scholarly journals Unusual 5'-regulatory structure and regulation of the murine Mlc1 gene: Lack of promoter-specific functional elements

2011 ◽  
Vol 2 (1) ◽  
pp. 11
Author(s):  
Darja Henseler ◽  
Jonathan D. Turner ◽  
Matthias Eckhardt ◽  
Maaike Van der Mark ◽  
Yanina Revsin ◽  
...  
Keyword(s):  
Reporter Gene ◽  
In Silico ◽  
Promoter Activity ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Regulatory Structure ◽  
Luciferase Reporter Gene ◽  
Promoter Regions ◽  
Potential Promoter

<!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:HyphenationZone>21</w:HyphenationZone> <w:PunctuationKerning /> <w:ValidateAgainstSchemas /> <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid> <w:IgnoreMixedContent>false</w:IgnoreMixedContent> <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText> <w:Compatibility> <w:BreakWrappedTables /> <w:SnapToGridInCell /> <w:WrapTextWithPunct /> <w:UseAsianBreakRules /> <w:DontGrowAutofit /> </w:Compatibility> <w:BrowserLevel>MicrosoftInternetExplorer4</w:BrowserLevel> </w:WordDocument> </xml><![endif]--><!--[if gte mso 9]><xml> <w:LatentStyles DefLockedState="false" LatentStyleCount="156"> </w:LatentStyles> </xml><![endif]--><!--[if gte mso 10]> <mce:style><! /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Normale Tabelle"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:""; mso-padding-alt:0pt 5.4pt 0pt 5.4pt; mso-para-margin:0pt; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;} --> <!--[endif]--> <p class="MsoNormal" style="text-align: justify;"><span style="color: black;" lang="EN-GB">The <em>MLC1</em> gene is involved in an autosomal recessive neurological disorder, megalencephalic leucoencephalopathy with subcortical cysts (MLC), which is characterized by macrocephaly during the first year of life and swollen white matter (leucoencephaly). Variants of <em>MLC1</em> have also been associated with psychiatric disorders such as schizophrenia, major depression and bipolar disorder. Currently, little is known about the encoded protein (MLC1). Judging from its similarity to other known proteins, it may serve as a trans-membrane transporter. However, the function of the encoded protein and its gene regulation has not been investigated successfully so far. We investigated the 5’ region of the murine <em>Mlc1</em> with respect to regulatory elements for gene expression. A promoter search and an <em>in silico</em> analysis were conducted. Luciferase reporter gene constructs with potential promoter regions were created to study promoter activity <em>in vitro</em>. We found two alternative first exons for the murine <em>Mlc1</em> but were not able to detect any promoter activity for the investigated reporter gene constructs in different cell lines, thus pointing to the presence of essential <em>cis</em>-acting elements far outside of the region. <em>In silico </em>analysis indicated an uncommon promoter structure for <em>Mlc1</em>, with CCAAT-boxes representing the only noticeable elements. </span></p>

scholarly journals Cloning and Functional Analysis of Rat Tweety-Homolog 1 Gene Promoter

2021 ◽  
Author(s):  
Malgorzata Gorniak-Walas ◽  
Karolina Nizinska ◽  
Katarzyna Lukasiuk
Keyword(s):  
Transcriptional Regulation ◽  
Reporter Gene ◽  
Hippocampal Neurons ◽  
Gene Promoter ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay

AbstractTweety-homolog 1 protein (Ttyh1) is abundantly expressed in neurons in the healthy brain, and its expression is induced under pathological conditions. In hippocampal neurons in vitro, Ttyh1 was implicated in the regulation of primary neuron morphology. However, the mechanisms that underlie transcriptional regulation of the Ttyh1 gene in neurons remain elusive. The present study sought to identify the promoter of the Ttyh1 gene and functionally characterize cis-regulatory elements that are potentially involved in the transcriptional regulation of Ttyh1 expression in rat dissociated hippocampal neurons in vitro. We cloned a 592 bp rat Ttyh1 promoter sequence and designed deletion constructs of the transcription factors specificity protein 1 (Sp1), E2F transcription factor 3 (E2f3), and achaete-scute homolog 1 (Ascl1) that were fused upstream of a luciferase reporter gene in pGL4.10[luc2]. The luciferase reporter gene assay showed the possible involvement of Ascl1, Sp1, and responsive cis-regulatory elements in Ttyh1 expression. These findings provide novel information about Ttyh1 gene regulation in neurons.

