Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication

ABSTRACTEbolavirus (EBOV) is an RNA virus that is known to cause severe hemorrhagic fever in humans and other primates.EBOV successfully enters and replicates in many cell types. This replication is dependent on the virus successfully coopting a number of cellular factors. Many of these factors are currently unidentified but represent potential targets for antiviral therapeutics. Here we show that cellular polyamines are critical for EBOV replication. We found that small-molecule inhibitors of polyamine synthesis block gene expression driven by the viral RNA-dependent RNA polymerase. Short hairpin RNA (shRNA) knockdown of the polyamine pathway enzyme spermidine synthase also resulted in reduced EBOV replication. These findings led us to further investigate spermidine, a polyamine that is essential for the hypusination of eukaryotic initiation factor 5A (eIF5A). Blocking the hypusination of eIF5A (and thereby inhibiting its function) inhibited both EBOV gene expression and viral replication. The mechanism appears to be due to the importance of hypusinated eIF5A for the accumulation of VP30, an essential component of the viral polymerase. The same reduction in hypusinated eIF5A did not alter the accumulation of other viral polymerase components. This action makes eIF5A function an important gate for proper EBOV polymerase assembly and function through the control of a single virus protein.IMPORTANCEEbolavirus (EBOV) is one of the most lethal human pathogens known. EBOV requires host factors for replication due to its small RNA genome. Here we show that the host protein eIF5A in its activated form is necessary for EBOV replication. We further show that the mechanism is through the accumulation of a single EBOV protein, VP30. To date, no other host proteins have been shown to interfere with the translation or stability of an EBOV protein. Activated eIF5A is the only protein in the cell known to contain the specific modification of hypusine; therefore, this pathway is a target for drug development. Further investigation into the mechanism of eIF5A interaction with VP30 could provide insight into therapeutics to combat EBOV.

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Native Structure-Based Peptides as Potential Protein–Protein Interaction Inhibitors of SARS-CoV-2 Spike Protein and Human ACE2 Receptor

Molecules ◽

10.3390/molecules26082157 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2157

Author(s):

Norbert Odolczyk ◽

Ewa Marzec ◽

Maria Winiewska-Szajewska ◽

Jarosław Poznański ◽

Piotr Zielenkiewicz

Keyword(s):

Drug Development ◽

Protein Interactions ◽

Rna Virus ◽

Antiviral Drug ◽

Host Protein ◽

Host Cells ◽

Cell Surface Receptor ◽

Short Peptides ◽

Virus Protein ◽

Positive Strand Rna

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a positive-strand RNA virus that causes severe respiratory syndrome in humans, which is now referred to as coronavirus disease 2019 (COVID-19). Since December 2019, the new pathogen has rapidly spread globally, with over 65 million cases reported to the beginning of December 2020, including over 1.5 million deaths. Unfortunately, currently, there is no specific and effective treatment for COVID-19. As SARS-CoV-2 relies on its spike proteins (S) to bind to a host cell-surface receptor angiotensin-converting enzyme-2(ACE2), and this interaction is proved to be responsible for entering a virus into host cells, it makes an ideal target for antiviral drug development. In this work, we design three very short peptides based on the ACE2 sequence/structure fragments, which may effectively bind to the receptor-binding domain (RBD) of S protein and may, in turn, disrupt the important virus-host protein–protein interactions, blocking early steps of SARS-CoV-2 infection. Two of our peptides bind to virus protein with affinity in nanomolar range, and as very short peptides have great potential for drug development.

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P0658MATRIX AND FLOW MODULATE GENE EXPRESSION IN A 3D VASCULARISED TUBULE MODEL

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0658 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Julie Williams ◽

Sanlin Robinson ◽

Babak Alaei ◽

Kimberly Homan ◽

Maryam Clausen ◽

...

