Internal Ribosome Entry Site Sequence Research Articles

Design and Synthesis of Circular RNA Expression Vectors.

The recent success of the synthetic mRNA-based anti-COVID-19 vaccines has demonstrated the broad potential of the mRNA platform for applications in medicine, thanks to the combined efforts of a small community that has vastly improved key determinants such as design and formulation of synthetic mRNA during the past three decades. However, the cost of production and sensitivity to enzymatic degradation are still limiting the broader application of synthetic mRNA for therapeutic applications. The increased interest in mRNA-based technologies has spurred a renaissance for circular RNA (circRNA), as the lack of free 5' and 3' ends substantially increases resistance against enzymatic degradation in biological systems and does not require expensive cap analogs, as translation is controlled by an Internal Ribosome Entry Site (IRES) sequence. Thus, it can be expected that circRNA will play an important role for future mRNA therapeutics. Here we provide a detailed guide to the production of synthetic circRNA.

Systematic comparison of nonviral gene delivery strategies for efficient co-expression of two transgenes in human mesenchymal stem cells

BackgroundHuman mesenchymal stem cells (hMSCs) are being researched for cell-based therapies due to a host of unique properties, however, genetic modification of hMSCs, accomplished through nonviral gene delivery, could greatly advance their therapeutic potential. Furthermore, expression of multiple transgenes in hMSCs could greatly advance their clinical significance for treatment of multifaceted diseases, as individual transgenes could be expressed that target separate pathogenic drivers of complex diseases. Expressing multiple transgenes can be accomplished by delivering multiple DNA vectors encoding for each transgene, or by delivering a single poly-cistronic vector that encodes for each transgene and accomplishes expression through either use of multiple promoters, an internal ribosome entry site (IRES), or a 2A peptide sequence. These different transgene expression strategies have been used to express multiple transgenes in various mammalian cells, however, they have not been fully evaluated in difficult-to-transfect primary cells, like hMSCs. This study systematically compared four transgene expression and delivery strategies for expression of two reporter transgenes in four donors of hMSCs from two tissue sources using lipid- and polymer-mediate transfection, as follows: (i) delivery of separate DNA vectors in separate nanoparticles; (ii) delivery of separate DNA vectors combined in the same nanoparticle; (iii) delivery of a bi-cistronic DNA vector with an IRES sequence via nanoparticles; and (iv) delivery of a bi-cistronic DNA vector with a dual 2A peptide sequence via nanoparticles.ResultsOur results indicate that expression of two transgenes in hMSCs, independent of expression or delivery strategy, is inefficient compared to expressing a single transgene. However, delivery of separate DNA vectors complexed in the same nanoparticle, or delivery of a bi-cistronic DNA vector with a dual 2A peptide sequence, significantly increased the number of hMSCs expressing both transgenes compared to other conditions tested.ConclusionSeparate DNA vectors delivered in the same nanoparticle and bi-cistronic DNA vectors with dual 2A peptide sequences are highly efficient at simultaneously expressing two transgenes in multiple donors of hMSCs from different tissue sources. The data presented in this work can guide the development of hMSC transfection systems for delivery of multiple transgenes, with the goal of producing clinically relevant, genetically modified hMSCs.

A precise and efficient circular RNA synthesis system based on a ribozyme derived from Tetrahymena thermophila.

Classic strategies for circular RNA (circRNA) preparation always introduce large numbers of linear transcripts or extra nucleotides to the circularized product. In this study, we aimed to develop an efficient system for circRNA preparation based on a self-splicing ribozyme derived from an optimized Tetrahymena thermophila group Ⅰ intron. The target RNA sequence was inserted downstream of the ribozyme and a complementary antisense region was added upstream of the ribozyme to assist cyclization. Then, we compared the circularization efficiency of ribozyme or flanking intronic complementary sequence (ICS)-mediated methods through the DNMT1, CDR1as, FOXO3, and HIPK3 genes and found that the efficiency of our system was remarkably higher than that of flanking ICS-mediated method. Consequently, the circularized products mediated by ribozyme are not introduced with additional nucleotides. Meanwhile, the overexpressed circFOXO3 maintained its biological functions in regulating cell proliferation, migration, and apoptosis. Finally, a ribozyme-based circular mRNA expression system was demonstrated with a split green fluorescent protein (GFP) using an optimized Coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) sequence, and this system achieved successful translation of circularized mRNA. Therefore, this novel, convenient, and rapid engineering RNA circularization system can be applied for the functional study and large-scale preparation of circular RNA in the future.

