Functional Analyses of Bovine Foamy Virus-Encoded miRNAs Reveal the Importance of a Defined miRNA for Virus Replication and Host–Virus Interaction

In addition to regulatory or accessory proteins, some complex retroviruses gain a repertoire of micro-RNAs (miRNAs) to regulate and control virus–host interactions for efficient replication and spread. In particular, bovine and simian foamy viruses (BFV and SFV) have recently been shown to express a diverse set of RNA polymerase III-directed miRNAs, some with a unique primary miRNA double-hairpin, dumbbell-shaped structure not known in other viruses or organisms. While the mechanisms of expression and structural requirements have been studied, the functional importance of these miRNAs is still far from understood. Here, we describe the in silico identification of BFV miRNA targets and the subsequent experimental validation of bovine Ankyrin Repeat Domain 17 (ANKRD17) and Bax-interacting factor 1 (Bif1) target genes in vitro and, finally, the suppression of ANKRD17 downstream genes in the affected pathway. Deletion of the entire miRNA cassette in the non-coding part of the U3 region of the long terminal repeats attenuated replication of corresponding BFV mutants in bovine cells. This repression can be almost completely trans-complemented by the most abundant miRNA BF2-5p having the best scores for predicted and validated BFV miRNA target genes. Deletion of the miRNA cassette does not grossly affect particle release and overall particle composition.

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Bovine Foamy Virus Transactivator BTas Interacts with Cellular RelB To Enhance Viral Transcription

Journal of Virology ◽

10.1128/jvi.01036-10 ◽

2010 ◽

Vol 84 (22) ◽

pp. 11888-11897 ◽

Cited By ~ 14

Author(s):

Jian Wang ◽

Juan Tan ◽

Hongyan Guo ◽

Qicheng Zhang ◽

Rui Jia ◽

...

Keyword(s):

Foamy Virus ◽

Nuclear Factor Κb ◽

Luciferase Reporter ◽

Viral Transcription ◽

Bovine Foamy Virus ◽

Intracellular Parasites ◽

Positive Feedback Circuit ◽

Cellular Machinery

ABSTRACT Viruses are obligate intracellular parasites that depend on cellular machinery for their efficient transcription and replication. In a previous study we reported that bovine foamy virus (BFV) is able to activate the nuclear factor κB (NF-κB) pathway through the action of its transactivator BTas to enhance viral transcription. However, the mechanism used by NF-κB to enhance BFV transcription remains elusive. To address this question, we employed a yeast two-hybrid assay to screen for BTas-interacting proteins. We found that RelB, a member of NF-κB protein family, interacts with BTas. We confirmed the putative RelB-BTas interaction in vitro and in vivo and identified the protein regions responsible for the RelB-BTas interaction. Using a luciferase reporter assay, we next showed that RelB enhances BFV transcription (BTas-induced long terminal repeat [LTR] transactivation) and that this process requires both the localization of the RelB-BTas interaction in the nucleus and the Rel homology domain of RelB. The knockdown of the cellular endogenous RelB protein using small interfering RNA (siRNA) significantly attenuated BTas-induced LTR transcription. The results of chromatin immunoprecipitation (ChIP) analysis showed that endogenous RelB binds to the viral LTR in BFV-infected cells. Together, these results suggest that BFV engages the RelB protein as a cotransactivator of BTas to enhance viral transcription. In addition, our findings indicate that BFV infection upregulates cellular RelB expression through BTas-induced NF-κB activation. Thus, this study demonstrates the existence of a positive-feedback circuit in which BFV utilizes the host's NF-κB pathway through the RelB protein for efficient viral transcription.

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Abstract WMP80: Micro RNA-Therapy in Murine Thromboembolic Stroke

Stroke ◽

10.1161/str.48.suppl_1.wmp80 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Kumar Vaibhav ◽

Shannon Williams ◽

Sumbul Fatima ◽

Babak Baban ◽

Krishnan M Dhandapani ◽

...