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Impact Analysis ◽

Cell Types ◽

Drug Uptake ◽

Shear Stresses ◽

The Matrix ◽

And Function

Abstract Background and Aims Questions abound regarding the translation of in vitro 2D cell culture systems to the human setting. This is especially true of the kidney in which there is a complex hierarchical structure and a multitude of cell types. While it is well accepted that extracellular matrix plays a large part in directing cellular physiology emerging research has highlighted the importance of shear stresses and flow rates too. To fully recapitulate the normal gene expression and function of a particular renal cell type how important is it to completely reconstitute their in vivo surroundings? Method To answer this question, we have cultured proximal tubular (PT) epithelial cells in a 3-dimensional channel embedded within an engineered extracellular matrix (ECM) under physiological flow that is colocalised with an adjacent channel lined with renal microvascular endothelial cells that mimic a peritubular capillary. Modifications to the system were made to allow up to 12 chips to be run in parallel in an easily handleable form. After a period of maturation under continuous flow, both cell types were harvested for RNAseq analyses. RNA expression data was compared with cells cultured under static 2-dimensional conditions on plastic or the engineered ECM. Additionally, the perfusion of glucose through this 3D vascularised PT model has been investigated in the presence and absence of known diabetes modulating agents. Results PCA of RNAseq data showed that a) static non-coated, b) static matrix-coated and c) flow matrix-coated conditions separated into 3 distinct groups, while cell co-culture had less impact. Analysis of transcriptomic signatures showed that many genes were modulated by the matrix with additional genes influenced under flow conditions. Several of these genes, classified as transporters, are of particular importance when using this model to assess drug uptake and safety implications. Co-culture regulated some interesting genes, but fewer than anticipated. Preliminary experiments are underway to monitor glucose uptake and transport between tubules under different conditions. Conclusion We have developed a medium throughput system in which matrix and flow modulate gene expression. This system can be used to study the physiology of molecular cross-talk between cells. Ongoing analysis will further consider relevance to human physiology.

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Tanycyte-independent control of hypothalamic leptin signaling

10.1101/528794 ◽

2019 ◽

Author(s):

Sooyeon Yoo ◽

David Cha ◽

Dong Won Kim ◽

Thanh V. Hoang ◽

Seth Blackshaw

Keyword(s):

Gene Expression ◽

Single Molecule ◽

Leptin Receptor ◽

Cell Types ◽

Leptin Signaling ◽

And Function ◽

Induced Changes ◽

The Brain ◽

Specific Deletion

AbstractLeptin is secreted by adipocytes to regulate appetite and body weight. Recent studies have reported that tanycytes actively transport circulating leptin across the brain barrier into the hypothalamus, and are required for normal levels of hypothalamic leptin signaling. However, direct evidence for leptin receptor (LepR) expression is lacking, and the effect of tanycyte-specific deletion of LepR has not been investigated. In this study, we analyze the expression and function of the tanycytic LepR in mice. Using single-molecule fluorescent in situ hybridization (smfISH), RT-qPCR, single-cell RNA sequencing (scRNA-Seq), and selective deletion of the LepR in tanycytes, we are unable to detect expression of LepR in the tanycytes. Tanycyte-specific deletion of LepR likewise did not affect leptin-induced pSTAT3 expression in hypothalamic neurons, regardless of whether leptin was delivered by intraperitoneal or intracerebroventricular injection. Finally, we use activity-regulated scRNA-Seq (act-Seq) to comprehensively profile leptin-induced changes in gene expression in all cell types in mediobasal hypothalamus. Clear evidence for leptin signaling is only seen in endothelial cells and subsets of neurons, although virtually all cell types show leptin-induced changes in gene expression. We thus conclude that LepR expression in tanycytes is either absent or undetectably low, that tanycytes do not directly regulate hypothalamic leptin signaling through a LepR-dependent mechanism, and that leptin regulates gene expression in diverse hypothalamic cell types through both direct and indirect mechanisms.

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The rocaglate CR-31-B (-) inhibits SARS-CoV-2 replication at non-cytotoxic, low nanomolar concentrations in vitro and ex vivo

10.1101/2020.11.24.389627 ◽

2020 ◽

Author(s):

Christin Müller ◽

Wiebke Obermann ◽

Nadja Karl ◽

Hans-Guido Wendel ◽

Gaspar Taroncher-Oldenburg ◽

...

