Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “Virus”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 937 records · Page 52
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

TMEM2 binds to CSNK2A3 to inhibit HBV infection via activation of the JAK/STAT pathway

To investigate mechanisms that TMEM2 activation inhibits hepatitis B virus (HBV) infection in hepatocarcinoma (HCC) cells, co-immunoprecipitation (Co-IP) and mass spectrometry were used in screening interacting proteins for TMEM2. Levels of casein kinase 2 subunit α3 (CSNK2A3) in HCC cells were found to be inhibited or overexpressed using siRNAs and pcDNA3.1-CSNK2A3, respectively. Effect of CSNK2A3 expression on cell proliferation was analyzed using MTS, while its effect on HBV infection was measured using ddPCR and IHC. Western blotting and JAK inhibitor ruxolitinib were also used to determine whether TMEM2-regulated CSNK2A3 expression and HBV infection were affected by JAK-STAT signaling. Co-IP and mass spectrometry results showed that CSNK2A3 interacts with TMEM2. Moreover, overexpression of CSNK2A3 significantly inhibited cell proliferation, while inhibition of CSNK2A3 promoted proliferation of HCC cells. In addition, overexpression of CSNK2A3 was observed to significantly enhance HBV infection, while siRNA knockdown of CSNK2A3 inhibited HBV infection. Notably, effect of CSNK2A3 overexpression on HBV infection was suppressed by TMEM2 overexpression. Further mechanistic analyses have revealed that TMEM2 could antagonize the effects of CSNK2A3 on cell proliferation and HBV infection via JAK-STAT pathway activation. In conclusion, TMEM2 has been determined to bind to CSNK2A3 to inhibit HBV infection via activation of the JAK-STAT pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

MIG/CXCL9 exacerbates the progression of metabolic-associated fatty liver disease by disrupting Treg/Th17 balance

CD4{sup +}CD25{sup +} regulatory T (Treg) cells and Th17 cells play important roles in the progression of metabolic-associated fatty liver disease (MAFLD). However, the contribution of monokine induced by interferon-gamma (MIG)/CXCL9 to the Treg/Th17 imbalance in MAFLD is only partially understood. In the present study, we detected increased levels of MIG/CXCL9 and a Treg/Th17 imbalance in the setting of metabolic-associated steatohepatitis (MASH). Recombinant adeno-associated virus-mediated gene transfer and silencing of MIG/CXCL9 expression in mice alleviated MASH and increased the Treg/Th17 ratio. Furthermore, the percentage of Th17 cells, but not Treg cells, differentiated from splenic CD4{sup +} T cells was significantly increased by administration of MIG/CXCL9. MIG/CXCL9 also promoted Th17 cell proliferation, and its effects were dose dependent. Levels of phosphorylated c-Jun N-terminal kinase (JNK) decreased dramatically when MIG/CXCL9 was inhibited in a murine MASH model. In cultured Treg cells, phosphorylated JNK levels decreased dose-dependently in response to MIG/CXCL9 inhibition, but increased in cultured Th17 cells. This effect was blocked in the presence of a JNK inhibitor. These findings underline the fundamental importance of MIG/CXCL9 in maintaining the Treg/Th17 balance in MAFLD and provide the foundations for a novel approach to preventing and treating MAFLD.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Integrin alpha 6 as a stemness driver is a novel promising target for HPV (+) head and neck squamous cell carcinoma

Although the incidence rates of head and neck squamous cell carcinoma (HNSCC) associated with human papilloma virus (HPV) infection have recently been on the rise, the underlying mechanism of its tumorigenesis remains largely unknown. Here, we investigated whether HNSCC cells with high expression of integrin alpha 6 (ITGα6), one of the HPV receptors, have a preference during HPV infection. In addition, we examined the gain or loss of function of the ITGα6 gene in HPV + ve HNSCC cells, as well as its prognostic value in patients with HNSCC. HPV pseudovirus was found to be more infective, with HNSCC cells featuring an overexpressed ITGα6 gene compared to the control cells. Overexpression and suppression of ITGα6 respectively increases and decreases stemness phenotypes of HPV + ve HNSCC cells. Furthermore, ITGα6 can regulate stemness by partially mediating AKT pathway in HPV + ve HNSCC cells. Finally, patients with HPV + ve HNSCC had a poor prognosis in cases of elevated ITGα6 expression; however, the expression levels of ITGα6 did not influence the survival rates of HPV-negative HNSCC patients. In conclusion, ITGα6 can serve as a potential therapeutic target for HPV + ve HNSCC cancer-like stem cells (CSCs).