Tactics targeting circular mRNA biosynthesis.

Faced with the development of mRNA technology in the field of medicine and vaccine, circular mRNA (circmRNA) becomes a strong alternative to mRNA for its circular secondary structure and higher stability. At present, the synthesis of circmRNAs has been realized by ligating linear mRNA precursors and is limited by poor efficiency. To solve this challenge, this study started with ribozyme catalysis and enzymatic reaction to explore different circmRNA biosynthesis strategies. In terms of ribozyme method, by screening different group I intron self-splicing system sequences, the sequence from thymidylate synthase (Td) gene of phage T4 showed the highest ligation efficiency. In terms of enzyme method, with the help of 20-bp homologous arm, T4 Rnl 2 was determined as the ligation method with the highest ligation efficiency. By comparing the two ligation methods, the expression level of circmRNA ligated by T4 Rnl 2 was 86% higher than that ligated by Td ribozyme. Based on these ligation methods, the screening results of internal ribosome entry site (IRES) sequences showed that mud crab dicistrovirus IRES was an IRES sequence with high ribosome binding ability and could be widely used in circmRNAs for efficient and stable translation in mammalian cells. These results should provide positive guidance for the industrial production of circmRNAs and the development of mRNA vaccines. Eventually, circmRNAs could widely function in the field of biomedicine.

In vivo genome editing at the albumin locus to treat methylmalonic acidemia

Methylmalonic acidemia (MMA) is a metabolic disorder most commonly caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Although adeno-associated viral (AAV) gene therapy has been effective at correcting the disease phenotype in MMA mouse models, clinical translation may be impaired by loss of episomal transgene expression and magnified by the need to treat patients early in life. To achieve permanent correction, we developed a dual AAV strategy to express a codon-optimized MMUT transgene from Alb and tested various CRISPR-Cas9 genome-editing vectors in newly developed knockin mouse models of MMA. For one target site in intron 1 of Alb, we designed rescue cassettes expressing MMUT behind a 2A-peptide or an internal ribosomal entry site sequence. A second guide RNA targeted the initiator codon, and the donor cassette encompassed the proximal albumin promoter in the 5′ homology arm. Although all editing approaches were therapeutic, targeting the start codon of albumin allowed the use of a donor cassette that also functioned as an episome and after homologous recombination, even without the expression of Cas9, as an integrant. Targeting the albumin locus using these strategies would be effective for other metabolic disorders where early treatment and permanent long-term correction are needed.

RNA-responsive elements for eukaryotic translational control.

The ability to control translation of endogenous or exogenous RNAs in eukaryotic cells would facilitate a variety of biotechnological applications. Current strategies are limited by low fold changes in transgene output and the size of trigger RNAs (trRNAs). Here we introduce eukaryotic toehold switches (eToeholds) as modular riboregulators. eToeholds contain internal ribosome entry site sequences and form inhibitory loops in the absence of a specific trRNA. When the trRNA is present, eToeholds anneal to it, disrupting the inhibitory loops and allowing translation. Through optimization of RNA annealing, we achieved up to 16-fold induction of transgene expression in mammalian cells. We demonstrate that eToeholds can discriminate among viral infection status, presence or absence of gene expression and cell types based on the presence of exogenous or endogenous RNA transcripts.

Retraction Note: Small interfering RNA targeted to stem-loop II of the 5\u2032 untranslated region effectively inhibits expression of six HCV genotypes

