔ185) both alone and in complex with a methylated DNA substrate at 1.68 and 2.27 Å resolution, respectively. The structures reveal that TgΔ185 consists of a YT521-B homology (YTH) domain, which is commonly found in eukaryotic proteins that bind methylated RNA and is structurally unrelated to the E. coli McrB DNA-binding domain. Structural superposition and co-crystallization further show that TgΔ185 shares a conserved aromatic cage with other YTH domains, which forms the binding pocket for a flipped-out base. Mutational analysis of this aromatic cage supports its role in conferring specificity for the methylated adenines, whereas an extended basic surface present in TgΔ185 facilitates its preferential binding to duplex DNA rather than RNA. Together, these findings establish a new binding mode and specificity among McrB homologs and expand the biological roles of YTH domains.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Rapid and efficient in planta genome editing in sorghum using foxtail mosaic virus‐mediated sgRNA delivery

SUMMARY The requirement of in vitro tissue culture for the delivery of gene editing reagents limits the application of gene editing to commercially relevant varieties of many crop species. To overcome this bottleneck, plant RNA viruses have been deployed as versatile tools for in planta delivery of recombinant RNA. Viral delivery of single‐guide RNAs (sgRNAs) to transgenic plants that stably express CRISPR‐associated (Cas) endonuclease has been successfully used for targeted mutagenesis in several dicotyledonous and few monocotyledonous plants. Progress with this approach in monocotyledonous plants is limited so far by the availability of effective viral vectors. We engineered a set of foxtail mosaic virus (FoMV) and barley stripe mosaic virus (BSMV) vectors to deliver the fluorescent protein AmCyan to track viral infection and movement in Sorghum bicolor . We further used these viruses to deliver and express sgRNAs to Cas9 and Green Fluorescent Protein (GFP) expressing transgenic sorghum lines, targeting Phytoene desaturase ( PDS ), Magnesium‐chelatase subunit I ( MgCh ), 4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate reductase , orthologs of maize Lemon white1 ( Lw1 ) or GFP . The recombinant BSMV did neither infect sorghum nor deliver or express AmCyan and sgRNAs. In contrast, the recombinant FoMV systemically spread throughout sorghum plants and induced somatic mutations with frequencies reaching up to 60%. This mutagenesis led to visible phenotypic changes, demonstrating the potential of FoMV for in planta gene editing and functional genomics studies in sorghum.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Compilation and utilization of a sorghum transcriptome compendium for gene regulatory network analysis and crop trait engineering

Sorghum bicolor (Sorghum) is a drought and heat tolerant C4 grass crop used to produce grain, forage, biofuels, and other bioproducts. Genetic improvement of sorghum hybrid crops is aided by a large and diverse germplasm, sorghum's diploid inbreeding genetics, and a relatively small genome that has facilitated genomic research. Over the past 20 years, the sorghum research community characterized the cytogenetic and recombinant landscapes of sorghum's 10 chromosomes, sequenced and annotated the sorghum genome, and used that information to identify genes/alleles that modulate flowering time, plant height, seed shattering, and other important traits. More recently, >1000 RNA-seq transcriptome profiles were collected from 15 sorghum genotypes to help understand the genetic basis of variation in growth and development of sorghum stems, tillers, roots, and leaves, and the regulation of biosynthetic pathways that produce epicuticular wax, dhurrin, and RFOs, compounds that contribute to sorghum's resilience. Transcriptome studies were designed to identify differentially expressed genes that are co-expressed during development or in response to a treatment to enable construction of gene regulatory networks. Co-expression and network analysis identified transcription factors and their cognate binding sites in target gene promoters and signaling pathways that modulate gene regulatory networks providing gene editing targets for further trait optimization. RNA-seq data from >20 experiments targeting sorghum organs, tissues, cell types, developmental stages, and responses to environmental conditions (i.e., diel, day-length, shading, water-deficit, temperature) has been compiled in a sorghum transcriptome compendium. The goal of this resource paper is to describe compendium content, accessibility, and a compendium data analysis pipeline and to illustrate the types of information that can be derived from the compendium with a focus on the elucidation of gene regulatory networks useful for guiding the improvement of sorghum traits through gene editing.