Keyword(s):

Target Genes ◽

Infarct Volume ◽

Protein Translation ◽

Micro Rna ◽

Thromboembolic Stroke ◽

Gene Expressions ◽

Mirna Array ◽

Micro Rnas ◽

Pai 1

Background: Micro RNAs (miRNAs) could target multiple mRNAs, repressing the protein translation. We report acute changes in humoral miRNAome in a murine thromboembolic stroke model (eMCAo), and demonstrate the benefits of miRNA therapy in improving cerebral blood flow (CBF). Methods: Non-biased micro RNA (miRNA) array and bioinformatics analysis was performed in plasma collected at 4h post-eMCAo from male mice (C57/B6, 16-weeks). Individual PCR for miRNAs was also performed in brain tissues at 24h post-eMCAo. Moreover, frozen human plasma samples collected at ~4.5h post-stroke were also used for miRNA analysis. Finally, the miRNA mimic that was predicted to target genes of our interest was also tested in vivo and in vitro . Results: Principal component analysis (PCA) of the miRNA-array showed ~68% variance in the humoral miRNAome 4h after eMCAo in mice, and a significant change in Stroke vs. Sham groups (Cut off value >2 fold; p<0.05). Of interest, the hairpin precursor of miR-449b was downregulated (~2.35 fold, p<0.05) at 4h post-eMCAo, while the mature miR-449b was also significantly reduced at 24h post-eMCAo. Mature miR-449b was significantly reduced in human stroke plasma, too. In human brain endothelial cells, miR-449b mimic downregulated gene expressions of both plasminogen activator inhibitor (PAI-1) and alpha 2- antiplasmin (α-AP) only in hypoxia but not during normoxia. Therefore, we finally tested the cholesterol-conjugated miR-449b mimic in the murine eMCAo model. Pre-treatment with miR-449b mimic (8 mg/kg bwt) increased the absolute CBF and reduced edema (as determined by MRI), and also improved the neurological outcomes and reduced % infarct volume (p<0.05). Results: The miR-449b mimic could be a possible therapy to suppress aberrant gene expressions of PAI-1 and α-AP, which will allow more spontaneous reperfusion and benefits from low dose tPA.

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Circulating micro-RNAs Differentially Expressed in Korean Alzheimer’s Patients with Brain Aβ Accumulation Activate Amyloidogenesis

10.21203/rs.3.rs-944590/v1 ◽

2021 ◽

Author(s):

Sakulrat Mankhong ◽

Sujin Kim ◽

Sohee Moon ◽

Seong-Hye Choi ◽

Hyo-Bum Kwak ◽

...

Keyword(s):

Target Genes ◽

Clinical Performance ◽

Interaction Network ◽

Differentially Expressed ◽

Diagnostic Utility ◽

Cross Sectional ◽

Positron Emission ◽

Micro Rnas ◽

Vitro Analysis

Abstract BACKGROUNDA role for extracellular vesicles (EVs) enriched with micro-RNAs (miRNAs) has been proposed in Alzheimer’s disease (AD) pathogenesis, leading to the discovery of blood miRNAs as biomarkers of AD. However, the diagnostic utility of specific miRNAs is not consistent. This study aimed to discover blood miRNAs that are differentially expressed in Korean AD patients, evaluate their clinical performance in plasma or plasma EVs, and investigate their role in amyloidogenesis. METHODSBlood from 15 (7 cognitively normal [CN] and 8 AD) out of 262 subjects (59 CN, 105 mild cognitive impairment [MCI], 98 AD) and 8 Parkinson’s disease (PD) patients was used to discover miRNAs differentially expressed in AD. We evaluated the clinical performance of these miRNAs in plasma of a subgroup of 100 subjects (51 CN, 22 MCI, 27 AD) and in plasma EVs isolated from the total cohort in a cross-sectional design. The effects of miRNAs on amyloid b (Ab) production and expression of their target genes were investigated in neuronal culture systems. RESULTSAmong 17 upregulated, and one downregulated miRNAs in AD (>2-fold), three upregulated miRNAs (miR-122-5p, miR-210-3p, and miR-590-5p) that were differentially expressed in AD compared with CN or PD were selected. Diagnostic utility for discrimination of AD or MCI from CN of the selected miRNAs in plasma or plasma EV was not high. Nevertheless, the levels of three miRNAs in plasma or plasma EVs of subjects who were Ab positive on positron emission tomography (PET) were significantly higher than those from subjects who were Ab-PET negative. Furthermore, the selected miRNAs induced Ab production through activation of b-cleavage of amyloid precursor protein and downregulated their target genes. Pathway enrichment and protein interaction network analysis of target genes of the miRNAs further supported the roles of the selected miRNAs in amyloidogenesis. CONCLUSIONThe diagnostic utility of circulating miR-122-5p, miR-210-3p, and miR-590-5p to discriminate AD from CN was modest. However, these miRNAs were highly expressed in patients with amyloid accumulation, which was supported by in vitro analysis of amyloidogenesis. Our results suggest that blood-based miRNA biomarkers may play a role in amyloidogenesis during AD onset and progression.