The antiviral action of interferon alpha targets the 5' untranslated region (UTR) used by hepatitis C virus (HCV) to translate protein by an internal ribosome entry site (IRES) mechanism. Although this sequence is highly conserved among different clinical strains, approximately half of chronically infected hepatitis C patients do not respond to interferon therapy. Therefore, development of small interfering RNA (siRNA) targeted to the 5'UTR to inhibit IRES mediated translation may represent an alternative approach that could circumvent the problem of interferon resistance. Four different plasmid constructs were prepared for intracellular delivery of siRNAs targeting the stem loop II-III of HCV 5' UTR. The effect of siRNA production on IRES mediated translation was investigated using chimeric clones between the gene for green fluorescence protein (GFP) and IRES sequences of six different HCV genotypes. The siRNA targeted to stem loop II effectively mediated degradation of HCV IRES mRNA and inhibited GFP expression in the case of six different HCV genotypes, where as siRNAs targeted to stem loop III did not. Furthermore, intracytoplasmic expression of siRNA into transfected Huh-7 cells efficiently degraded HCV genomic RNA and inhibited core protein expression from infectious full-length infectious clones HCV 1a and HCV 1b strains. These in vitro studies suggest that siRNA targeted to stem-loop II is highly effective inhibiting IRES mediated translation of the major genotypes of HCV. Stem-loop II siRNA may be a good target for developing an intracellular immunization strategy based antiviral therapy to inhibit hepatitis C virus strains that are not inhibited by interferon.

Development of a Novel Tetracycline-Inducible Kiss1-Cre Mouse Line for Temporally Controlled Gene Deletion in Kisspeptin Neurons

The tetracycline (Tet)-controlled inducible system is the most widely used reversible system for transgenic expression in mice. Previously, we generated a GnRH-CreTeR mouse model, using a first-generation Tet-inducible system to temporally induce expression of Cre recombinase in GnRH neurons. Recently, the Tet-inducible system has undergone several modifications to significantly reduce previous limitations that include leaky background expression and lower sensitivity to tetracycline induction. Therefore, we have developed a novel mouse model bearing a Tet-inducible kisspeptin-Cre allele (iKiss-Cre mouse) that will enable temporal control over the selective deletion of genes from Kiss1 neurons. This temporally controlled gene deletion will eliminate a longstanding technical limitation of conventional steroid receptor knockout models in which steroid regulation of the axis is confounded by steroid developmental and organizational effects in the reproductive axis. Two mouse lines were generated. The first line targets kisspeptin neurons with a third generation Tet-inducible reverse tetracycline transactivator (rtTA, Tet-On 3G) expressed under the control of the Kiss1 allele. Using CRISPR-Cas9 technology, we inserted a cassette containing an internal ribosome entry site (IRES) sequence followed by the rtTA downstream of the Kiss1 coding region as was previously done using Cre recombinase. Transcription of the recombinant Kiss1 allele yields a bicistronic messenger RNA, from which both kisspeptin and rtTA are independently translated. The second mouse line, TRE-Cre mice, was constructed to express Cre recombinase under control of the PTRE3G promoter. The PTRE3G promoter is bidirectional to allow simultaneous monitoring of Cre expression and a second florescent protein reporter (ZsGreen1). The Kiss1-rtTA only binds to and activates the PTRE3G promoter in the presence of doxycycline (an analog of tetracycline). Offspring from breeding of these mouse lines, iKiss-Cre, result in a system capable of generating Tet-induced expression of Cre recombinase in kisspeptin neurons. In order to document specificity and sensitivity of this system, we performed immunofluorescent staining and observed colocalization of kisspeptin with Cre recombinase in brain sections of iKiss-Cre mice only after doxycycline treatment. We will use this model to investigate negative feedback actions of E2 on the adultpulse generator after E2 exerts its organizational actions on maturing kisspeptin neurons. We expect that this mouse model will become a major tool used by the neuroendocrine community and serve as proof of principle for development of similar inducible knockout models employing the current generation of inducible methodologies.

Identification of the internal ribosome entry sites in the 5'‑untranslated region of the c‑fos gene