Keyword(s):

Antiviral Activity ◽

Ex Vivo ◽

Rna Virus ◽

Mrna Translation ◽

Hemorrhagic Fever ◽

Airway Epithelial Cells ◽

Initiation Factor ◽

Protein Accumulation ◽

Hemorrhagic Fever Virus

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a betacoronavirus in the subgenus Sarbecovirus causes a respiratory disease with varying symptoms referred to as coronavirus disease 2019 (COVID-19) and is responsible for a pandemic that started in early 2020. With no vaccines or effective antiviral treatments available, and infection and fatality numbers continuing to increase globally, the quest for novel therapeutic solutions remains an urgent priority. Rocaglates, a class of plant-derived cyclopenta[b]benzofurans, exhibit broad-spectrum antiviral activity against positive- and negative-sense RNA viruses. This compound class inhibits eukaryotic initiation factor 4A (eIF4A)-dependent mRNA translation initiation, resulting in strongly reduced viral RNA translation. The synthetic rocaglate CR-31-B (-) has previously been shown to inhibit the replication of human coronaviruses, such as HCoV-229E and MERS-CoV, as well as Zika-, Lassa-, Crimean Congo hemorrhagic fever virus in primary cells. Here, we assessed the antiviral activity of CR-31-B (-) against SARS-CoV-2 using both in vitro and ex vivo cell culture models. In African green monkey Vero E6 cells, CR-31-B (-) inhibited SARS-CoV-2 replication with an EC50 of ~1.8 nM. In line with this, viral protein accumulation and replication/transcription complex formation were found to be strongly reduced by this compound. In an ex vivo infection system using human airway epithelial cells, CR-31-B (-) was found to cause a massive reduction of SARS-CoV-2 titers by about 4 logs to nearly non-detectable levels. The data reveal a potent anti-SARS-CoV-2 activity by CR-31-B (-), corroborating previous results obtained for other coronaviruses and supporting the idea that rocaglates may be used in first-line antiviral intervention strategies against novel and emerging RNA virus outbreaks.

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Molecular Assessment of Epiretinal Membrane: Activated Microglia, Oxidative Stress and Inflammation

Antioxidants ◽

10.3390/antiox9080654 ◽

2020 ◽

Vol 9 (8) ◽

pp. 654 ◽

Cited By ~ 1

Author(s):

Sushma Vishwakarma ◽

Rishikesh Kumar Gupta ◽

Saumya Jakati ◽

Mudit Tyagi ◽

Rajeev Reddy Pappuru ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Epiretinal Membrane ◽

Hypoxia Inducible Factor ◽

Gene Expression Signature ◽

Cell Types ◽

Activated Microglia ◽

Inflammatory Conditions ◽

New Information ◽

And Function

Fibrocellular membrane or epiretinal membrane (ERM) forms on the surface of the inner limiting membrane (ILM) in the inner retina and alters the structure and function of the retina. ERM formation is frequently observed in ocular inflammatory conditions, such as proliferative diabetic retinopathy (PDR) and retinal detachment (RD). Although peeling of the ERM is used as a surgical intervention, it can inadvertently distort the retina. Our goal is to design alternative strategies to tackle ERMs. As a first step, we sought to determine the composition of the ERMs by identifying the constituent cell-types and gene expression signature in patient samples. Using ultrastructural microscopy and immunofluorescence analyses, we found activated microglia, astrocytes, and Müller glia in the ERMs from PDR and RD patients. Moreover, oxidative stress and inflammation associated gene expression was significantly higher in the RD and PDR membranes as compared to the macular hole samples, which are not associated with inflammation. We specifically detected differential expression of hypoxia inducible factor 1-α (HIF1-α), proinflammatory cytokines, and Notch, Wnt, and ERK signaling pathway-associated genes in the RD and PDR samples. Taken together, our results provide new information to potentially develop methods to tackle ERM formation.