RNA-seq↗
DOE OSTI2015

TU-CD-BRB-12: Radiogenomics of MRI-Guided Prostate Cancer Biopsy Habitats

Purpose: Diagnostic prostate biopsies are subject to sampling bias. We hypothesize that quantitative imaging with multiparametric (MP)-MRI can more accurately direct targeted biopsies to index lesions associated with highest risk clinical and genomic features. Methods: Regionally distinct prostate habitats were delineated on MP-MRI (T2-weighted, perfusion and diffusion imaging). Directed biopsies were performed on 17 habitats from 6 patients using MRI-ultrasound fusion. Biopsy location was characterized with 52 radiographic features. Transcriptome-wide analysis of 1.4 million RNA probes was performed on RNA from each habitat. Genomics features with insignificant expression values (<0.25) and interquartile range <0.5 were filtered, leaving total of 212 genes. Correlation between imaging features, genes and a 22 feature genomic classifier (GC), developed as a prognostic assay for metastasis after radical prostatectomy was investigated. Results: High quality genomic data was derived from 17 (100%) biopsies. Using the 212 ‘unbiased’ genes, the samples clustered by patient origin in unsupervised analysis. When only prostate cancer related genomic features were used, hierarchical clustering revealed samples clustered by needle-biopsy Gleason score (GS). Similarly, principal component analysis of the imaging features, found the primary source of variance segregated the samples into high (≥7) and low (6) GS. Pearson’s correlation analysis of genes with significant expression showed two main patterns of gene expression clustering prostate peripheral and transitional zone MRI features. Two-way hierarchical clustering of GC with radiomics features resulted in the expected groupings of high and low expressed genes in this metastasis signature. Conclusions: MP-MRI-targeted diagnostic biopsies can potentially improve risk stratification by directing pathological and genomic analysis to clinically significant index lesions. As determinant lesions are more reliably identified, targeting with radiotherapy should improve outcome. This is the first demonstration of a link between quantitative imaging features (radiomics) with genomic features in MRI-directed prostate biopsies. The research was supported by NIH- NCI R01 CA 189295 and R01 CA 189295; E Davicioni is partial owner of GenomeDx Biosciences, Inc. M Takhar, N Erho, L Lam, C Buerki and E Davicioni are current employees at GenomeDx Biosciences, Inc.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Structural Analysis of a Viral Ovarian Tumor Domain Protease from the Crimean-Congo Hemorrhagic Fever Virus in Complex with Covalently Bonded Ubiquitin

Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne, negative-sense, single-stranded RNA [ssRNA(-)] nairovirus that produces fever, prostration, and severe hemorrhages in humans. With fatality rates for CCHF ranging up to 70% based on several factors, CCHF is considered a dangerous emerging disease. Originally identified in the former Soviet Union and the Congo, CCHF has rapidly spread across large sections of Europe, Asia, and Africa. Recent reports have identified a viral homologue of the ovarian tumor protease superfamily (vOTU) within its L protein. This protease has subsequently been implicated in downregulation of the type I interferon immune response through cleavage of posttranslational modifying proteins ubiquitin (Ub) and the Ub-like interferon-simulated gene 15 (ISG15). Additionally, homologues of vOTU have been suggested to perform similar roles in the positive-sense, single-stranded RNA [ssRNA(+)] arteriviruses. By utilizing X-ray crystallographic techniques, the structure of vOTU covalently bound to ubiquitin propylamine, a suicide substrate of the enzyme, was elucidated to 1.7 {angstrom}, revealing unique structural elements that define this new subclass of the OTU superfamily. In addition, kinetic studies were carried out with aminomethylcoumarin (AMC) conjugates of monomeric Ub, ISG15, and NEDD8 (neural precursor cell expressed, developmentally downregulated 8) substrates in order to provide quantitative insights into vOTU's preference for Ub and Ub-like substrates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The protein structurome of Orthornavirae and its dark matter

Metatranscriptomics is uncovering more and more diverse families of viruses with RNA genomes comprising the viral kingdom Orthornavirae in the realm Riboviria. Thorough protein annotation and comparison are essential to get insights into the functions of viral proteins and virus evolution. In addition to sequence- and hmm profile-based methods, protein structure comparison adds a powerful tool to uncover protein functions and relationships. We constructed an Orthornavirae “structurome” consisting of already annotated as well as unannotated (“dark matter”) proteins and domains encoded in viral genomes. We used protein structure modeling and similarity searches to illuminate the remaining dark matter in hundreds of thousands of orthornavirus genomes. The vast majority of the dark matter domains showed either “generic” folds, such as single α-helices, or no high confidence structure predictions. Nevertheless, a variety of lineage-specific globular domains that were new either to orthornaviruses in general or to particular virus families were identified within the proteomic dark matter of orthornaviruses, including several predicted nucleic acid-binding domains and nucleases. In addition, we identified a case of exaptation of a cellular nucleoside monophosphate kinase as an RNA-binding protein in several virus families. Notwithstanding the continuing discovery of numerous orthornaviruses, it appears that all the protein domains conserved in large groups of viruses have already been identified. The rest of the viral proteome seems to be dominated by poorly structured domains including intrinsically disordered ones that likely mediate specific virus-host interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Transcriptomic data sets examining several stress responses in Zymomonas mobilis strains