Μελέτη της επίδρασης της ελευρωπαΐνης στην ομοιοστασία των λιπιδίων

2017 ◽  
Author(s):  
Φωτεινή Μάλλιου
Keyword(s):  
Reporter Gene ◽  
In Silico ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Gene Assay

Η Ελευρωπαΐνη, το κύριο πολυφαινολικό συστατικό της ελιάς, παρουσιάζει αντιοξειδωτικές και υπολιπιδαιμικές ιδιότητες. Ο ενεργοποιημένος υποδοχέας επαγωγής του πολλαπλασιασμού των υπεροξεισωμάτων τύπου α (PPARα), διαδραματίζει καίριο ρόλο στον έλεγχο του μεταβολισμού των λιπιδίων και στην ενεργειακή ομοιαστασία του κυττάρου. Η συγκεκριμένη έρευνα εστιάζει στους μηχανισμούς της υπολιπιδαιμικής δράσης της Ελευρωπαΐνης με έμφαση στο ρόλο της Ελευρωπαΐνης στην ενεργοποίηση του PPARα. Για τον σκοπό αυτό, έγινε αξιολόγηση in silico της ικανότητας της Ελευρωπαΐνης να προσδένεται στον PPARα. Θεωρητικά μοντέλα πρόσδεσης στην κρυσταλλική δομή του PPARα με τη χρήση Μοριακής Προσομοίωσης Πρόσδεσης, επιβεβαιώνουν την υπόθεσή μας ότι η Ελευρωπαΐνη είναι αγωνιστής του PPARα. Επιπλέον, διερευνήθηκε in vitro με το Luciferase reporter gene assay η ικανότητα της Ελευρωπαΐνης να προσδένεται στον υποδοχέα PPARα και να τον ενεργοποιεί. Τα αποτελέσματα από την in silico και την in vitro μελέτη δείχνουν σαφώς ότι η ελευρωπαΐνη ενεργοποιεί τον PPARα. Στη συνέχεια έγινε in vivo διερεύνηση της ενεργοποίησης του PPARα από την Ελευρωπαΐνη και αξιολόγηση της επίδρασής της στο λιπιδαιμικό προφίλ των πειραματόζωων. Αγωγή αρσενικών μυών άγριου τύπου (SV129 Wild Type) με Ελευρωπαΐνη σε δοσολογία 100mg/kg, p.o, τα οποία ακολούθησαν τυπική δίαιτα για τρωκτικά, για 6 εβδομάδες, είχε ως αποτέλεσμα επαγωγή του Pparα και των γονιδίων-στόχων του στο ήπαρ, πιθανώς μέσω ενεργοποίησης του PI3K/AKT/p70S6K σηματοδοτικού μονοπατιού. Αυτή η επίδραση της Ελευρωπαΐνης φαίνεται να σχετίζεται άμεσα με σημαντική μείωση των επιπέδων των TGs του ορού και της ολικής χοληστερόλης, γιατί η Ελευρωπαΐνη δεν είχε καμία επίδραση σε αυτούς τους λιπιδαιμικούς δείκτες σε διαγονιδιακούς Pparα null μύες. Στην κατεύθυνση της διερεύνησης της επίδρασης της Ελευρωπαΐνης σε ηπατικούς παράγοντες και σε παράγοντες, που εκφράζονται στο λευκό λιπώδη, κρίσιμους για την ομοιοστασία των Τριγλυκεριδίων, διαπιστώθηκαν τα ακόλουθα: 1) Ενεργοποίηση της ορμονο-ευαίσθητης λιπάσης (HSL) στο λευκό λιπώδη ιστό (W.A.T.) των άγριου τύπου μυών, 2) επαγωγή ποικίλλων ηπατικών παραγόντων, που συμμετέχουν στη σύνθεση, τη μεταφορά, τον μεταβολισμό και την απέκκριση των τριγλυκεριδίων. Αυτή η ενεργοποίηση της HSL στον W.A.T. και των ηπατικών παραγόντων, που συμμετέχουν στην ομοιοστασία των λιπιδίων, είναι πιθανόν να ενισχύει την επαγόμενη από την Ελευρωπαΐνη μείωση των επιπέδων των τριγλυκεριδίων και της χοληστερόλης στον ορό. Συνοψίζοντας, φαίνεται ότι η Ελευρωπαΐνη μειώνει τα τριγλυκερίδια και την ολική χοληστερόλη του ορού σε μύες μέσω ενεργοποίησης του PPARα. Τα δεδομένα αυτής της μελέτης δείχνουν επίσης, ότι στην υπολιπιδαιμική δράση της Ελευρωπαΐνης συμμετέχει και η ενεργοποίηση της HSL στο λευκό λιπώδη ιστό καθώς και η επαγωγή ηπατικών γονιδίων, που διαδραματίζουν βασικό ρόλο την ομοιοστασία των τριγλυκεριδίων, δηλαδή τη σύνθεση, την μεταφορά, τον μεταβολισμό και την κάθαρσή τους. Συμπερασματικά, η μελέτη έδειξε τις υπολιπιδαιμικές δυνατότητες της Ελευρωπαΐνης σε μύες και αποσαφήνισε σε σημαντικό βαθμό τους μηχανισμούς της υπολιπιδαιμικής δράσης της, φωτίζοντας κυρίως τον ρόλο του PPARα. Επειδή η διατήρηση της ομοιοστασίας των λιπιδίων είναι μια πολύπλοκη διαδικασία, που ρυθμίζεται από πολλούς παράγοντες, πιστεύουμε ότι πιθανώς εμπλέκονται και άλλοι μηχανισμοί στην δράση της Ελευρωπαΐνης, η διερεύνηση των οποίων είναι αντικείμενο μελλοντικών μελετών.