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Thoc1 Deficiency Compromises Gene Expression Necessary for Normal Testis Development in the Mouse

Molecular and Cellular Biology ◽

10.1128/mcb.01633-08 ◽

2009 ◽

Vol 29 (10) ◽

pp. 2794-2803 ◽

Cited By ~ 26

Author(s):

Xiaoling Wang ◽

Meenalakshmi Chinnam ◽

Jianmin Wang ◽

Yanqing Wang ◽

Xiaojing Zhang ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Rna Processing ◽

Cell Types ◽

Specific Gene ◽

Mammalian Development ◽

Expression Of Genes ◽

Normal Differentiation ◽

And Function

ABSTRACT Accumulating evidence suggests that regulation of RNA processing through an RNP-driven mechanism is important for coordinated gene expression. This hypothesis predicts that defects in RNP biogenesis will adversely affect the elaboration of specific gene expression programs. To explore the role of RNP biogenesis on mammalian development, we have characterized the phenotype of mice hypomorphic for Thoc1. Thoc1 encodes an essential component of the evolutionarily conserved TREX complex. TREX accompanies the elongating RNA polymerase II and facilitates RNP assembly and recruitment of RNA processing factors. Hypomorphic Thoc1 mice are viable despite significantly reduced Thoc1 expression in the tissues examined. While most tissues of Thoc1-deficient mice appear to develop and function normally, gametogenesis is severely compromised. Male infertility is associated with a loss in spermatocyte viability and abnormal endocrine signaling. We suggest that loss of spermatocyte viability is a consequence of defects in the expression of genes required for normal differentiation of cell types within the testes. A number of the genes affected appear to be direct targets for regulation by Thoc1. These findings support the notion that Thoc1-mediated RNP assembly contributes to the coordinated expression of genes necessary for normal differentiation and development in vivo.

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Identification of residues in Lassa virus glycoprotein 1 involved in receptor switch

10.1101/2021.04.30.442226 ◽

2021 ◽

Author(s):

Jiao Guo ◽

Xiaoying Jia ◽

Yang Liu ◽

Junyuan Cao ◽

Gengfu Xiao ◽

...

Keyword(s):

Binding Affinity ◽

Rna Virus ◽

Treatment Options ◽

Hemorrhagic Fever ◽

Cell Types ◽

Lymphocytic Choriomeningitis ◽

Lassa Virus ◽

Viral Glycoprotein ◽

Mutant Proteins ◽

Alpha Dystroglycan

Lassa virus (LASV) is an enveloped, negative-sense RNA virus that causes Lassa hemorrhagic fever, for which there are limited treatment options. Successful LASV entry requires the viral glycoprotein 1 (GP1) to undergo a receptor switch from its primary receptor alpha-dystroglycan (α-DG) to its endosomal receptor lysosome-associated membrane protein 1 (LAMP1). A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch. To test the hypothesis that other non-conserved residues also contribute to receptor switch, we constructed a series of GP1 mutant proteins and tested them for binding to LAMP1. Four residues, L84, K88, L107, and H170, were identified as critical for receptor switch. Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus residue (L84N, K88E, L10F, and H170S) reduced the binding affinity of GP1LASV for LAMP1. Moreover, all the mutations caused decreases in GPC-mediated membrane fusion at both pH 4.5 and 5.2. The infectivity of pseudotyped viruses bearing either GPCL84N or GPCK88E decreased sharply in multiple cell types, whereas L107F and H170S had only mild effects on infectivity. Notably, in LAMP1 knockout cells, all four mutants showed reduced pseudovirus infectivity. Using biolayer light interferometry assay, we found that all four mutants had decreased binding affinity to LAMP1, in the order L84N > L107F > K88E > H170S.

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Predicting CTCF-mediated chromatin interactions by integrating genomic and epigenomic features

10.1101/215871 ◽

2017 ◽

Cited By ~ 2

Author(s):

Yan Kai ◽

Jaclyn Andricovich ◽

Zhouhao Zeng ◽

Jun Zhu ◽

Alexandros Tzatsos ◽

...