We used RNA-seq to compare gene expression from Zymomonas mobilis ZM4 grown under various conditions: aerobic ± paraquat, anaerobic ± hydrogen peroxide, or an iron chelator. We analyzed two mutant strains lacking predicted transcription factors (ZMO_0442 and ZMO_1411) grown under aerobic or anaerobic conditions. We report the RNA-seq data from these experiments.

Bacterial Stress Response↗
DOE OSTI2018

Transcriptome Analysis of Neisseria gonorrhoeae during Natural Infection Reveals Differential Expression of Antibiotic Resistance Determinants between Men and Women

Neisseria gonorrhoeae is a bacterial pathogen responsible for the sexually transmitted infection gonorrhea. Emergence of antimicrobial resistance (AMR) of N. gonorrhoeae worldwide has resulted in limited therapeutic choices for this infection. Men who seek treatment often have symptomatic urethritis; in contrast, gonococcal cervicitis in women is usually minimally symptomatic, but may progress to pelvic inflammatory disease. Previously, we reported the first analysis of gonococcal transcriptome expression determined in secretions from women with cervical infection. Here, we defined gonococcal global transcriptional responses in urethral specimens from men with symptomatic urethritis and compared these with transcriptional responses in specimens obtained from women with cervical infections and in vitro-grown N. gonorrhoeae isolates. This is the first comprehensive comparison of gonococcal gene expression in infected men and women. RNA sequencing analysis revealed that 9.4% of gonococcal genes showed increased expression exclusively in men and included genes involved in host immune cell interactions, while 4.3% showed increased expression exclusively in women and included phage-associated genes. Infected men and women displayed comparable antibiotic-resistant genotypes and in vitro phenotypes, but a 4-fold higher expression of the Mtr efflux pump-related genes was observed in men. These results suggest that expression of AMR genes is programed genotypically and also driven by sex-specific environments. Collectively, our results indicate that distinct N. gonorrhoeae gene expression signatures are detected during genital infection in men and women. We propose that therapeutic strategies could target sex-specific differences in expression of antibiotic resistance genes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Transcriptome architecture of the three main lineages of agrobacteria

ABSTRACT Agrobacteria are a diverse, polyphyletic group of prokaryotes with multipartite genomes capable of transferring DNA into the genomes of host plants, making them an essential tool in plant biotechnology. Despite their utility in plant transformation, genome-wide transcriptional regulation is not well understood across the three main lineages of agrobacteria. Transcription start sites (TSSs) are a necessary component of gene expression and regulation. In this study, we used differential RNA-seq and a TSS identification algorithm optimized on manually annotated TSS, then validated with existing TSS to identify thousands of TSS with nucleotide resolution for representatives of each lineage. We extend upon the 356 TSSs previously reported in Agrobacterium fabrum C58 by identifying 1,916 TSSs. In addition, we completed genomes and phenotyping of Rhizobium rhizogenes C16/80 and Allorhizobium vitis T60/94, identifying 2,650 and 2,432 TSSs, respectively. Parameter optimization was crucial for an accurate, high-resolution view of genome and transcriptional dynamics, highlighting the importance of algorithm optimization in genome-wide TSS identification and genomics at large. The optimized algorithm reduced the number of TSSs identified internal and antisense to the coding sequence on average by 90.5% and 91.9%, respectively. Comparison of TSS conservation between orthologs of the three lineages revealed differences in cell cycle regulation of ctrA as well as divergence of transcriptional regulation of chemotaxis-related genes when grown in conditions that simulate the plant environment. These results provide a framework to elucidate the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. IMPORTANCE Transcription start sites (TSSs) are fundamental for understanding gene expression and regulation. Agrobacteria, a group of prokaryotes with the ability to transfer DNA into the genomes of host plants, are widely used in plant biotechnology. However, the genome-wide transcriptional regulation of agrobacteria is not well understood, especially in less-studied lineages. Differential RNA-seq and an optimized algorithm enabled identification of thousands of TSSs with nucleotide resolution for representatives of each lineage. The results of this study provide a framework for elucidating the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. The optimized algorithm also highlights the importance of parameter optimization in genome-wide TSS identification and genomics at large.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A Structural Perspective on the Alphavirus Life Cycle

