A Versatile Plant Rhabdovirus-Based Vector for Gene Silencing, miRNA Expression and Depletion, and Antibody Production

Plant virus vectors are ideal tools for delivery of genetic cargo into host cells for functional genomics studies and protein overexpression. Although a vast number of plant virus vectors have been developed for different purposes, the utility of a particular virus vector is generally limited. Here, we report a multipurpose plant rhabdovirus-based vector system suitable for a wide range of applications in Nicotiana benthamiana. We engineered sonchus yellow net rhabdovirus (SYNV)-based gene silencing vectors through expressing a sense, antisense, or double-stranded RNAs of target genes. Robust target gene silencing was also achieved with an SYNV vector expressing a designed artificial microRNA. In addition, ectopic expression of a short tandem target mimic RNA using the SYNV vector led to a significant depletion of the target miR165/166 and caused abnormal leaf development. More importantly, SYNV was able to harbor two expression cassettes that permitted simultaneous RNA silencing and overexpression of large reporter gene. This dual capacity vector also enabled systemic expression of a whole-molecule monoclonal antibody consisting of light and heavy chains. These results highlight the utility of the SYNV vector system in gene function studies and agricultural biotechnology and provide a technical template for developing similar vectors of other economically important plant rhabdoviruses.

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MicroRNA miR-27 Inhibits Adenovirus Infection by Suppressing the Expression of SNAP25 and TXN2

Journal of Virology ◽

10.1128/jvi.00159-17 ◽

2017 ◽

Vol 91 (12) ◽

Cited By ~ 12

Author(s):

Mitsuhiro Machitani ◽

Fuminori Sakurai ◽

Keisaku Wakabayashi ◽

Kosuke Nakatani ◽

Masashi Tachibana ◽

...

Keyword(s):

Cell Cycle ◽

Gene Silencing ◽

Cell Cycle Arrest ◽

Target Genes ◽

Host Cells ◽

Microarray Gene Expression ◽

Posttranscriptional Gene Silencing ◽

Gene Therapies ◽

Cycle Arrest ◽

Efficient Suppression

ABSTRACT Recent studies have reported that host microRNAs (miRNAs) regulate infections by several types of viruses via various mechanisms and that inhibition of the miRNA processing factors enhances or prevents viral infection. However, it has not been clarified whether these effects of miRNAs extend to adenovirus (Ad) infection. Here we show that miR-27a and -b efficiently inhibit infection with an Ad via the downregulation of SNAP25 and TXN2, which are members of the SNARE proteins and the thioredoxin family, respectively. Approximately 80% reductions in Ad genomic copy number were found in cells transfected with miR-27a/b mimics, whereas there were approximately 2.5- to 5-fold larger copy numbers of the Ad genome following transfection with miR-27a/b inhibitors. Microarray gene expression analysis and in silico analysis demonstrated that SNAP25 and TXN2 are target genes of miR-27a/b. A reporter assay using plasmids containing the 3′ untranslated regions of the SNAP25 and TXN2 genes showed that miR-27a/b directly suppressed SNAP25 and TXN2 expression through posttranscriptional gene silencing. Knockdown of SNAP25 led to a significant inhibition of Ad entry into cells. Knockdown of TXN2 induced cell cycle arrest at G1 phase, leading to a reduction in Ad replication. In addition, overexpression of Ad-encoded small noncoding RNAs (VA-RNAs) restored the miR-27a/b-mediated reduction in infection level with a VA-RNA-lacking Ad mutant due to the VA-RNA-mediated inhibition of miR-27a/b expression. These results indicate that miR-27a and -b suppress SNAP25 and TXN2 expression via posttranscriptional gene silencing, leading to efficient suppression of Ad infection. IMPORTANCE Adenovirus (Ad) is widely used as a platform for replication-incompetent Ad vectors (Adv) and replication-competent oncolytic Ad (OAd) in gene therapy and virotherapy. Regulation of Ad infection is highly important for efficient gene therapies using both Adv and OAd. In this study, we demonstrate that miR-27a and -b, which are widely expressed in host cells, suppress SNAP25 and TXN2 expression through posttranscriptional gene silencing. Suppression of SNAP25 and TXN2 expression leads to inhibition of Ad entry into cells and to cell cycle arrest, respectively, leading to efficient suppression of Ad infection. Our findings provide important clues to the improvement of gene therapies using both Adv and OAd.

