Rluc Gene Research Articles

Establishment of a reverse genetics system for duck Tembusu virus to study virulence and screen antiviral genes

Recently, a newly emerged avian flavivirus, duck Tembusu virus (TMUV), was identified as the causative agent of a serious duck viral disease in Asia. Its rapid spread and expanded host range have raised substantial concerns regarding its potential threat to non-avian hosts, including humans. In this study, we report an infectious cDNA clone for a clinical strain CQW1 isolated from Southwest China, which is representative of the disease outbreak in the Chinese mainland. We generated a full-length cDNA clone pACYC FL-TMUV, which is infectious, and this cDNA clone-derived recombinant TMUV (rTMUV) showed comparative growth kinetics in both BHK21 cells and DEF cells compared with parental TMUV (pTMUV). In addition, rTMUV also showed the same high virulence in 9-day-old duck embryos as that in pTMUV, suggesting that rTMUV possessed similar properties to the natural virus both in vitro and in vivo. Based on the cDNA-clone, we first generated a reporter TMUV (TMUV-RLuc) carrying a Renilla luciferase (RLuc) gene. The luciferase kinetics of TMUV-RLuc were determined both in BHK21 and DEF cells. It seems that TMUV-RLuc grew well in vitro; however, the insertion of the RLuc gene attenuated viral replication in vitro. The higher viral titres of TMUV-RLuc were observed in BHK21 compared with that in DEF cells. The antiviral effects of exogenous-expressed duck RIG-I, MDA5, STING, MAVS, TBK1, IFNα and IFNγ were studied in vitro by using TMUV-RLuc. Our reverse genetics system will provide a multicomponent platform for the pathogenesis study of duck TMUV and the development of molecular countermeasures against duck TMUV infection.

Natural Killer Cell (NK-92MI)-Based Therapy for Pulmonary Metastasis of Anaplastic Thyroid Cancer in a Nude Mouse Model.

Natural killer (NK) cells represent the third largest population of lymphocytes, and they play an important role in immune surveillance against tumors. The lungs are a common metastatic site for anaplastic thyroid cancer (ATC), and metastasis is one of the most frequent causes of mortality in this type of cancer. In the current study, we evaluated the effects of NK cell-based immunotherapy for pulmonary metastasis of ATC and determined how it affects the effector molecules of NK cells. Human NK cells (NK-92MI) were retrovirally transduced to express the effluc gene. Human ATC cells (CAL-62) were transduced with the effluc and Rluc genes. The cytotoxicity of NK cells against CAL-62 cells was assessed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay system. Pulmonary metastases of ATC were developed by i.v. injection of CAL-62, and metastasis growth was monitored using bioluminescence imaging (BLI). To treat the metastases, five million NK-92MI cells were injected twice into the caudal vein of nude mice. To assess the targetability of NK cells to ATC tumors, NK-92MI cells expressing the effluc gene (NK/F) were administered through the tail vein of nude mice with a pulmonary metastasis or tumor xenograft. BLI was subsequently performed at 1, 3, 24, and 48 h. NK/F and CAL-62 cells expressing the effluc or Rluc gene (CAL-62/F, CAL-62/R) were successfully established. Expression of the effluc and Rluc genes in NK/F, CAL-62/F, and CAL-62/R cells was verified by RT-polymerase chain reaction, western blotting, and luciferase assay. After coculture of NK-92MI and CAL-62/F cells for 24 h, the BLI signal intensity of CAL-62/F cells proportionally decreased with the number of cocultured NK cells. An ATC pulmonary metastasis mouse model was successfully generated, and NK cells significantly inhibited the growth of the metastasis (p < 0.01). The NK/F cells exhibited targetability to the pulmonary metastasis and tumor xenograft in the mouse model. The results of present study suggest that NK cells are able to target ATC tumors and that NK cell-based immunotherapy may serve as an effective therapeutic approach for pulmonary metastases of ATC.

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

Human 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 the Renilla luciferase (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 replication in 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 IC50 of 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.

Stabilization of luciferase from Renilla reniformis using random mutations.