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Aging-associated stem/progenitor cell dysfunction in the salivary glands of mice

Highlights: • The function of CD133-positive cells changes with age and is expected to be one of the causative factors associated with salivary gland aging. Although stem cell aging leads to a decline in tissue homeostasis and regenerative capacity, it remains unclear whether salivary gland stem cell function changes during this process. However, the salivary glands are gradually replaced by connective tissue during aging. Here, we show a decline in the stem cell ability of CD133-positive stem/progenitor cells in the salivary glands of aged mice. The CD133-positive cells were isolated from young, adult, and aged mice. The number of CD133-positive cells was significantly decreased in aged mice. They also showed a lower sphere formation capacity compared to young and adult mice. RNA sequencing revealed that CD133-positive cells in aged mice exhibited lower gene expression of several aging-related genes, including FoxO3a, than those in young and adult mice. Salivary gland cells infected with a recombinant lentivirus encoding the FoxO3a gene showed a reduction in oxidative stress induced by hydrogen peroxide compared with those infected with a control virus. Thus, FoxO3a may inhibit stem cell aging via oxidative stress.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Role of karyopherin nuclear transport receptors in nuclear transport by nuclear trafficking peptide

Nuclear trafficking peptide (NTP), a cell-penetrating peptide (CPP) composed of 10 amino acids (aa) (RIFIHFRIGC), has potent nuclear trafficking activity. Recently, we established a protein-based cell engineering system by using NTP, but it remained elusive how NTP functions as a CPP with nuclear orientation. In the present study, we identified importin subunit β1 (IMB1) and transportin 1 (TNPO1) as cellular proteins underlying the activity of NTP. These karyopherin nuclear transport receptors were identified as candidate molecules by liquid chromatography/mass spectrometry analysis, and downregulation of each protein by small interfering RNA significantly reduced NTP activity (P < 0.01). Biochemical analyses revealed that NTP bound directly to both molecules, and the forced expression of an IMB1 fragment (296–516 aa) or TNPO1 fragment (1–297 aa), which both contain binding sites to NTP, reduced nuclear NTP-green fluorescent protein (GFP) levels when it was added to cell culture medium. NTP is derived from viral protein R (Vpr) of human immunodeficiency virus-1, and Vpr enters the nucleus and exerts pleiotropic functions. Notably, Vpr bound directly to IMB1 and TNPO1, and its function was significantly impaired by the forced expression of the 296–516-aa fragment of IMB1 and 1–297-aa fragment of TNPO1. Interestingly, NTP completely blocked the physical association of Vpr with IMB1 and TNPO1. Although the nuclear localization mechanism of Vpr remains unknown, our data suggest that NTP functions as a novel nuclear localization signal of Vpr.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Higher-Order VLP-Based Protein Macromolecular Framework Structures Assembled via Coiled-Coil Interactions

Hierarchical organization is one of the fundamental features observed in biological systems that allows for efficient and effective functioning. Virus-like particles (VLPs) are elegant examples of a hierarchically organized supramolecular structure, where many subunits are self-assembled to generate the functional cage-like architecture. Utilizing VLPs as building blocks to construct two- and three-dimensional (3D) higher-order structures is an emerging research area in developing functional biomimetic materials. VLPs derived from P22 bacteriophages can be repurposed as nanoreactors by encapsulating enzymes and modular units to build higher-order catalytic materials via several techniques. In this study, we have used coiled-coil peptide interactions to mediate the P22 interparticle assembly into a highly stable, amorphous protein macromolecular framework (PMF) material, where the assembly does not depend on the VLP morphology, a limitation observed in previously reported P22 PMF assemblies. Many encapsulated enzymes lose their optimum functionalities under the harsh conditions that are required for the P22 VLP morphology transitions. Therefore, the coiled-coil-based PMF provides a fitting and versatile platform for constructing functional higher-order catalytic materials compatible with sensitive enzymes. Finally, we have characterized the material properties of the PMF and utilized the disordered PMF to construct a biocatalytic 3D material performing single- and multistep catalysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Substrate Partitioning into Protein Macromolecular Frameworks for Enhanced Catalytic Turnover

Spatial partitioning of chemical processes is an important attribute of many biological systems, the effect of which is reflected in the high efficiency of enzymes found within otherwise chaotic cellular environments. Barriers, often provided through the formation of compartments or phase segregation, gate the access of macromolecules and small molecules within the cell and provide an added level of metabolic control. Taking inspiration from nature, we have designed virus-like particles (VLPs) as nanoreactor compartments that sequester enzyme catalysts and have used these as building blocks to construct 3D protein macromolecular framework (PMF) materials, which are structurally characterized using small-angle X-ray scattering (SAXS). Additionally, the highly charged PMFs form a separate phase in suspension, and by tuning the ionic strength, we show positively charged molecules preferentially partition into the PMF, while negatively charged molecules are excluded. This molecular partitioning was exploited to tune the catalytic activity of enzymes enclosed within the individual particles in the PMF, the results of which showed that positively charged substrates had turnover rates that were 8500× faster than their negatively charged counterparts. Moreover, the catalytic PMF led to cooperative behavior resulting in charge dependent trends opposite to those observed with individual P22 nanoreactor particles.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Structures of flavivirus RNA promoters suggest two binding modes with NS5 polymerase

