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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-ΔI177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals

The African swine fever virus (ASFV) mutant ASFV-G-ΔI177L is a safe and efficacious vaccine which induces protection against the challenge of its parental virus, the Georgia 2010 isolate. Although a genetic DIVA (differentiation between infected and vaccinated animals) assay has been developed for this vaccine, still there is not a serological DIVA test for differentiating between animals vaccinated with ASFV-G-ΔI177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-ΔI177L mutant having deleted the EP402R gene, which encodes for the viral protein responsible for mediating the hemadsorption of swine erythrocytes. The resulting virus, ASFV-G-ΔI177L/ΔEP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-ΔI177L. Domestic pigs intramuscularly (IM) inoculated with either 10 2 or 10 6 HAD 50 of ASFV-G-ΔI177L/ΔEP402R remained clinically normal, when compared with a group of mock-vaccinated animals, indicating the absence of residual virulence. Interestingly, an infectious virus could not be detected in the blood samples of the ASFV-G-ΔI177L/ΔEP402R-inoculated animals in either group at any of the time points tested. Furthermore, while all of the mock-inoculated animals presented a quick and lethal clinical form of ASF after the intramuscular inoculation challenge with 10 2 HAD 50 of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-ΔI177L/ΔEP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-ΔI177L/ΔEP402R-inoculated pigs developed strong virus-specific antibody responses against viral antigens, reaching maximum levels at 28 days post inoculation. Importantly, all of the sera collected at that time point in the ASFV-G-ΔI177L/ΔEP402R-inoculated pigs did not react in a direct ELISA coated with the recombinant EP402R protein. Conversely, the EP402R protein was readily recognized by the pool of sera from the animals immunized with recombinant live attenuated vaccine candidates ASFV-G-ΔI177L, ASFV-G-ΔMGF, or ASFV-G-Δ9GL/ΔUK. Therefore, ASFV-G-ΔI177L/ΔEP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Cryo-EM structure of RNA-induced tau fibrils reveals a small C-terminal core that may nucleate fibril formation

In neurodegenerative diseases including Alzheimer’s and amyotrophic lateral sclerosis, proteins that bind RNA are found in aggregated forms in autopsied brains. Evidence suggests that RNA aids nucleation of these pathological aggregates; however, the mechanism has not been investigated at the level of atomic structure. Here, we present the 3.4-Å resolution structure of fibrils of full-length recombinant tau protein in the presence of RNA, determined by electron cryomicroscopy (cryo-EM). The structure reveals the familiar in-register cross-β amyloid scaffold but with a small fibril core spanning residues Glu391 to Ala426, a region disordered in the fuzzy coat in all previously studied tau polymorphs. RNA is bound on the fibril surface to the positively charged residues Arg406 and His407 and runs parallel to the fibril axis. The fibrils dissolve when RNase is added, showing that RNA is necessary for fibril integrity. While this structure cannot exist simultaneously with the tau fibril structures extracted from patients’ brains, it could conceivably account for the nucleating effects of RNA cofactors followed by remodeling as fibrils mature.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

SPLIT: Stable Protein Coacervation Using a Light Induced Transition

Protein coacervates serve as hubs to concentrate and sequester proteins and nucleotides and thus function as membraneless organelles to manipulate cell physiology. We have engineered a coacervating protein to create tunable, synthetic membraneless organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl, which cleaves in response to 405 nm light. We developed a fusion protein containing a solubilizing maltose-binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellularsized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. Finally, the methods described here provide novel strategies for inducing protein phase separation using light.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2024

The crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins

Abstract RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron–electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.

Biochemistry & Molecular Biology↗
DOE OSTI2022

Chromosome-Level Genome Assembly of Mentha longifolia L. Reveals Gene Organization Underlying Disease Resistance and Essential Oil Traits

Mentha longifolia (L.) Huds., a wild, diploid mint species, has been developed as a model for mint genetic and genomic research to aid breeding efforts that target Verticillium wilt disease resistance and essential oil monoterpene composition. Here, we present a near-complete, chromosome-scale mint genome assembly for M. longifolia USDA accession CMEN 585. This new assembly is an update of a previously published genome draft, with dramatic improvements. A total of 42,107 protein-coding genes were annotated and placed on 12 chromosomal scaffolds. One hundred fifty-three genes contained conserved sequence domains consistent with nucleotide binding site-leucine-rich-repeat (NBS-LRR) plant disease resistance genes. Homologs of genes implicated in Verticillium wilt resistance in other plant species were also identified. Multiple paralogs of genes putatively involved in p-menthane monoterpenoid biosynthesis were identified and several cases of gene clustering documented. Heterologous expression of candidate genes, purification of recombinant target proteins, and subsequent enzyme assays allowed us to identify the genes underlying the pathway that leads to the most abundant monoterpenoid volatiles. Here, the bioinformatic and functional analyses presented here are laying the groundwork for using marker-assisted selection in improving disease resistance and essential oil traits in mints.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Development of HEK-293 Cell Lines Constitutively Expressing Flaviviral Antigens for Use in Diagnostics