Alphaviruses are mosquito-borne, enveloped viruses with a positive-sense, single-stranded RNA genome. Alphaviruses enter host cells via receptor-mediated endocytosis, using various cellular surface receptors such as matrix remodeling-associated protein 8 (MXRA8), low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3), and very low-density lipoprotein receptor (VLDLR), which facilitate binding to the viral glycoproteins. Following entry, viral proteins are expressed and nonstructural proteins assemble into replication complexes in host cells, driving RNA synthesis and genome replication. Viral assembly occurs at the plasma membrane, where nascent virions bud from the host cell in a process driven by capsid and spike proteins. Recent combinatorial structural studies have provided detailed molecular insights into various steps of the alphavirus life cycle. These structural insights into the alphavirus life cycle enhance our understanding of viral replication and assembly, with significant implications for antiviral strategies and the development of alphavirus-based vaccine vectors.

RNA virus↗
DOE OSTI2022

A transcriptome software comparison for the analyses of treatments expected to give subtle gene expression responses

Background: In this comparative study we evaluate the performance of four software tools: DNAstar-D (DESeq2), DNAstar-E (edgeR), CLC Genomics and Partek Flow for identification of differentially expressed genes (DEGs) using a transcriptome of E. coli. The RNA-seq data are from the effect of below-background radiation 5.5 nGy total dose (0.2nGy/hr) on E. coli grown shielded from natural radiation 655 m below ground in a pre-World War II steel vault. The gene expression response to three supplemented sources of radiation designed to mimic natural background, 1952 – 5720 nGy in total dose (71–208 nGy/hr), are compared to this “radiation-deprived” treatment. In addition, RNA-seq data of Caenorhabditis elegans nematode from similar radiation treatments was analyzed by three of the software packages. Results: In E. coli, the four software programs identified one of the supplementary sources of radiation (KCl) to evoke about 5 times more transcribed genes than the minus-radiation treatment (69–114 differentially expressed genes, DEGs), and so the rest of the analyses used this KCl vs “Minus” comparison. After imposing a 30-read minimum cutoff, one of the DNAStar options shared two of the three steps (mapping, normalization, and statistic) with Partek Flow (they both used median of ratios to normalize and the DESeq2 statistical package), and these two programs identified the highest number of DEGs in common with each other (53). In contrast, when the programs used different approaches in each of the three steps, between 31 and 40 DEGs were found in common. Regarding the extent of expression differences, three of the four programs gave high fold-change results (15–178 fold), but one (DNAstar’s DESeq2) resulted in more conservative fold-changes (1.5–3.5). In a parallel study comparing three qPCR commercial validation software programs, these programs also gave variable results as to which genes were significantly regulated. Similarly, the C. elegans analysis showed exaggerated fold-changes in CLC and DNAstar’s edgeR while DNAstar-D was more conservative. Conclusions: Regarding the extent of expression (fold-change), and considering the subtlety of the very low level radiation treatments, in E. coli three of the four programs gave what we consider exaggerated fold-change results (15 – 178 fold), but one (DNAstar’s DESeq2) gave more realistic fold-changes (1.5–3.5). When RT-qPCR validation comparisons to transcriptome results were carried out, they supported the more conservative DNAstar-D’s expression results. When another model organism’s (nematode) response to these radiation differences was similarly analyzed, DNAstar-D also resulted in the most conservative expression patterns. Therefore, we would propose DESeq2 (“DNAstar-D”) as an appropriate software tool for differential gene expression studies for treatments expected to give subtle transcriptome responses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

BSMV-mediated genome editing exhibits host-specific heritability: germline transmission in barley and somatic edits in Nicotiana benthamiana