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A Limited Set of Human MicroRNA Is Deregulated in Follicular Thyroid Carcinoma

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2006-0693 ◽

2006 ◽

Vol 91 (9) ◽

pp. 3584-3591 ◽

Cited By ~ 206

Author(s):

Frank Weber ◽

Rosemary E. Teresi ◽

Christoph E. Broelsch ◽

Andrea Frilling ◽

Charis Eng

Keyword(s):

Thyroid Carcinoma ◽

Target Genes ◽

Follicular Thyroid Carcinoma ◽

Follicular Adenoma ◽

Aberrant Expression ◽

Small Noncoding Rnas ◽

Downstream Target ◽

Micro Rnas

Abstract Context: Although the pathogenesis of follicular thyroid carcinoma (FTC) and its relation to follicular adenoma (FA) remains unclear, detailed understanding of FTC carcinogenesis would facilitate addressing the scientific and clinical challenges, given that there are morphological and molecular similarities between FTC and the frequently occurring FA. Micro-RNAs (miRNAs) are a new class of small, noncoding RNAs implicated in development and cancer and may lend novel clues to FTC genesis. For the latter process, a deregulated miRNA can orchestrate the aberrant expression of several hundred target genes. Objective: The objective of the study was to identify deregulated miRNAs in FTC. Design: We used two high-density expression arrays to identify miRNAs and their target genes that are differentially expressed between FTC and FA. Validation was done by quantitative RT-PCR. We further functionally characterized the effect of deregulated miRNAs in vitro using HEK293T, FTC133, and K5 cell lines. Patients: In total, 45 primary thyroid samples (23 FTC, 20 FA, four normal control thyroid) were analyzed. Results: Two specific miRNAs, miR-197 and miR-346, were significantly overexpressed in FTC. In vitro overexpression of either miRNA induced proliferation, whereas inhibition led to growth arrest. Overexpression of miR-197 and miR-346 repressed the expression of their predicted target genes in vitro and in vivo. Conclusions: Our observations show that miR-197 and miR-346 contribute to FTC carcinogenesis. Both miRNAs and their target genes might potentially provide for novel molecular markers and act as novel targets for treatment by interference, which could potentially normalize the deregulated profile of many downstream target genes.

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Bovine Foamy Virus: Shared and Unique Molecular Features In Vitro and In Vivo

Viruses ◽

10.3390/v11121084 ◽

2019 ◽

Vol 11 (12) ◽

pp. 1084 ◽

Cited By ~ 2

Author(s):

Magdalena Materniak-Kornas ◽

Juan Tan ◽

Anke Heit-Mondrzyk ◽

Agnes Hotz-Wagenblatt ◽

Martin Löchelt

Keyword(s):

Animal Studies ◽

Foamy Virus ◽

Companion Animals ◽

Population Level ◽

Medical Problems ◽

Bovine Foamy Virus ◽

Molecular Features ◽

Unclear Etiology

The retroviral subfamily of Spumaretrovirinae consists of five genera of foamy (spuma) viruses (FVs) that are endemic in some mammalian hosts. Closely related species may be susceptible to the same or highly related FVs. FVs are not known to induce overt disease and thus do not pose medical problems to humans and livestock or companion animals. A robust lab animal model is not available or is a lab animal a natural host of a FV. Due to this, research is limited and often focused on the simian FVs with their well-established zoonotic potential. The authors of this review and their groups have conducted several studies on bovine FV (BFV) in the past with the intention of (i) exploring the risk of zoonotic infection via beef and raw cattle products, (ii) studying a co-factorial role of BFV in different cattle diseases with unclear etiology, (iii) exploring unique features of FV molecular biology and replication strategies in non-simian FVs, and (iv) conducting animal studies and functional virology in BFV-infected calves as a model for corresponding studies in primates or small lab animals. These studies gained new insights into FV-host interactions, mechanisms of gene expression, and transcriptional regulation, including miRNA biology, host-directed restriction of FV replication, spread and distribution in the infected animal, and at the population level. The current review attempts to summarize these findings in BFV and tries to connect them to findings from other FVs.