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California TRV-based VIGS vectors mediate gene silencing at elevated temperatures but with greater growth stunting

BMC Plant Biology ◽

10.1186/s12870-021-03324-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jamilur Rahman ◽

Ian T. Baldwin ◽

Klaus Gase

Keyword(s):

Gene Silencing ◽

Host Plant ◽

Target Genes ◽

Elevated Temperatures ◽

Tobacco Rattle Virus ◽

Vector System ◽

Nicotiana Attenuata ◽

Cdna Clones ◽

Virus Induced Gene Silencing ◽

In Planta

Abstract Background Tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS), a widely used functional genomics tool, requires growth temperatures typically lower than those of the plant’s native environment. Enabling VIGS under native conditions in the field according to applicable safety regulations could be a revolutionary advance for ecological research. Results Here, we report the development of an enhanced thermal tolerant VIGS vector system based on a TRV California isolate. cDNA clones representing the whole viral genome were sequenced and used to construct separate binary plant transformation vectors for functional elements of RNA1 (6765 nt) and RNA2 (3682 nt). VIGS of target genes was induced by transient transformation of the host plant with both vectors or by treating the host plant with sap from already VIGS induced plants. In Nicotiana attenuata the silencing efficiency of the PDS (phytoene desaturase) gene was 90% at 28 °C and 78% at 30 °C. Silencing at these temperatures was more prominent and durable than silencing induced by the widely used TRV PpK20-based pBINTRA6/pTV00 system, but was associated with a viral phenotype. Differences in the suppressor protein and RNA dependent RNA polymerase sequences between the TRV California isolate and PpK20 may be the reason for their different thermal tolerance. Conclusions The new TRV California-based VIGS vectors induce gene silencing in Nicotiana attenuata at higher temperatures than the existing pBINTRA6/pTV00 vector system, but cause greater growth defects. The new vector system opens up an avenue to study genes functions in planta under field conditions.

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The reovirus σ3 protein inhibits NF-κB-dependent antiviral signaling

10.1101/2021.10.05.463132 ◽

2021 ◽

Author(s):

Andrew J McNamara ◽

Austin D Brooks ◽

Pranav Danthi

Keyword(s):

Target Genes ◽

Immune Surveillance ◽

Ectopic Expression ◽

Innate Immune ◽

Host Cells ◽

Common Mechanism ◽

Type I ◽

Antiviral Signaling ◽

Reovirus Infection ◽

Distinct Mechanism

Viral antagonism of innate immune pathways is a common mechanism by which viruses evade immune surveillance. Infection of host cells with reovirus leads to the blockade of NF-κB, a key transcriptional regulator of the host's innate immune response. One mechanism by which reovirus infection results in inhibition of NF-κB is through a diminishment in levels of upstream activators, IKKβ and NEMO. Here, we demonstrate a second, distinct mechanism by which reovirus blocks NF-κB. We report that expression of a single viral protein, σ3, is sufficient to inhibit expression of NF-κB target genes. Further, σ3-mediated blockade of NF-κB occurs without changes to IKK levels or activity. Expression of only a subset of NF-κB target genes is reduced. Among NF-κB targets, the expression of type I interferon is significantly diminished by σ3 expression. Correspondingly, ectopic expression of σ3 enhances viral replication. Expression of NF-κB target genes varies following infection with closely related reovirus strains. Our genetic analysis identifies that these differences are controlled by polymorphisms in the amino acid sequence of σ3. This work identifies a new role for reovirus σ3 as a viral antagonist of the NF-κB-dependent antiviral pathways.