Flaviviruses use a ~70 nucleotide stem-loop structure called stem-loop A (SLA) at the 5' end of the RNA genome as a promoter for RNA synthesis. Flaviviral polymerase NS5 specifically recognizes SLA to initiate RNA synthesis and methylate the 5' guanosine cap. We report the crystal structures of dengue (DENV) and Zika virus (ZIKV) SLAs. DENV and ZIKV SLAs differ in the relative orientations of their top stem-loop helices to bottom stems, but both form an intermolecular three-way junction with a neighboring SLA molecule. To understand how NS5 engages SLA, we determined the SLA-binding site on NS5 and modeled the NS5-SLA complex of DENV and ZIKV. Our results show that the gross conformational differences seen in DENV and ZIKV SLAs can be compensated by the differences in the domain arrangements in DENV and ZIKV NS5s. We describe two binding modes of SLA and NS5 and propose an SLA-mediated RNA synthesis mechanism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural understanding of non-nucleoside inhibition in an elongating herpesvirus polymerase

All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A functional map of HIV-host interactions in primary human T cells

Human Immunodeficiency Virus (HIV) relies on host molecular machinery for replication. Systematic attempts to genetically or biochemically define these host factors have yielded hundreds of candidates, but few have been functionally validated in primary cells. Here, we target 426 genes previously implicated in the HIV lifecycle through protein interaction studies for CRISPR-Cas9-mediated knock-out in primary human CD4+ T cells in order to systematically assess their functional roles in HIV replication. We achieve efficient knockout (>50% of alleles) in 364 of the targeted genes and identify 86 candidate host factors that alter HIV infection. 47 of these factors validate by multiplex gene editing in independent donors, including 23 factors with restrictive activity. Both gene editing efficiencies and HIV-1 phenotypes are highly concordant among independent donors. Importantly, over half of these factors have not been previously described to play a functional role in HIV replication, providing numerous novel avenues for understanding HIV biology. These data further suggest that host-pathogen protein-protein interaction datasets offer an enriched source of candidates for functional host factor discovery and provide an improved understanding of the mechanics of HIV replication in primary T cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

A cocrystal structure of dengue capsid protein in complex of inhibitor

Dengue virus (DENV) was listed as a top 10 public health threat by the World Health Organization in 2019. No clinically approved anti-DENV drug is currently available. Here we report the high-resolution cocrystal structure (1.5 Å) of the DENV-2 capsid protein in complex with an inhibitor that potently suppresses DENV-2 but not other DENV serotypes. The inhibitor induces a “kissing” interaction between two capsid dimers. The inhibitor-bound capsid tetramers are assembled inside virions, resulting in defective uncoating of nucleocapsid when infecting new cells. Resistant DENV-2 emerges through one mutation that abolishes hydrogen bonds in the capsid structure, leading to a loss of compound binding. Structure-based analysis has defined the amino acids responsible for the inhibitor’s inefficacy against other DENV serotypes. The results have uncovered an antiviral mechanism through inhibitor-induced tetramerization of the viral capsid and provided essential structural and functional knowledge for rational design of panserotype DENV capsid inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication

For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent cryoelectron microscopy (cryo-EM) of nodavirus RNA replication complexes revealed that the viral double-stranded RNA replication template is coiled inside a 30- to 90-nm invagination of the outer mitochondrial membrane, whose necked aperture to the cytoplasm is gated by a 12-fold symmetric, 35-nm diameter “crown” complex that contains multifunctional viral RNA replication protein A. Here we report optimizing cryo-EM tomography and image processing to improve crown resolution from 33 to 8.5 Å. This resolves the crown into 12 distinct vertical segments, each with 3 major subdomains: A membrane-connected basal lobe and an apical lobe that together comprise the ~19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at ~35-nm diameter. Despite widely varying replication vesicle diameters, the resulting two rings of membrane interaction sites constrain the vesicle neck to a highly uniform shape. Labeling protein A with a His-tag that binds 5-nm Ni-nanogold allowed cryo-EM tomography mapping of the C terminus of protein A to the apical lobe, which correlates well with the predicted structure of the C-proximal polymerase domain of protein A. These and other results indicate that the crown contains 12 copies of protein A arranged basally to apically in an N-to-C orientation. Moreover, the apical polymerase localization has significant mechanistic implications for template RNA recruitment and (-) and (+)RNA synthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Immunological Correlates of the HIV-1 Replication-Competent Reservoir Size