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Cell-type- and region-specific modulation of cocaine seeking by micro-RNA-1 in striatal projection neurons

Molecular Psychiatry ◽

10.1038/s41380-021-01328-2 ◽

2021 ◽

Author(s):

Benoit Forget ◽

Elena Martin Garcia ◽

Arthur Godino ◽

Laura Domingo Rodriguez ◽

Vincent Kappes ◽

...

Keyword(s):

Target Genes ◽

Micro Rna ◽

Cell Populations ◽

Projection Neurons ◽

Cell Type ◽

Mrna Targets ◽

Cocaine Seeking ◽

Micro Rnas

AbstractThe persistent and experience-dependent nature of drug addiction may result in part from epigenetic alterations, including non-coding micro-RNAs (miRNAs), which are both critical for neuronal function and modulated by cocaine in the striatum. Two major striatal cell populations, the striato-nigral and striato-pallidal projection neurons, express, respectively, the D1 (D1-SPNs) and D2 (D2-SPNs) dopamine receptor, and display distinct but complementary functions in drug-evoked responses. However, a cell-type-specific role for miRNAs action has yet to be clarified. Here, we evaluated the expression of a subset of miRNAs proposed to modulate cocaine effects in the nucleus accumbens (NAc) and dorsal striatum (DS) upon sustained cocaine exposure in mice and showed that these selected miRNAs were preferentially upregulated in the NAc. We focused on miR-1 considering the important role of some of its predicted mRNA targets, Fosb and Npas4, in the effects of cocaine. We validated these targets in vitro and in vivo. We explored the potential of miR-1 to regulate cocaine-induced behavior by overexpressing it in specific striatal cell populations. In DS D1-SPNs miR-1 overexpression downregulated Fosb and Npas4 and reduced cocaine-induced CPP reinstatement, but increased cue-induced cocaine seeking. In DS D2-SPNs miR-1 overexpression reduced the motivation to self-administer cocaine. Our results indicate a role of miR1 and its target genes, Fosb and Npas4, in these behaviors and highlight a precise cell-type- and region-specific modulatory role of miR-1, illustrating the importance of cell-specific investigations.

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In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission

Viruses ◽

10.3390/v7112907 ◽

2015 ◽

Vol 7 (11) ◽

pp. 5855-5874 ◽

Cited By ~ 8

Author(s):

Qiuying Bao ◽

Michaela Hipp ◽

Annette Hugo ◽

Janet Lei ◽

Yang Liu ◽

...

Keyword(s):

Foamy Virus ◽

Free Virus ◽

In Vitro Evolution ◽

Bovine Foamy Virus

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The Influence of Envelope C-Terminus Amino Acid Composition on the Ratio of Cell-Free to Cell-Cell Transmission for Bovine Foamy Virus

Viruses ◽

10.3390/v11020130 ◽

2019 ◽

Vol 11 (2) ◽

pp. 130 ◽

Cited By ~ 3

Author(s):

Suzhen Zhang ◽

Xiaojuan Liu ◽

Zhibin Liang ◽

Tiejun Bing ◽

Wentao Qiao ◽

...

Keyword(s):

High Titer ◽

Foamy Virus ◽

Evolution System ◽

Bovine Foamy Virus ◽

C Terminus ◽

Release Capacity ◽

Cell Transmission ◽

Extensive Cell ◽

Original Cell

Foamy viruses (FVs) have extensive cell tropism in vitro, special replication features, and no clinical pathogenicity in naturally or experimentally infected animals, which distinguish them from orthoretroviruses. Among FVs, bovine foamy virus (BFV) has undetectable or extremely low levels of cell-free transmission in the supernatants of infected cells and mainly spreads by cell-to-cell transmission, which deters its use as a gene transfer vector. Here, using an in vitro virus evolution system, we successfully isolated high-titer cell-free BFV strains from the original cell-to-cell transmissible BFV3026 strain and further constructed an infectious cell-free BFV clone called pBS-BFV-Z1. Following sequence alignment with a cell-associated clone pBS-BFV-B, we identified a number of changes in the genome of pBS-BFV-Z1. Extensive mutagenesis analysis revealed that the C-terminus of envelope protein, especially the K898 residue, controls BFV cell-free transmission by enhancing cell-free virus entry but not the virus release capacity. Taken together, our data show the genetic determinants that regulate cell-to-cell and cell-free transmission of BFV.