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Artificial microRNA (amiRNA) induced gene silencing in alfalfa (Medicago sativa)

Botany ◽

10.1139/cjb-2012-0166 ◽

2013 ◽

Vol 91 (2) ◽

pp. 117-122 ◽

Cited By ~ 5

Author(s):

Julian C. Verdonk ◽

Michael L. Sullivan

Keyword(s):

Gene Silencing ◽

Medicago Sativa ◽

Target Gene ◽

Target Genes ◽

Mirna Precursor ◽

Crop Species ◽

Forage Crop ◽

Ribonucleic Acids ◽

Endogenous Gene ◽

Medicago Sativa L

Gene silencing is a powerful technique that allows the study of the function of specific genes by selectively reducing their transcription. Several different approaches can be used, however they all have in common the artificial generation of single stranded small ribonucleic acids (RNAs) that are utilized by the endogenous gene silencing machinery of the organism. Artificial microRNAs (amiRNA) can be used to very specifically target genes for silencing because only a short sequence of 21 nucleotides of the gene of interest is used. Gene silencing via amiRNA has been developed for Arabidopsis thaliana (L.) Heynh. and rice using endogenous microRNA (miRNA) precursors and has been shown to also work effectively in other dicot species using the arabidopsis miRNA precursor. Here, we demonstrate that the arabidopsis miR319 precursor can be used to silence genes in the important forage crop species alfalfa (Medicago sativa L.) by silencing the expression of a transgenic beta-glucuronidase (GUSPlus) target gene.

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Ways into Understanding HIF Inhibition

Cancers ◽

10.3390/cancers13010159 ◽

2021 ◽

Vol 13 (1) ◽

pp. 159

Author(s):

Tina Schönberger ◽

Joachim Fandrey ◽

Katrin Prost-Fingerle

Keyword(s):

Protein Interactions ◽

Target Genes ◽

Protein Protein Interactions ◽

Low Oxygen ◽

Drug Candidates ◽

Open Questions ◽

Wide Range ◽

Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

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A surrogate virus neutralization test to quantify antibody-mediated inhibition of SARS-CoV-2 in finger stick dried blood spot samples

Scientific Reports ◽

10.1038/s41598-021-94653-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Amelia E. Sancilio ◽

Richard T. D’Aquila ◽

Elizabeth M. McNally ◽

Matthew P. Velez ◽

Michael G. Ison ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Venous Blood ◽

Host Cells ◽

Dried Blood Spot ◽

Blood Collection ◽

Widespread Application ◽

Live Virus ◽

Dilution Series ◽

Wide Range ◽

Blood Spot

AbstractThe spike protein of SARS-CoV-2 engages the human angiotensin-converting enzyme 2 (ACE2) receptor to enter host cells, and neutralizing antibodies are effective at blocking this interaction to prevent infection. Widespread application of this important marker of protective immunity is limited by logistical and technical challenges associated with live virus methods and venous blood collection. To address this gap, we validated an immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction in a single drop of capillary whole blood, collected on filter paper as a dried blood spot (DBS) sample. Samples are eluted overnight and incubated in the presence of spike antigen and ACE2 in a 96-well solid phase plate. Competitive immunoassay with electrochemiluminescent label is used to quantify neutralizing activity. The following measures of assay performance were evaluated: dilution series of confirmed positive and negative samples, agreement with results from matched DBS-serum samples, analysis of results from DBS samples with known COVID-19 status, and precision (intra-assay percent coefficient of variation; %CV) and reliability (inter-assay; %CV). Dilution series produced the expected pattern of dose–response. Agreement between results from serum and DBS samples was high, with concordance correlation = 0.991. Analysis of three control samples across the measurement range indicated acceptable levels of precision and reliability. Median % surrogate neutralization was 46.9 for PCR confirmed convalescent COVID-19 samples and 0.1 for negative samples. Large-scale testing is important for quantifying neutralizing antibodies that can provide protection against COVID-19 in order to estimate the level of immunity in the general population. DBS provides a minimally-invasive, low cost alternative to venous blood collection, and this scalable immunoassay-based method for quantifying inhibition of the spike-ACE2 interaction can be used as a surrogate for virus-based assays to expand testing across a wide range of settings and populations.