We expressed luciferase (RLuc) from Renilla reniformis in Escherichia coli RLuc was purified using a Ni-NTA column and subsequently characterized. It was unstable in acidic solutions and at 30°C. To increase the stability of RLuc, the Rluc gene was randomly mutated using error-prone polymerase chain reaction. E. coli harboring the mutated gene was screened by detecting luminescence on a plate containing the substrate coelenterazine at 34°C. Three mutants, i.e. N264SS287P, N178D and F116LI137V, were obtained. The solubilities and specific activities of these mutants were higher than those of the wild type. Furthermore, the N264SS287P mutant maintained stability at a temperature approximately 5°C higher than that of the wild type, while denaturation of the F116LI137V mutant started at a temperature that was 5°C lower than the wild type, and ended at a temperature that was 7°C higher. We examined the obtained mutations using thermal shift assays and a computer program Coot in this study.

Duck hepatitis A virus serotype 1 minigenome: a model for studying the viral 3'UTR effect on viral translation.

To date, the genetic replication and translation mechanisms as well as the pathogenesis of duck hepatitis A virus type 1 (DHAV-1) have not been adequately characterized due to the lack of a reliable and efficient cell culture system. Although the full-length infections clone system is the best platform to manipulate the virus, it is relatively difficult to assemble this system due to the lack of a suitable cell line. It has been proven that the minigenome system an efficient reverse genetics system for the study of RNA viruses. In some cases, it can be used to displace the infectious clone of RNA viruses. Here, we generated a minigenome for DHAV-1 with two luciferase reporter genes, firefly luciferase (Fluc) and Renilla luciferase (Rluc). The Rluc gene was used as a reference gene for the normalization of the Fluc gene expression in transfected cells, which provided a platform for studying the regulatory mechanisms of DHAV-1. Furthermore, to investigate the role of DHAV-3'UTR in the regulation of viral protein translation, deletions in the 3'UTR were introduced into the DHAV-1 minigenome. Luciferase activity, an indicator of virus translation, was then determined. These results showed that a minigenome system for DHAV-1 was successfully constructed for the first time and that the complete or partial deletion of the DHAV-3'UTR did not affect the expression level of the reporter gene, indicating that DHAV-1 translation may not be modulated by the viral genomic 3'UTR sequence.

Distinct Allostery Induced in the Cyclic GMP-binding, Cyclic GMP-specific Phosphodiesterase (PDE5) by Cyclic GMP, Sildenafil, and Metal Ions

The activity of many proteins orchestrating different biological processes is regulated by allostery, where ligand binding at one site alters the function of another site. Allosteric changes can be brought about by either a change in the dynamics of a protein, or alteration in its mean structure. We have investigated the mechanisms of allostery induced by chemically distinct ligands in the cGMP-binding, cGMP-specific phosphodiesterase, PDE5. PDE5 is the target for catalytic site inhibitors, such as sildenafil, that are used for the treatment of erectile dysfunction and pulmonary hypertension. PDE5 is a multidomain protein and contains two N-terminal cGMP-specific phosphodiesterase, bacterial adenylyl cyclase, FhLA transcriptional regulator (GAF) domains, and a C-terminal catalytic domain. Cyclic GMP binding to the GAFa domain and sildenafil binding to the catalytic domain result in conformational changes, which to date have been studied either with individual domains or with purified enzyme. Employing intramolecular bioluminescence resonance energy transfer, which can monitor conformational changes both in vitro and in intact cells, we show that binding of cGMP and sildenafil to PDE5 results in distinct conformations of the protein. Metal ions bound to the catalytic site also allosterically modulated cGMP- and sildenafil-induced conformational changes. The sildenafil-induced conformational change was temperature-sensitive, whereas cGMP-induced conformational change was independent of temperature. This indicates that different allosteric ligands can regulate the conformation of a multidomain protein by distinct mechanisms. Importantly, this novel PDE5 sensor has general physiological and clinical relevance because it allows the identification of regulators that can modulate PDE5 conformation in vivo.