Chromatin Conformation and a Distal Regulatory Element Activate the Human Erythroid Ankyrin-1 Promoter.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 803-803
Author(s):  
Ashley N. Owen ◽  
Robert I. Liem ◽  
Andre M. Pilon ◽  
Patrick G. Gallagher ◽  
David M. Bodine
Keyword(s):  
Reporter Gene ◽  
Regulatory Element ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Dnase I ◽  
Luciferase Reporter ◽  
Erythroid Cells ◽  
Luciferase Reporter Gene ◽  
Human And Mouse ◽  
In Erythroid Cells

Abstract Ankyrin forms the bridge between the spectrin/actin network of the erythrocyte membrane skeleton and the red cell membrane by binding to both β-spectrin and band 3. The erythrocyte ankyrin promoter (Ank-1E) is active only in erythroid cells, while two other Ank-1 promoters located 20 kb downstream and 40 kb upstream of Ank-1E are active in the cerebellum and muscle cells respectively. We have been studying the mechanism by which the Ank-1E promoter becomes active in erythroid cells by studying the cis acting regulatory elements and the chromatin structure of the Ank-1 promoter region. We have previously shown that the sequences between −296 and −15 of the Ank-1E promoter are fully sufficient for erythroid specific, copy number dependent uniform expression of reporter genes in transgenic mice. We have also mapped a DNase I Hypersensitive site (5′HS) between −300 and −100 of the human and mouse Ank-1E promoters in human K562 and mouse fetal liver cells. Both the mouse and human 5′HS are capable of preventing the silencing of a β-globin/GFP reporter gene in K562 cells, establishing that they function as barrier elements. Consistent with this observation, the human and mouse 5′HS are hyperacetylated in erythroid cells. The chromatin 10 kb 5′ to the 5′HS is DNase I resistant (associated with inactive chromatin) in human and mouse erythroid and non-erythroid cells. Approximately 6 kb 3′ to 5′HS are two adjacent HS (3′HS1, 3′HS2). Beyond 3′HS2 the chromatin is also DNase I resistant in both human and mouse erythroid and non-erythroid cells. Between 5′HS and 3′HS1 the 6kb region is DNase I sensitive (active) in erythroid cells but not in other cell types. We hypothesized that this 6 kb region contains regulatory elements that activate the Ank-1E promoter. To screen for regulatory elements we isolated overlapping segments of a 10 kb region extending from 2 kb upstream of 5′HS to 2 kb downstream of 3′HS2. We inserted these fragments into a plasmid vector containing the Ank-1E promoter linked to a luciferase reporter gene and transfected these constructs into K562 cells. A single region up regulated Ank-1E/luciferase expression. This region mapped to a 211bp segment that included 3′HS1, but did not include 3′HS2. A fragment containing only 3′HS2 did not up regulate an Ank-1E/luciferase reporter gene, but 3′HS2 was capable of preventing the silencing of a β-globin/Green Fluorescent Protein reporter gene in K562 cells, demonstrating barrier activity. The region around 3′HS1 and 2 was also a site of histone hyperacetylation. The sequence of the 211 bp fragment