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Epigenetic Changes in Host Ribosomal DNA Promoter Induced by an Asymptomatic Plant Virus Infection

Biology ◽

10.3390/biology9050091 ◽

2020 ◽

Vol 9 (5) ◽

pp. 91 ◽

Cited By ~ 1

Author(s):

Miryam Pérez-Cañamás ◽

Elizabeth Hevia ◽

Carmen Hernández

Keyword(s):

Gene Silencing ◽

Ribosomal Dna ◽

Plant Virus ◽

Dna Methyltransferase ◽

Cytosine Methylation ◽

De Novo ◽

Methylation Pattern ◽

Transcriptional Gene Silencing ◽

Post Transcriptional Gene Silencing ◽

The Impact

DNA cytosine methylation is one of the main epigenetic mechanisms in higher eukaryotes and is considered to play a key role in transcriptional gene silencing. In plants, cytosine methylation can occur in all sequence contexts (CG, CHG, and CHH), and its levels are controlled by multiple pathways, including de novo methylation, maintenance methylation, and demethylation. Modulation of DNA methylation represents a potentially robust mechanism to adjust gene expression following exposure to different stresses. However, the potential involvement of epigenetics in plant-virus interactions has been scarcely explored, especially with regard to RNA viruses. Here, we studied the impact of a symptomless viral infection on the epigenetic status of the host genome. We focused our attention on the interaction between Nicotiana benthamiana and Pelargonium line pattern virus (PLPV, family Tombusviridae), and analyzed cytosine methylation in the repetitive genomic element corresponding to ribosomal DNA (rDNA). Through a combination of bisulfite sequencing and RT-qPCR, we obtained data showing that PLPV infection gives rise to a reduction in methylation at CG sites of the rDNA promoter. Such a reduction correlated with an increase and decrease, respectively, in the expression levels of some key demethylases and of MET1, the DNA methyltransferase responsible for the maintenance of CG methylation. Hypomethylation of rDNA promoter was associated with a five-fold augmentation of rRNA precursor levels. The PLPV protein p37, reported as a suppressor of post-transcriptional gene silencing, did not lead to the same effects when expressed alone and, thus, it is unlikely to act as suppressor of transcriptional gene silencing. Collectively, the results suggest that PLPV infection as a whole is able to modulate host transcriptional activity through changes in the cytosine methylation pattern arising from misregulation of methyltransferases/demethylases balance.

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Simultaneous silencing of two different Arabidopsis genes with a novel virus-induced gene silencing vector

Plant Methods ◽

10.1186/s13007-020-00701-6 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Kunxin Wu ◽

Yadan Wu ◽

Chunwei Zhang ◽

Yan Fu ◽

Zhixin Liu ◽

...

Keyword(s):