Understanding what shapes the latent human immunodeficiency virus type 1 (HIV-1) reservoir is critical for developing strategies for cure. Here, we measured frequency of persistent HIV-1 infection after 5 years of suppressive antiretroviral therapy initiated during chronic infection. Pretreatment CD8 + T-cell activation, nadir CD4 count, and CD4:CD8 ratio predicted reservoir size.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype

Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work, we developed a technology called VipariNama (ViN) in which vectors based on the tobacco rattle virus are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, ViN accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

An advanced workflow for single-particle imaging with the limited data at an X-ray free-electron laser

An improved analysis for single-particle imaging (SPI) experiments, using the limited data, is presented here. Results are based on a study of bacteriophage PR772 performed at the Atomic, Molecular and Optical Science instrument at the Linac Coherent Light Source as part of the SPI initiative. Existing methods were modified to cope with the shortcomings of the experimental data: inaccessibility of information from half of the detector and a small fraction of single hits. The general SPI analysis workflow was upgraded with the expectation-maximization based classification of diffraction patterns and mode decomposition on the final virus-structure determination step. The presented processing pipeline allowed us to determine the 3D structure of bacteriophage PR772 without symmetry constraints with a spatial resolution of 6.9 nm. The obtained resolution was limited by the scattering intensity during the experiment and the relatively small number of single hits.

72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS↗
DOE OSTI2020

The U.S. High-Performance Computing Consortium in the Fight Against COVID-19

U.S. computing leaders, including Department of Energy National Laboratories, have partnered with universities, government agencies, and the private sector to research responses to COVID-19, providing an unprecedented collection of resources that include some of the fastest computers in the world. For HPC users, these leadership machines will drive the AI to accelerate the discovery of promising treatments, enable at-scale simulations to understand the virus’s protein structure and attack mechanisms, and help inform policymakers to deploy resources effectively.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Lifelong cytomegalovirus and early‐LIFE irradiation synergistically potentiate age‐related defects in response to vaccination and infection

Abstract While whole‐body irradiation (WBI) can induce some hallmarks of immune aging, (re)activation of persistent microbial infection also occurs following WBI and may contribute to immune effects of WBI over the lifespan. To test this hypothesis in a model relevant to human immune aging, we examined separate and joint effects of lifelong latent murine cytomegalovirus (MCMV) and of early‐life WBI over the course of the lifespan. In late life, we then measured the response to a West Nile virus (WNV) live attenuated vaccine, and lethal WNV challenge subsequent to vaccination. We recently published that a single dose of non‐lethal WBI in youth, on its own, was not sufficient to accelerate aging of the murine immune system, despite widespread DNA damage and repopulation stress in hematopoietic cells. However, 4Gy sub‐lethal WBI caused manifest reactivation of MCMV. Following vaccination and challenge with WNV in the old age, MCMV‐infected animals experiencing 4Gy, but not lower, dose of sub‐lethal WBI in youth had reduced survival. By contrast, old irradiated mice lacking MCMV and MCMV‐infected, but not irradiated, mice were both protected to the same high level as the old non‐irradiated, uninfected controls. Analysis of the quality and quantity of anti‐WNV immunity showed that higher mortality in MCMV‐positive WBI mice correlated with increased levels of MCMV‐specific immune activation during WNV challenge. Moreover, we demonstrate that infection, including that by WNV, led to MCMV reactivation. Our data suggest that MCMV reactivation may be an important determinant of increased late‐life mortality following early‐life irradiation and late‐life acute infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Characterization of the SARS-CoV-2 B.1.621 (Mu) variant

The SARS-CoV-2 B.1.621 (Mu) variant emerged in January 2021 and was categorized as a variant of interest by the World Health Organization in August 2021. This designation prompted us to study the sensitivity of this variant to antibody neutralization. In a live virus neutralization assay with serum samples from individuals vaccinated with the Pfizer/BioNTech or Moderna mRNA vaccines, we measured neutralization antibody titers against B.1.621, an early isolate (spike 614D), and a variant of concern (B.1.351, Beta variant). We observed reduced neutralizing antibody titers against the B.1.621 variant (3.4- to 7-fold reduction, depending on the serum sample and time after the second vaccination) compared to the early isolate and a similar reduction when compared to B.1.351. Likewise, convalescent serum from hamsters previously infected with an early isolate neutralized B.1.621 to a lower degree. Despite this antibody titer reduction, hamsters could not be efficiently rechallenged with the B.1.621 variant, suggesting that the immune response to the first infection is adequate to provide protection against a subsequent infection with the B.1.621 variant.

59 BASIC BIOLOGICAL SCIENCES↗