containing 3′HS1 does not contain consensus sequences for any known erythroid-specific transcription factors, but does contain potential binding sites fro Sp1, AP-1 and E-box binding proteins. Using the Chromatin Conformation Capture assay we demonstrated that 5′HS and 3′HS1 and 2 are in close proximity in K562 chromatin, but are not closely associated in chromatin from other cell types. We propose that an erythroid-specific chromatin loop brings 3′HS1 and 2 into proximity with 5′HS, adjacent to the Ank-1E promoter. This interaction translocates the positive regulatory element in 3′HS1 to the Ank-1E promoter allowing the Ank-1E promoter to become active in erythroid cells.

scholarly journals Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening

2005 ◽  
Vol 49 (9) ◽  
pp. 3776-3783 ◽  
Author(s):  
Ashutosh ◽  
Suman Gupta ◽  
Ramesh ◽  
Shyam Sundar ◽  
Neena Goyal
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Field Isolates

ABSTRACT Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.

scholarly journals Hormonal regulation of the human sterol 27-hydroxylase gene CYP27A1

2003 ◽  
Vol 372 (2) ◽  
pp. 529-534 ◽  
Author(s):  
Zufan ARAYA ◽  
Wanjin TANG ◽  
Kjell WIKVALL
Keyword(s):  
Reporter Gene ◽  
Hormonal Regulation ◽  
Promoter Activity ◽  
Luciferase Activity ◽  
Luciferase Reporter ◽  
Signal Transduction Pathways ◽  
Cytochrome P450 Enzyme ◽  
Luciferase Reporter Gene ◽  
Response Elements ◽  
P450 Enzyme

The mitochondrial sterol 27-hydroxylase (CYP27A1) is a multifunctional cytochrome P450 enzyme that catalyses important hydroxylations in the biosynthesis of bile acids and bioactivation of vitamin D3. Previous results [Babiker, Andersson, Lund, Xiu, Deeb, Reshef, Leitersdorf, Diczfalusy and Björkhem (1997) J. Biol. Chem. 272, 26253–26261] suggest that CYP27A1 plays an important role in cholesterol homoeostasis and affects atherogenesis. In the present study, the regulation of the human CYP27A1 gene by growth hormone (GH), insulin-like growth factor-1 (IGF-1), dexamethasone, thyroid hormones and PMA was studied. HepG2 cells were transfected transiently with luciferase reporter gene constructs containing DNA fragments flanking the 5′-region of the human CYP27A1 gene. GH, IGF-1 and dexamethasone increased the promoter activity by 2–3-fold, whereas thyroxine (T4) and PMA repressed the activity significantly when measured with luciferase activity expressed in the cells. The endogenous CYP27A1 enzyme activity in the cells was stimulated by GH, IGF-1 and dexamethasone, whereas T4 and PMA inhibited the activity. Experiments with progressive deletion/luciferase reporter gene constructs indicated that the response elements for GH may be localized in a region upstream to position −1094 bp. The putative response elements for dexamethasone were mapped to positions between −792 and −1095 bp. The −451 bp fragment of the human CYP27A1 gene was found to confer the activation by IGF-1, and the inhibition by T4 and PMA. Results of the present study suggest that CYP27A1 is regulated in human cells by hormones and signal-transduction pathways.