MiR-301a as an NF-κB activator in pancreatic cancer cells

NF-κB is constitutively activated in most human pancreatic adenocarcinoma, which is a deadly malignancy with a 5-year survival rate of about 5%. In this work, we investigate whether microRNAs (miRNAs) contribute to NF-κB activation in pancreatic cancer. We demonstrate that miR-301a down-regulates NF-κB-repressing factor (Nkrf) and elevates NF-κB activation. As NF-κB promotes the transcription of miR-301a, our results support a positive feedback loop as a mechanism for persistent NF-κB activation, in which miR-301a represses Nkrf to elevate NF-κB activity, which in turn promotes miR-301a transcription. Nkrf was found down-regulated and miR-301a up-regulated in human pancreatic adenocarcinoma tissues. Moreover, miR-301a inhibition or Nkrf up-regulation in pancreatic cancer cells led to reduced NF-κB target gene expression and attenuated xenograft tumour growth, indicating that miR-301a overexpression contributes to NF-κB activation. Revealing this novel mechanism of NF-κB activation by an miRNA offers new avenues for therapeutic interventions against pancreatic cancer.

Influence of two kinds of SV40 poly (A) signals on upstream gene expression

Objective To investigate the regulatory effects of two kinds of SV40 poly(A) signals on the upstream gene expression in three cell lines,so as to provide theoretical evidence for selection of poly(A) of vectors.Methods A dual luciferase reporter vector Dual-Luc was constructed,and two SV40 poly(A) signal sequences were inserted in the downstream of the R-Luc gene separately.Then the two diverse dual luciferase reporter gene vectors Dual-Luc2 and Dual-Luc3 were transfected into 293,L-02,and HeLa cells.The relative quantities of the target gene (F-Luc) to control gene (R-Luc) were measured by Dual-GloTM Luciferase Assay System and Reverse Transcription-Polymerase Chain Reaction (RT-PCR).Results The two Dual-Luciferase vectors (Dual-Luc2 and Dual-Luc3) were successfully constructed.Dual-GloTM Luciferase Assay showed that the mean F-Luc/R-Luc values were 3.25±0.43 and 3.03±0.14 in Dual-Luc2 and Dual-Luc3 transfected 293 cells(P0.05),6.16±0.39 and 3.83±0.39 in L-02 cells(P0.05),and 1.21±0.10 and 0.66±0.02 in HeLa cells(P0.05),respectively.The results of RT-PCR were consistent with those of luciferase assay.Conclusion The two kinds of SV40 poly(A) signals have different regulatory effects on the upstream gene expression in different cell lines.SV40 poly(A) signals regulate the upstream gene expression at the transcriptional stage.

Secreted production of Renilla luciferase in Bacillus subtilis

Luciferase (Rluc) from the soft coral Renilla reniformis has been widely used as a bioluminescent reporter, and its secreted production has been solely performed in mammalian cells thus far. To make the production more efficient, a series of approaches was attempted to overproduce Rluc extracellularly in Bacillus subtilis. First, Cys124 in the Rluc gene was substituted with Ala. The mutant gene was subsequently incorporated into a pUB110/R6K-based plasmid, consequently, fusing with the P43 promoter and the sacB signal peptide. With the nitrogen-rich medium, B. subtilis strain bearing the plasmid became able to secret a detectable amount of Rluc. Moreover, the secretion signal for the Rluc gene was replaced by the aprN leader peptide with or without the propeptide. The result led to a more than twofold increase in the secreted Rluc. Finally, by enhancing the transcription of the Rluc gene implementing the P43 and spac tandem promoter, it resulted in the secreted Rluc with a yield of 100 mg/L. Overall, this study illustrates a potential strategy for improving the secretion efficiency of heterologous proteins in B. subtilis.

Suppression of ΔbipAPhenotypes inEscherichia coliby Abolishment of Pseudouridylation at Specific Sites on the 23S rRNA

The BipA protein of Escherichia coli has intriguing similarities to the elongation factor subfamily of GTPases, including EF-Tu, EF-G, and LepA. In addition, phenotypes of a bipA deletion mutant suggest that BipA is involved in regulation of a variety of pathways. These two points have led to speculation that BipA may be a novel regulatory protein that affects efficient translation of target genes through direct interaction with the ribosome. We isolated and characterized suppressors of the cold-sensitive growth phenotype exhibited by DeltabipA strains and identified insertion mutations in rluC. The rluC gene encodes a pseudouridine synthase responsible for pseudouridine modification of 23S rRNA at three sites, all located near the peptidyl transferase center. Deletion of rluC not only suppressed cold sensitivity but also alleviated the decrease in capsule synthesis exhibited by bipA mutants, suggesting that the phenotypic effects of BipA are manifested through an effect on the ribosome. The suppressor effect is specific to rluC, as deletion of other rlu genes did not relieve cold sensitivity, and further, more than a single pseudouridine residue is involved, as alteration of single residues did not produce suppressors. These results are consistent with a role for BipA in either the structure or the function of the ribosome and imply that wild-type ribosomes are dependent on BipA for efficient expression of target mRNAs and that the lack of pseudouridylation at these three sites renders the ribosomes BipA independent.