Keyword(s):

Gene Expression ◽

Long Range ◽

Zinc Finger Protein ◽

Cell Types ◽

Learning Framework ◽

Chromatin Interactions ◽

Long Range Interactions ◽

Extensive Cell ◽

Cell Type Specific ◽

And Function

AbstractThe CCCTC-binding zinc finger protein (CTCF)-mediated network of long-range chromatin interactions is important for genome organization and function. Although this network has been considered largely invariant, we found that it exhibits extensive cell-type-specific interactions that contribute to cell identity. Here we present Lollipop—a machine-learning framework—which predicts CTCF-mediated long-range interactions using genomic and epigenomic features. Using ChIA-PET data as benchmark, we demonstrated that Lollipop accurately predicts CTCF-mediated chromatin interactions both within and across cell-types, and outperforms other methods based only on CTCF motif orientation. Predictions were confirmed computationally and experimentally by Chromatin Conformation Capture (3C). Moreover, our approach reveals novel determinants of CTCF-mediated chromatin wiring, such as gene expression within the loops. Our study contributes to a better understanding about the underlying principles of CTCF-mediated chromatin interactions and their impact on gene expression.

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Transcriptome Network Analysis Identifies CXCL13-CXCR5 Signaling Modules in the Prostate Tumor Immune Microenvironment

Scientific Reports ◽

10.1038/s41598-019-46491-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Adaugo Q. Ohandjo ◽

Zongzhi Liu ◽

Eric B. Dammer ◽

Courtney D. Dill ◽

Tiara L. Griffen ◽

...

Keyword(s):

Gene Expression ◽

Network Analysis ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Expression Signature ◽

Cell Types ◽

Immune Microenvironment ◽

Tumor Immune Microenvironment ◽

Quality Checks ◽

And Function

Abstract The tumor immune microenvironment (TIME) consists of multiple cell types that contribute to the heterogeneity and complexity of prostate cancer (PCa). In this study, we sought to understand the gene-expression signature of patients with primary prostate tumors by investigating the co-expression profiles of patient samples and their corresponding clinical outcomes, in particular “disease-free months” and “disease reoccurrence”. We tested the hypothesis that the CXCL13-CXCR5 axis is co-expressed with factors supporting TIME and PCa progression. Gene expression counts, with clinical attributes from PCa patients, were acquired from TCGA. Profiles of PCa patients were used to identify key drivers that influence or regulate CXCL13-CXCR5 signaling. Weighted gene co-expression network analysis (WGCNA) was applied to identify co-expression patterns among CXCL13-CXCR5, associated genes, and key genetic drivers within the CXCL13-CXCR5 signaling pathway. The processing of downloaded data files began with quality checks using NOISeq, followed by WGCNA. Our results confirmed the quality of the TCGA transcriptome data, identified 12 co-expression networks, and demonstrated that CXCL13, CXCR5 and associated genes are members of signaling networks (modules) associated with G protein coupled receptor (GPCR) responsiveness, invasion/migration, immune checkpoint, and innate immunity. We also identified top canonical pathways and upstream regulators associated with CXCL13-CXCR5 expression and function.

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Retinal miRNA Functions in Health and Disease

Genes ◽

10.3390/genes10050377 ◽

2019 ◽

Vol 10 (5) ◽

pp. 377 ◽

Cited By ~ 14

Author(s):

Marta Zuzic ◽

Jesus Eduardo Rojo Arias ◽

Stefanie Gabriele Wohl ◽

Volker Busskamp

Keyword(s):

Gene Expression ◽

Visual Impairment ◽

Current Knowledge ◽

Cell Types ◽

Therapeutic Agents ◽

Retinal Cell ◽

Mrna Targets ◽

Health And Disease ◽

Cell Type Specific ◽

And Function

The health and function of our visual system relies on accurate gene expression. While many genetic mutations are associated with visual impairment and blindness, we are just beginning to understand the complex interplay between gene regulation and retinal pathologies. MicroRNAs (miRNAs), a class of non-coding RNAs, are important regulators of gene expression that exert their function through post-transcriptional silencing of complementary mRNA targets. According to recent transcriptomic analyses, certain miRNA species are expressed in all retinal cell types, while others are cell type-specific. As miRNAs play important roles in homeostasis, cellular function, and survival of differentiated retinal cell types, their dysregulation is associated with retinal degenerative diseases. Thus, advancing our understanding of the genetic networks modulated by miRNAs is central to harnessing their potential as therapeutic agents to overcome visual impairment. In this review, we summarize the role of distinct miRNAs in specific retinal cell types, the current knowledge on their implication in inherited retinal disorders, and their potential as therapeutic agents.

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