Plant RNA virus–mediated guide RNA (gRNA) delivery represents a transformative advance in genome editing technologies. Unlike conventional transformation methods that rely on labor-intensive tissue culture and regeneration for each individual gRNA delivery, viral vectors can rapidly and systemically transmit gRNAs into pre-established Cas-expressing plants, providing an accelerated route for functional genomics and trait discovery directly in planta . However, key design parameters, including subgenomic promoter choice, transcript architecture, and their effects on viral fitness and editing outcomes, remain to be elucidated for most viral platforms. We developed five Barley stripe mosaic virus (BSMV) vectors, each with distinct subgenomic promoter elements to drive single gRNA expression. These were initially evaluated in Cas9-expressing transgenic Nicotiana benthamiana plants targeting the Phytoene desaturase ( PDS ) gene to compare their editing efficiencies. Single gRNAs expressed under the duplicated γb subgenomic promoter or when fused directly to the γb genome achieved the highest mutation frequencies (up to 90% at 60 days post-inoculation), whereas β1- and β2-driven sgRNAs produced delayed and reduced editing. Thus, promoter selection critically determines gRNA accumulation and the efficacy of BSMV-mediated genome editing. The top-performing design was then applied to Cas9-expressing barley ( Hordeum vulgare ) targeting HvCMF7 (conferring green-white variegation) and HvGW2.1 (impacts grain width and weight). BSMV spread systemically throughout barley, inducing somatic and heritable mutations at frequencies up to 100%, with virus-free edited progeny. In contrast, despite robust somatic editing in N. benthamiana, no heritable mutations were detected indicating species-dependent limitations in germline transmission. Our systematic comparison of subgenomic promoter architectures establishes clear design principles for optimizing viral vector–mediated delivery. Promoter choice and transcript structure critically shape editing efficiency and viral stability. The host-specific boundary for germline editing, defined by efficient heritable editing in barley but not N. benthamiana , highlights where BSMV offers advantages and where alternative vectors or hybrid strategies are required, guiding rational platform selection for diverse crop species and applications. Collectively, these findings establish BSMV as a promising next-generation vector for rapid, tissue culture–free, and transformation-independent genome editing in cereals and other recalcitrant monocots.

barley↗
DOE OSTI2016

A divalent cation-dependent variant of the glmS ribozyme with stringent Ca 2+ selectivity co-opts a preexisting nonspecific metal ion-binding site

Ribozymes use divalent cations for structural stabilization, as catalytic cofactors, or both. Because of the prominent role of Ca 2+ in intracellular signaling, engineered ribozymes with stringent Ca 2+ selectivity would be important in biotechnology. The wild-type glmS ribozyme (glmSWT) requires glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. Previously, a glmS ribozyme variant with three adenosine mutations (glm SAAA ) was identified, which dispenses with GlcN6P and instead uses, with little selectivity, divalent cations as cofactors for site-specific RNA cleavage. We now report a Ca 2+ -specific ribozyme (glmSCa) evolved from glm SAAA that is >10,000 times more active in Ca 2+ than Mg 2+ , is inactive in even 100 mM Mg2+, and is not responsive to GlcN6P. This stringent selectivity, reminiscent of the protein nuclease from Staphylococcus, allows rapid and selective ribozyme inactivation using a Ca 2+ chelator such as EGTA. Because glmSCa functions in physiologically relevant Ca 2+ concentrations, it can form the basis for intracellular sensors that couple Ca 2+ levels to RNA cleavage. Biochemical analysis of glmSCa reveals that it has co-opted for selective Ca 2+ binding a nonspecific cation-binding site responsible for structural stabilization in glmSWT and glmSAAA. Fine-tuning of the selectivity of the cation site allows repurposing of this preexisting molecular feature.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Structural basis for ligand binding to the guanidine-II riboswitch

The guanidine-II riboswitch, also known as mini-ykkC, is a conserved mRNA element with more than 800 examples in bacteria. It consists of two stem–loops capped by identical, conserved tetraloops that are separated by a linker region of variable length and sequence. Like the guanidine-I riboswitch, it controls the expression of guanidine carboxylases and SugE-like genes. The guanidine-II riboswitch specifically binds free guanidinium cations and functions as a translationally controlled on-switch. Here we report the structure of a P2 stem–loop from the Pseudomonas aeruginosa guanidine-II riboswitch aptamer bound to guanidine at 1.57 Å resolution. The hairpins dimerize via the conserved tetraloop, which also contains the binding pocket. Two guanidinium molecules bind near the dimerization interface, one in each tetraloop. The guanidinium cation is engaged in extensive hydrogen bonding to the RNA. Contacts include the Hoogsteen face of a guanine base and three nonbridging phosphate oxygens. Cation–π interactions and ionic interactions also stabilize ligand binding. Finally, the guanidine-II riboswitch utilizes the same recognition strategies as the guanidine-I riboswitch while adopting an entirely different and much smaller RNA fold.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A multiscale model of the action of a capsid assembly modulator for the treatment of chronic hepatitis B