Flaviviruses are important human pathogens worldwide. Diagnostic testing for these viruses is difficult because many of the pathogens require specialized biocontainment. To address this issue, we generated 39 virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells of 13 different flaviviruses, including dengue, yellow fever, Japanese encephalitis, West Nile, St. Louis encephalitis, Zika, Rocio, Ilheus, Usutu, and Powassan viruses. Antigen secretion was stable for at least 10 cell passages, as measured by enzyme-linked immunosorbent assays and immunofluorescence assays. Thirty-five cell lines (90%) had stable antigen expression over 10 passages, with three of these cell lines (7%) increasing in antigen expression and one cell line (3%) decreasing in antigen expression. Antigen secretion in the HEK-293 cell lines was higher than in previously developed COS-1 cell line counterparts. These antigens can replace current antigens derived from live or inactivated virus for safer use in diagnostic testing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Experimental and Computational Insights into the Structural Dynamics of the Fc Fragment of IgG1 Subtype from Biosimilar VEGF‐Trap

The constant fragment (Fc) of the immunoglobulin G1 (IgG1) subtype is increasingly recognized as a crucial scaffold in the development of advanced therapeutics due to its enhanced specificity, efficacy, and extended half‐life. A prime example is VEGF‐Trap (Aflibercept), a recombinant fusion protein that merges the Fc region of the IgG1 subtype with the binding domains of vascular endothelial growth factor receptors (VEGFR)‐1 and VEGFR‐2. The Fc region's role in N‐glycosylation is particularly important, as it significantly influences protein stability. Herein, the first near‐physiological temperature structures of the N‐glycan‐bound Fc fragment of IgG1 subtype from a biosimilar VEGF‐Trap are presented, determined using the SPring‐8 Angstrom Compact free electron LAser (SACLA) and the Turkish Light Source (Turkish DeLight). Comparative analysis with cryogenic structures, including existing data, reveals alternate conformations within the glycan‐binding pocket. Furthermore, molecular dynamics simulations indicate the presence of a high degree of structural plasticity, explaining how the protein adapts its structure through conformational changes. The observed structural fluctuations/conformational changes demonstrate the effect of N‐glycans on protein stability. These findings offer new insights into the molecular basis of Fc‐mediated functions and provide valuable information for the design of next‐generation therapeutics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

The exo-β- N -acetylmuramidase NamZ from Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases

Endo-β-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that catalyze an endo-lytic cleavage of peptidoglycan; i.e., they hydrolyze the β-1,4-glycosidic bonds connecting N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In contrast, little is known about exo-β-N-acetylmuramidases, which catalyze an exo-lytic cleavage of β-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We now report that ybbC of B. subtilis, renamed namZ, encodes the reported exo-β-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover and recycling. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl β-MurNAc and the peptidoglycan-derived disaccharide MurNAc-β-1,4-GlcNAc. Together with the exo-β-N-acetylglucosaminidase NagZ and the exo-muramoyl-L-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. A structure model of NamZ, built on the basis of two crystal structures of putative orthologs from Bacteroides fragilis, revealed a two-domain structure including a Rossmann-fold-like domain that constitutes a unique glycosidase fold. Thus, NamZ, a member of the DUF1343 protein family of unknown function, is now classified as the founding member of a new family of glycosidases (CAZy GH171). NamZ-like peptidoglycan hexosaminidases are mainly present in the phylum Bacteroidetes and less frequently found in individual genomes within Firmicutes (Bacilli, Clostridia), Actinobacteria, and γ-proteobacteria.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

PHOTO‐SENSITIVE LEAF ROLLING 1 encodes a polygalacturonase that modifies cell wall structure and drought tolerance in rice

Summary The biosynthesis and modification of cell wall composition and structure are controlled by hundreds of enzymes and have a direct consequence on plant growth and development. However, the majority of these enzymes has not been functionally characterised. Rice mutants with leaf‐rolling phenotypes were screened in a field. Phenotypic analysis under controlled conditions was performed for the selected mutant and the relevant gene was identified by map‐based cloning. Cell wall composition was analysed by glycome profiling assay. We identified a photo‐sensitive leaf rolling 1 ( psl1 ) mutant with ‘napping’ (midday depression of photosynthesis) phenotype and reduced growth. The PSL1 gene encodes a cell wall‐localised polygalacturonase (PG), a pectin‐degrading enzyme. psl1 with a 260‐bp deletion in its gene displayed leaf rolling in response to high light intensity and/or low humidity. Biochemical assays revealed PG activity of recombinant PSL1 protein. Significant modifications to cell wall composition in the psl1 mutant compared with the wild‐type plants were identified. Such modifications enhanced drought tolerance of the mutant plants by reducing water loss under osmotic stress and drought conditions. Taken together, PSL1 functions as a PG that modifies cell wall biosynthesis, plant development and drought tolerance in rice.