A Ferritin-responsive Internal Ribosome Entry Site Regulates Folate Metabolism

Cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme levels are elevated by the expression of the heavy chain ferritin (H ferritin) cDNA in cultured cells without corresponding changes in mRNA levels, resulting in enhanced folate-dependent de novo thymidylate biosynthesis and impaired homocysteine remethylation. In this study, the mechanism whereby H ferritin regulates cSHMT expression was determined. cSHMT translation is shown to be regulated by an H ferritin-responsive internal ribosome entry site (IRES) located within the cSHMT mRNA 5'-untranslated region (5'-UTR). The cSHMT 5'-UTR exhibited IRES activity during in vitro translation of bicistronic mRNA templates, and in MCF-7 and HeLa cells transfected with bicistronic mRNAs. IRES activity was depressed in H ferritin-deficient mouse embryonic fibroblasts and elevated in cells expressing the H ferritin cDNA. H ferritin was shown to interact with the mRNA-binding protein CUGBP1, a protein known to interact with the alpha and beta subunits of eukaryotic initiation factor eIF2. Small interference RNA-mediated depletion of CUGBP1 decreased IRES activity from bicistronic templates that included the cSHMT 3'-UTR in the bicistronic construct. The identification of this H ferritin-responsive IRES represents a mechanism that accounts for previous observations that H ferritin regulates folate metabolism.

Identification of a Novel Internal Ribosome Entry Site in Giardiavirus That Extends to Both Sides of the Initiation Codon

In Giardia lamblia, enhanced translation of luciferase mRNA, flanked between the 5'-untranslated region (UTR) and 3 '-end of giardiavirus transcript, requires the presence of the initial 264-nucleotide (nt) viral capsid-coding region. By introducing the transcripts of dicistronic viral constructs into Giardia, we demonstrated that the 264-nt downstream region alone is insufficient to function as an internal ribosome entry site (IRES) without including a portion of the 5 '-UTR as well. Deletion analysis showed that efficient internal initiation requires the last 253 nts (nts 114-367) of the 5 '-UTR in combination with the downstream 264 nts. Specific mutations that disrupted the predicted secondary structural elements in either the 5 '-UTR or the 264-nt capsid-coding region completely abolished the IRES-mediated translation of downstream cistron, suggesting that the IRES activity requires the presence of these structures in both regions. Mutations that abolished translation of the first cistron did not, however, affect the IRES-mediated translation of the second cistron, indicating that this IRES-mediated translation is independent of the translation of the upstream cistron. This is, to our knowledge, the first reported identification of a viral IRES with an estimated size of 517 nts that extends to both sides of the initiation site.

The rluC Gene of Escherichia coli Codes for a Pseudouridine Synthase That Is Solely Responsible for Synthesis of Pseudouridine at Positions 955, 2504, and 2580 in 23 S Ribosomal RNA

Escherichia coli ribosomal RNA contains 10 pseudouridines, one in the 16 S RNA and nine in the 23 S RNA. Previously, the gene for the synthase responsible for the 16 S RNA pseudouridine was identified and cloned, as was a gene for a synthase that makes a single pseudouridine in 23 S RNA. The yceC open reading frame of E. coli is one of a set of genes homologous to these previously identified ribosomal RNA pseudouridine synthases. In this work, the gene was cloned, overexpressed, and shown to code for a pseudouridine synthase able to react with in vitro transcripts of 23 S ribosomal RNA. Deletion of the gene and analysis of the 23 S RNA from the deletion strain for the presence of pseudouridine at its nine known sites revealed that this synthase is solely responsible in vivo for the synthesis of three of the nine pseudouridine residues, at positions 955, 2504, and 2580. Therefore, this gene has been renamed rluC. Despite the absence of one-third of the normal complement of pseudouridines, there was no change in the exponential growth rate in either LB or M-9 medium at temperatures ranging from 24 to 42 degrees C. From this work and our previous studies, we have now identified three synthases that account for 50% of the pseudouridines in the E. coli ribosome.