Gene Silencing ◽

Double Mutant ◽

Target Genes ◽

Rna Binding ◽

Protein Product ◽

Antiviral Defense ◽

Virus Induced Gene Silencing ◽

Argonaute 2 ◽

Plant Genes ◽

Silencing Pathways

Abstract Background Virus-induced gene silencing (VIGS) is a useful tool for functional characterizations of plant genes. However, the penetrance of VIGS varies depending on the genes to be silenced, and has to be evaluated by examining the transcript levels of target genes. Results In this report, we report the development of a novel VIGS vector that permits a preliminary assessment of the silencing penetrance. This new vector is based on an attenuated variant of Turnip crinkle virus (TCV) known as CPB that can be readily used in Arabidopsis thaliana to interrogate genes of this model plant. A CPB derivative, designated CPB1B, was produced by inserting a 46 nucleotide section of the Arabidopsis PHYTOENE DESATURASE (PDS) gene into CPB, in antisense orientation. CPB1B induced robust PDS silencing, causing easily visible photobleaching in systemically infected Arabidopsis leaves. More importantly, CPB1B can accommodate additional inserts, derived from other Arabidopsis genes, causing the silencing of two or more genes simultaneously. With photobleaching as a visual marker, we adopted the CPB1B vector to validate the involvement of DICER-LIKE 4 (DCL4) in antiviral defense against TCV. We further revealed the involvement of ARGONAUTE 2 (AGO2) in PDS silencing and antiviral defense against TCV in dcl2drb4 double mutant plants. These results demonstrated that DOUBLE-STRANDED RNA-BINDING PROTEIN 4 (DRB4), whose protein product (DRB4) commonly partners with DCL4 in the antiviral silencing pathway, was dispensable for PDS silencing induced by CPB1B derivative in dcl2drb4 double mutant plants. Conclusions The CPB1B-based vector developed in this work is a valuable tool with visualizable indicator of the silencing penetrance for interrogating Arabidopsis genes, especially those involved in the RNA silencing pathways.

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Host-vector systems

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1989.0061 ◽

1989 ◽

Vol 324 (1224) ◽

pp. 477-485 ◽

Cited By ~ 2

Keyword(s):

Genetic Manipulation ◽

Pharmaceutical Products ◽

Host Cells ◽

Animal Cells ◽

Vector Systems ◽

Wide Range ◽

Novel Host ◽

Basic Concepts ◽

Foreign Genes ◽

New Generation

In 1980 it was only possible to express foreign genes in bacteria and a few easily cultured animal cells. During the subsequent eight years specialized vectors have been developed to allow the genetic manipulation of a wide range of both prokaryotes and eukaryotes. One of the major goals of biotechnology in 1980 was to use host cells as ‘factories’ for the production of proteins that were only available in minute quantities from natural sources. This has already lead to a new generation of pharmaceutical products. Advances in our understanding of host-vector systems have defined new goals. The basic concepts of expression vector design will be illustrated. Some of the new goals are discussed with particular reference to the exploitation of novel host-vector systems to develop vaccines and anti-viral agents against AIDS.

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G protein-coupled odorant receptors underlie mechanosensitivity in mammalian olfactory sensory neurons

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1418515112 ◽

2014 ◽

Vol 112 (2) ◽

pp. 590-595 ◽

Cited By ~ 43

Author(s):

Timothy Connelly ◽

Yiqun Yu ◽

Xavier Grosmaitre ◽

Jue Wang ◽

Lindsey C. Santarelli ◽

...

Keyword(s):

G Protein ◽

Sensory Neurons ◽

Ectopic Expression ◽

Host Cells ◽

Odorant Receptors ◽

Olfactory Sensory Neurons ◽

Mechanical Stimuli ◽

Genetic Ablation ◽

Mechanical Responses ◽

G Protein Coupled

Mechanosensitive cells are essential for organisms to sense the external and internal environments, and a variety of molecules have been implicated as mechanical sensors. Here we report that odorant receptors (ORs), a large family of G protein-coupled receptors, underlie the responses to both chemical and mechanical stimuli in mouse olfactory sensory neurons (OSNs). Genetic ablation of key signaling proteins in odor transduction or disruption of OR–G protein coupling eliminates mechanical responses. Curiously, OSNs expressing different OR types display significantly different responses to mechanical stimuli. Genetic swap of putatively mechanosensitive ORs abolishes or reduces mechanical responses of OSNs. Furthermore, ectopic expression of an OR restores mechanosensitivity in loss-of-function OSNs. Lastly, heterologous expression of an OR confers mechanosensitivity to its host cells. These results indicate that certain ORs are both necessary and sufficient to cause mechanical responses, revealing a previously unidentified mechanism for mechanotransduction.

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