scholarly journals Endothelial-specific gene expression directed by the tie gene promoter in vivo

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1828-1835 ◽  
Author(s):  
J Korhonen ◽  
I Lahtinen ◽  
M Halmekyto ◽  
L Alhonen ◽  
J Janne ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Promoter Activity ◽  
Cultured Cells ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Luciferase Reporter Gene ◽  
Human And Mouse

The tie gene encodes a receptor tyrosine kinase that is expressed in the endothelium of blood vessels, particularly during embryonic development and angiogenesis in adults. We have cloned and characterized the mouse tie gene and isolated the human and mouse tie promoters. The promoter activities of human and mouse tie were analyzed using luciferase reporter gene constructs in transfected cell lines and beta-galactosidase constructs in transgenic mice. In transfection assays of cultured cells, both human and mouse promoter DNA fragments showed activity that was not restricted to endothelial cells. In contrast, in transgenic mice both promoters directed expression of the reporter gene to endothelial cells undergoing vasculogenesis and angiogenesis. In adult mice, tie promoter activity in lung and many vessels of the kidney was as high as in the vessels of the corresponding embryonic tissues, whereas in the heart, brain and liver, tie promoter activity was downregulated and restricted to coronaries, cusps, capillaries, and arteries. Our results show that the endothelial cell-type specificity of the tie promoter in vivo can be transferred to heterologous genes by using relatively short promoter fragments. The tie promoter, thus, has useful properties for potential gene therapy.

scholarly journals Isoproterenol and cAMP regulation of the human brain natriuretic peptide gene involves Src and Rac

2000 ◽  
Vol 278 (6) ◽  
pp. E1115-E1123 ◽  
Author(s):  
Quan He ◽  
Guiyun Wu ◽  
Margot C. Lapointe
Keyword(s):  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Kinase Inhibitor ◽  
Regulatory Elements ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Dominant Negative Mutant ◽  
Luciferase Reporter Gene ◽  
Src Tyrosine Kinase

Brain natriuretic peptide (BNP) gene expression and chronic activation of the sympathetic nervous system are characteristics of the development of heart failure. We studied the role of the β-adrenergic signaling pathway in regulation of the human BNP (hBNP) promoter. An hBNP promoter (−1818 to +100) coupled to a luciferase reporter gene was transferred into neonatal cardiac myocytes, and luciferase activity was measured as an index of promoter activity. Isoproterenol (ISO), forskolin, and cAMP stimulated the promoter, and the β2-antagonist ICI 118,551 abrogated the effect of ISO. In contrast, the protein kinase A (PKA) inhibitor H-89 failed to block the action of cAMP and ISO. Pertussis toxin (PT), which inactivates Gαi, inhibited ISO- and cAMP-stimulated hBNP promoter activity. The Src tyrosine kinase inhibitor PP1 and a dominant-negative mutant of the small G protein Rac also abolished the effect of ISO and cAMP. Finally, we studied the involvement of M-CAT-like binding sites in basal and inducible regulation of the hBNP promoter. Mutation of these elements decreased basal and cAMP-induced activity. These data suggest that β-adrenergic regulation of hBNP is PKA independent, involves a Gαi-activated pathway, and targets regulatory elements in the proximal BNP promoter.

scholarly journals Peroxisome-Proliferator Receptor γ Represses Hepatic Sex Hormone-Binding Globulin Expression