Zhang, Guangheng↗
DOE OSTI2022

Identification of xylan arabinosyl 2- O -xylosyltransferases catalyzing the addition of 2- O -xylosyl residue onto arabinosyl side chains of xylan in grass species

Grass xylan, the major hemicellulose in both primary and secondary cell walls, is heavily decorated with α-1,3-linked arabinofuranosyl (Araf) residues that may be further substituted at O-2 with xylosyl (Xyl) or Araf residues. Although xylan 3-O-arabinosyltransferases (XATs) catalyzing 3-O-Araf addition onto xylan have been characterized, glycosyltransferases responsible for the transfer of 2-O-Xyl or 2-O-Araf onto 3-O-Araf residues of xylan to produce the Xyl-Araf and Araf-Araf disaccharide side chains remain to be identified. In this report, we showed that a rice GT61 member, named OsXAXT1 (xylan arabinosyl 2-O-xylosyltransferase 1) herein, was able to mediate the addition of Xyl-Araf disaccharide side chains onto xylan when heterologously co-expressed with OsXAT2 in the Arabidopsis gux1/2/3 (glucuronic acid substitution of xylan 1/2/3) triple mutant that lacks any glycosyl substitutions. Recombinant OsXAXT1 protein expressed in human embryonic kidney 293 cells exhibited a xylosyltransferase activity catalyzing the addition of Xyl from UDP-Xyl onto arabinosylated xylooligomers. Further, consistent with its function as a xylan arabinosyl 2-O-xylosyltransferase, CRISPR-Cas9-mediated mutations of the OsXAXT1 gene in transgenic rice plants resulted in a reduction in the level of Xyl-Araf disaccharide side chains in xylan. Furthermore, we revealed that XAXT1 close homologs from several other grass species, including switchgrass, maize, and Brachypodium, possessed the same functions as OsXAXT1, indicating functional conservation of XAXTs in grass species. Together, our findings establish that grass XAXTs are xylosyltransferases catalyzing Xyl transfer onto O-2 of Araf residues of xylan to form the Xyl-Araf disaccharide side chains, which furthers our understanding of genes involved in xylan biosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structure and antigenicity of the divergent human astrovirus VA1 capsid spike

Human astrovirus (HAstV) is a known cause of viral gastroenteritis in children worldwide, but HAstV can cause also severe and systemic infections in immunocompromised patients. There are three clades of HAstV: classical, MLB, and VA/HMO. While all three clades are found in gastrointestinal samples, HAstV-VA/HMO is the main clade associated with meningitis and encephalitis in immunocompromised patients. To understand how the HAstV-VA/HMO can infect the central nervous system, we investigated its sequence-divergent capsid spike, which functions in cell attachment and may influence viral tropism. Here we report the high-resolution crystal structures of the HAstV-VA1 capsid spike from strains isolated from patients with gastrointestinal and neuronal disease. The HAstV-VA1 spike forms a dimer and shares a core beta-barrel structure with other astrovirus capsid spikes but is otherwise strikingly different, suggesting that HAstV-VA1 may utilize a different cell receptor, and an infection competition assay supports this hypothesis. Furthermore, by mapping the capsid protease cleavage site onto the structure, the maturation and assembly of the HAstV-VA1 capsid is revealed. Finally, comparison of gastrointestinal and neuronal HAstV-VA1 sequences, structures, and antigenicity suggests that neuronal HAstV-VA1 strains may have acquired immune escape mutations. Overall, our studies on the HAstV-VA1 capsid spike lay a foundation to further investigate the biology of HAstV-VA/HMO and to develop vaccines and therapeutics targeting it.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Construction of chicken infectious anemia virus infectious clone and study on its pathogenicity