Chronic hepatitis B virus (HBV) infection is strongly associated with increased risk of liver cancer and cirrhosis. While existing treatments effectively inhibit the HBV life cycle, viral rebound frequently occurs following treatment interruption. Consequently, functional cure rates of chronic HBV infection remain low and there is increased interest in a novel treatment modality, capsid assembly modulators (CAMs). Here, we develop a multiscale mathematical model of CAM treatment in chronic HBV infection. By fitting the model to participant data from a phase I trial of the first-generation CAM vebicorvir, we estimate the drug’s dose-dependent effectiveness and identify the physiological mechanisms that drive the observed biphasic decline in HBV DNA and RNA, and mechanistic differences between HBeAg-positive and negative infection. Finally, we demonstrate analytically and numerically that the relative change of HBV RNA more accurately reflects the antiviral effectiveness of a CAM than the relative change in HBV DNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Comparing variability in diagnosis of upper respiratory tract infections in patients using syndromic, next generation sequencing, and PCR-based methods

Early and accurate diagnosis of respiratory pathogens and associated outbreaks can allow for the control of spread, epidemiological modeling, targeted treatment, and decision making–as is evident with the current COVID-19 pandemic. Many respiratory infections share common symptoms, making them difficult to diagnose using only syndromic presentation. Yet, with delays in getting reference laboratory tests and limited availability and poor sensitivity of point-of-care tests, syndromic diagnosis is the most-relied upon method in clinical practice today. Here, we examine the variability in diagnostic identification of respiratory infections during the annual infection cycle in northern New Mexico, by comparing syndromic diagnostics with polymerase chain reaction (PCR) and sequencing-based methods, with the goal of assessing gaps in our current ability to identify respiratory pathogens. Of 97 individuals that presented with symptoms of respiratory infection, only 23 were positive for at least one RNA virus, as confirmed by sequencing. Whereas influenza virus (n = 7) was expected during this infection cycle, we also observed coronavirus (n = 7), respiratory syncytial virus (n = 8), parainfluenza virus (n = 4), and human metapneumovirus (n = 1) in individuals with respiratory infection symptoms. Four patients were coinfected with two viruses. In 21 individuals that tested positive using PCR, RNA sequencing completely matched in only 12 (57%) of these individuals. Few individuals (37.1%) were diagnosed to have an upper respiratory tract infection or viral syndrome by syndromic diagnostics, and the type of virus could only be distinguished in one patient. Thus, current syndromic diagnostic approaches fail to accurately identify respiratory pathogens associated with infection and are not suited to capture emerging threats in an accurate fashion. We conclude there is a critical and urgent need for layered agnostic diagnostics to track known and unknown pathogens at the point of care to control future outbreaks.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Global Analysis of Chlorella variabilis NC64A mRNA Profiles during the Early Phase of Paramecium bursaria Chlorella Virus-1 Infection

The PBCV-1/Chlorella variabilis NC64A system is a model for studies on interactions between viruses and algae. Here we present the first global analyses of algal host transcripts during the early stages of infection, prior to virus replication. During the course of the experiment stretching over 1 hour, about a third of the host genes displayed significant changes in normalized mRNA abundance that either increased or decreased compared to uninfected levels. The population of genes with significant transcriptional changes gradually increased until stabilizing at 40 minutes post infection. Functional categories including cytoplasmic ribosomal proteins, jasmonic acid biosynthesis and anaphase promoting complex/cyclosomes had a significant excess in upregulated genes, whereas spliceosomal snRNP complexes and the shikimate pathway had significantly more down-regulated genes, suggesting that these pathways were activated or shut-down in response to the virus infection. Lastly, we examined the expression of C. varibilis RNA polymerase subunits, as PBCV-1 transcription depends on host RNA polymerases. Two subunits were up-regulated, RPB10 and RPC34, suggesting that they may function to support virus transcription. These results highlight genes and pathways, as well as overall trends, for further refinement of our understanding of the changes that take place during the early stages of viral infection.

59 BASIC BIOLOGICAL SCIENCES↗