Endocrinology ◽  
2009 ◽  
Vol 150 (5) ◽  
pp. 2183-2189 ◽  
Author(s):  
David M. Selva ◽  
Geoffrey L. Hammond
Keyword(s):  
Hepg2 Cells ◽  
Promoter Activity ◽  
Binding Activity ◽  
Sex Hormone Binding Globulin ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Luciferase Reporter Gene ◽  
Pparγ Agonist ◽  
Gene Assay

Plasma SHBG production by the liver is influenced by its metabolic state, and hepatocyte nuclear factor-4α regulates SHBG expression in response to changes in lipogenesis. Peroxisome-proliferator receptors (PPARs) also regulate glucose homeostasis and fatty acid metabolism. The human SHBG promoter contains a PPAR-response element (PPAR-RE), and plasma SHBG levels increase in polycystic ovarian syndrome patients treated with the PPARγ agonist, rosiglitazone. In addition, plasma SHBG levels are associated with a genetic polymorphism in the PPARγ-2 coding sequence that alters its transcriptional activity. Therefore, we set out to determine whether PPARγ influences hepatic production of SHBG by using human HepG2 hepatoblastoma cells as an in vitro model. Surprisingly, treatment of HepG2 cells with rosiglitazone reduced SHBG production and SHBG promoter activity (as assessed in a luciferase reporter gene assay) by 20–25%, whereas the PPARγ antagonist, GW9662, increased both by 2- to 3-fold. The effects of PPARγ agonists and antagonists on SHBG promoter activity were substantially diminished when the PPAR-RE in the SHBG promoter was mutated. A PPARγ small interfering RNA also increased SHBG production by HepG2 cells as well as SHBG promoter activity, and the latter was accentuated by cotreatment with GW9662. Importantly, overexpression of a PPARγ-2 Pro12 variant in HepG2 cells was more effective at reducing SHBG promoter activity, when compared with PPARγ-2 Ala12, consistent with its superior PPAR-RE binding activity. We conclude that PPARγ represses human SHBG expression in liver cells, and that differences in PPARγ levels and activity contribute directly to variations in plasma SHBG levels.

scholarly journals Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene

2016 ◽  
Vol 60 (9) ◽  
pp. 5492-5503 ◽  
Author(s):  
Liang Shen ◽  
Yang Yang ◽  
Fei Ye ◽  
Gaoshan Liu ◽  
Marc Desforges ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Luciferase Reporter ◽  
Wild Type Virus ◽  
Upper Respiratory Tract ◽  
Rna Helicases ◽  
Luciferase Reporter Gene ◽  
Screening Assay ◽  
Content Type ◽  
Tract Infections

ABSTRACTHuman coronaviruses (HCoVs) cause 15 to 30% of mild upper respiratory tract infections. However, no specific antiviral drugs are available to prevent or treat HCoV infections to date. Here, we developed four infectious recombinant HCoVs-OC43 (rHCoVs-OC43) which express theRenillaluciferase (Rluc) reporter gene. Among these four rHCoVs-OC43, rOC43-ns2DelRluc (generated by replacing ns2 with the Rluc gene) showed robust luciferase activity with only a slight impact on its growth characteristics. Additionally, this recombinant virus remained stable for at least 10 passages in BHK-21 cells. rOC43-ns2DelRluc was comparable to its parental wild-type virus (HCoV-OC43-WT) with respect to the quantity of the antiviral activity of chloroquine and ribavirin. We showed that chloroquine strongly inhibited HCoV-OC43 replicationin vitro, with a 50% inhibitory concentration (IC50) of 0.33 μM. However, ribavirin showed inhibition of HCoV-OC43 replication only at high concentrations which may not be applicable to humans in clinical treatment, with an IC50of 10 μM. Furthermore, using a luciferase-based small interfering RNA (siRNA) screening assay, we identified double-stranded-RNA-activated protein kinase (PKR) and DEAD box RNA helicases (DDX3X) that exhibited antiviral activities, which were further verified by the use of HCoV-OC43-WT. Therefore, rOC43-ns2DelRluc represents a promising safe and sensitive platform for high-throughput antiviral screening and quantitative analysis of viral replication.

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