Chicken infectious anemia virus (CIAV) can be transmitted through contaminated live poultry vaccine. However, the pathogenicity of contaminated CIAV strains is rarely reported. Previously, the chickens showed the typical symptoms of anemia after using the attenuated live fowl pox virus (FPV) vaccine. Therefore, exogenous CIAV contamination was suspected. We detected anti-CIAV antibodies in SPF chicks vaccinated with the FPV vaccine. CIAV contamination was confirmed in the FPV vaccine, and the CIAV strain was named JS2020-FPV. This study aims to rescue JS2020-FPV by reverse genetic assays and investigate its pathogenicity. Firstly, double-copies infectious clone of JS2020-FPV was constructed. For the pathogenicity study, infectious clone of JS2020-FPV was used to inoculate 1-day-old SPF chicks. The typical symptoms of anemia were observed in the JS2020-PFV group 14 days post inoculation. The hematocrit and body weight of chicks in the JS2020-PFV group were significantly lower than those in the mock group. Notably, the thymus development index and antibody levels of NDV were lower in chicks in the JS2020-PFV group than those in the mock group. Different degrees of apoptosis of MSB1 and DF-1 were observed after inoculated with the JS2020-FPV VP3 recombinant fusion protein expressed by E. coli system, indicating that VP3 induced apoptosis in the transformed cells. Overall, the pathogenicity of the CIAV detected in the contaminated vaccine was confirmed by inoculating SPF chicks with the double-copies infectious DNA clone in this study. Our findings indicate that the dangers of vaccine contamination cannot be ignored.

Wang, Jinjin↗
DOE OSTI2020

The C962R ORF of African Swine Fever Strain Georgia Is Non-Essential and Not Required for Virulence in Swine

African swine fever virus (ASFV) is the causative agent of the African swine fever (ASF) epizootic currently affecting pigs throughout Eurasia, causing significant economic losses in the swine industry. The virus genome encodes for more than 160 genes, of which only a few have been studied in detail. Here we describe the previously uncharacterized ASFV open reading frame (ORF) C962R, a gene encoding for a putative NTPase. RNA transcription studies using infected swine macrophages demonstrate that the C962R gene is translated as a late virus protein. A recombinant ASFV lacking the C962R gene (ASFV-G-ΔC962R) demonstrates in vivo that the C962R gene is non-essential, since ASFV-G-ΔC962R has similar replication kinetics in primary swine macrophage cell cultures when compared to parental highly virulent field isolate Georgia2007 (ASFV-G). Experimental infection of domestic pigs with ASFV-G-ΔC962R produced a clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the C962R gene from the ASFV genome does not impact virulence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structure and Antigenicity of the Porcine Astrovirus 4 Capsid Spike

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine.

Virology↗
DOE OSTI2021

Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii

Recombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas . RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

PCP consensus protein/peptide alphavirus antigens stimulate broad spectrum neutralizing antibodies

Vaccines based on proteins and peptides may be safer and if calculated based on many sequences, more broad-spectrum than those designed based on single strains. Physicochemical Property Consensus (PCP con ) alphavirus (AV) antigens from the B-domain of the E2 envelope protein were designed, synthesized recombinantly and shown to be immunogenic (i.e. sera after inoculation detected the antigen in dotspots and ELISA). Antibodies in sera after inoculation with B-region antigens based on individual AV species (eastern or Venezuelan equine encephalitis (EEEV con , VEEV con ), or chikungunya (CHIKV con ) bound only their cognate protein, while those designed against multiple species (Mosaik con and EVC con ) recognized all three serotype specific antigens. The VEEV con and EEEV con sera only showed antiviral activity against their related strains (in plaque reduction neutralization assays (PRNT 50/80 ). Peptides designed to surface exposed areas of the E2-A-domain of CHIKV con were added to CHIKV con inocula to provide anti-CHIKV antibodies. EVC con , based on three different alphavirus species, combined with E2-A-domain peptides from AllAV con , a PCPcon of 24 diverse AV, generated broad spectrum, antiviral antibodies against VEEV, EEEV and CHIKV, AV with less than 35% amino acid identity to each other (>65% diversity). This is a promising start to a molecularly defined vaccine against all AV. A further study with these antigens can illuminate what areas are most important for a robust immune response, resistant to mutations in rapidly evolving viruses. The validated computational methods can also be used to design broad spectrum antigens against many other pathogen families.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Compositions and methods for making selenocysteine containing polypeptides

Non-naturally occurring tRNASec and methods of using them for recombinant expression of proteins engineered to include one or more selenocysteine residues are disclosed. The non-naturally occurring tRNASec can be used for recombinant manufacture of selenocysteine containing polypeptides encoded by mRNA without the requirement of an SECIS element. In some embodiments, selenocysteine containing polypeptides are manufactured by co-expressing a non-naturally occurring tRNASec a recombinant expression system, such as E. coli, with SerRS, EF-Tu, SelA, or PSTK and SepSecS, and an mRNA with at least one codon that recognizes the anticodon of the non-naturally occurring tRNASec.

Soll, Dieter↗