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Lentiviral interferon: A novel method for gene therapy in bladder cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.7_suppl.456 ◽

2019 ◽

Vol 37 (7_suppl) ◽

pp. 456-456

Author(s):

Sharada Mokkapati ◽

Jonathan J Duplisea ◽

Devin Plote ◽

Vikram M Narayan ◽

Amy Lim ◽

...

Keyword(s):

Gene Therapy ◽

Bladder Cancer ◽

Gene Delivery ◽

Cell Viability ◽

Interferon Alpha ◽

Target Genes ◽

Complete Response ◽

Blue Dye ◽

Control Virus

456 Background: Gene therapy for bladder cancer (BLCA) is rapidly evolving. We reported that intravesical adenoviral interferon-alpha (Ad-IFNα) produced a complete response in 35% of patients with BCG-unresponsive BLCA enrolled in a Phase II trial. Lentivirus (LV) is another potential vector for intravesical delivery of IFNα. Unlike the adenovirus, LV can infect non-dividing cells and integrate into the host’s genome, making it one of the most efficient gene delivery vectors. The objective of this study was to investigate lentiviral interferon-alpha (LV-IFNα) BLCA gene therapy in preclinical models. Methods: Murine BLCA cell lines were transduced in-vitro with LV-IFNα using a multiplicity of infection (MOI) of 2:1. IFNα levels were measured by ELISA. Cell viability was assessed using Trypan blue dye exclusion. qPCR was used to identify expression of IFNα target genes. A LV-βGalactosidase reporter construct was delivered intravesically, and urinary IFNα levels were measured in mice treated with LV-IFNα or control virus to assess gene transfer. To assess survival benefit, p53+/- C57/B6 mice were exposed to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) to induce CIS and then treated with LV-IFNα or control virus, and sacrificed when moribund. Results: Efficient LV-IFNα transduction of BLCA cells was observed at an MOI of 2:1, resulting in increased expression of IFNα and its target genes PDL-1, TRAIL, and IRF7 (p<0.001), and reduced cell viability vs. controls (p<0.001). Mechanistically, TRAIL dependent cytotoxicity in the LV-IFNα cells was rescued by Caspase 8 inhibition. βGal expression confirmed efficient transduction of murine urothelium. Urinary IFNα levels were elevated in mice receiving LV-IFNα compared with control virus. BBN mice treated with LV-IFNα had longer overall survival than mice treated with control virus (p=0.04). LV-IFNα induced intratumoral CD8+ T cell infiltration, high expression of PD-L1, and inhibited angiogenesis. Conclusions: LV-IFNα effectively upregulated IFNα target genes, was cytotoxic to murine BLCA cells, and improved the survival of BBN tumor-bearing mice. LV appears to be a promising vector for intravesical gene delivery.

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CHOP-Dependent Stress-Inducible Expression of a Novel Form of Carbonic Anhydrase VI

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.495 ◽

1999 ◽

Vol 19 (1) ◽

pp. 495-504 ◽

Cited By ~ 102

Author(s):

John Sok ◽

Xiao-Zhong Wang ◽

Nikoleta Batchvarova ◽

Masahiko Kuroda ◽

Heather Harding ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Target Gene ◽

Target Genes ◽

Direct Evidence ◽

Nuclear Protein ◽

Intracellular Form ◽

Carbonic Anhydrase Vi ◽

Responsive Element

ABSTRACT CHOP (also called GADD153) is a stress-inducible nuclear protein that dimerizes with members of the C/EBP family of transcription factors and was initially identified as an inhibitor of C/EBP binding to classic C/EBP target genes. Subsequent experiments suggested a role for CHOP-C/EBP heterodimers in positively regulating gene expression; however, direct evidence that this is the case has so far not been uncovered. Here we describe the identification of a positively regulated direct CHOP-C/EBP target gene, that encoding murine carbonic anhydrase VI (CA-VI). The stress-inducible form of the gene is expressed from an internal promoter and encodes a novel intracellular form of what is normally a secreted protein. Stress-induced expression of CA-VI is both CHOP and C/EBPβ dependent in that it does not occur in cells deficient in either gene. A CHOP-responsive element was mapped to the inducibleCA-VI promoter, and in vitro footprinting revealed binding of CHOP-C/EBP heterodimers to that site. Rescue of CA-VIexpression in c/ebpβ−/− cells by exogenous C/EBPβ and a shorter, normally inhibitory isoform of the protein known as LIP suggests that the role of the C/EBP partner is limited to targeting the CHOP-containing heterodimer to the response element and points to a preeminent role for CHOP in CA-VI induction during stress.

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