The Fos proto‑oncogene, activator protein‑1 (AP‑1) transcription factor subunit (<em>c‑fos</em>) gene, a member of the immediate early gene family, encodes c‑Fos, which is a subunit of the AP‑1 transcription factor. The present study aimed to investigate the mechanism by which the translation efficiency of <em>c‑fos</em> mRNA is upregulated when cellular protein synthesis is shut off. The result of western blotting revealed that the protein expression levels of c‑Fos were increased in rhabdomyosarcoma cells infected with enterovirus 71 (EV71) compared with uninfected cells. PCR was used to get the <em>c‑fos</em> 5'‑untranslated region (UTR). The luciferase assay of a bicistronic vector containing the <em>c‑fos</em> 5'UTR revealed that the <em>c‑fos</em> 5'UTR contains an internal ribosome entry site (IRES) sequence and a 175 nucleotide sequence (between 31 and 205 nt) that is essential for IRES activity. Analysis of potential IRES trans‑acting factors revealed that poly(C)‑binding protein 2 (PCBP2) negatively regulated the activity of the <em>c‑fos</em> IRES, whereas the La autoantigen (La) positively regulated its activity. The results of RNA‑protein immunoprecipitation demonstrated that both PCBP2 and La bound to the<em> c‑fos</em> 5'UTR. Furthermore, the IRES activity of <em>in vitro</em>‑transcribed <em>c‑fos</em> mRNA was upregulated during EV71 infection. The present study suggested a mechanism for the effect of viral infection on host genes, and provided a novel target for gene translation regulation.

NanoLuc Bioluminescence-Driven Photodynamic Activation of Cholecystokinin 1 Receptor with Genetically-Encoded Protein Photosensitizer MiniSOG.

In contrast to reversible activation by agonist, cholecystokinin 1 receptor (CCK1R) is permanently activated by singlet oxygen generated in photodynamic action, with sulphonated aluminium phthalocyanine or genetically encoded mini singlet oxygen generator (miniSOG) as photosensitizer. In these works, a halogen light source was used to power photodynamic action. For possible in vivo application of photodynamic CCK1R physiology, bearing a cumbersome light-delivery device connected to an external light source by experimental animals might interfere with their behavior. Therefore, in the present work, the possibility of bioluminescence-driven miniSOG photodynamic CCK1R activation was examined, as monitored by Fura-2 calcium imaging. In parallel experiments, it was found that, after plasma membrane (PM)-localized expression of miniSOGPM in AR4-2J cells, light irradiation with blue light-emitting diode (LED) (450 nm, 85 mW·cm−2, 1.5 min) induced persistent calcium oscillations that were blocked by CCK1R antagonist devazepide 2 nM. NanoLuc was expressed bicistronically with miniSOGPM via an internal ribosome entry site (IRES) sequence (pminiSOGPM-IRES-NanoLuc). The resultant miniSOGPM-IRES-NanoLuc-AR4-2J cells were found to generate strong bioluminescence upon addition of NanoLuc substrate coelenterazine. Strikingly, coelenterazine 5 microM was found to trigger long-lasting calcium oscillations (a hallmark for permanent CCK1R activation) in perifused miniSOGPM-IRES-NanoLuc-AR4-2J cells. These data indicate that NanoLuc bioluminescence can drive miniSOGPM photodynamic CCK1R activation, laying the foundation for its future in vivo applications.

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development.

Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. Cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. In a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. Most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. In the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. Despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. In this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. Further, this article discusses some recent progress in cell-free systems derived from Chinese hamster ovary and Sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. We particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. We also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development.

Screening for functional IRESes using \u03b1-complementation system of \u03b2-galactosidase in Pichia pastoris

BackgroundPichia pastoris is becoming a promising chassis cell for metabolic engineering and synthetic biology after its whole genome and transcriptome sequenced. However, the current systems for multigene co-expression in P. pastoris are not efficient. The internal ribosome entry site (IRES) has an ability to recruit the ribosome to initiate protein synthesis by cap-independent translation manner. This study seeks to screen IRES sequences that are functional in P. pastoris, which will allow P. pastoris to express multiple proteins in a single mRNA and increase its efficacy as a platform for metabolic engineering and synthetic biology.ResultsIn order to efficiently screen the IRES sequences, we first set out to create a screening system using LacZ gene. Due to the cryptic transcription of the LacZ gene, we established the α-complementation system of β-galactosidase in P. pastoris with the optimum length of the α-complementing peptide at ~ 92 amino acids. The optimal α-complementing peptide was then used as the second reporter to screen IRESes in the engineered GS115 expressing the corresponding ω-peptide. A total of 34 reported IRESes were screened. After ruling out all false positive or negative IRESes, only seven IRESes were functional in P. pastoris, which were from TEV, PVY, RhPV, TRV, KSHV, crTMV viruses and the 5′-UTR of the YAP1 gene of S. cerevisiae.ConclusionsWe showed here that α-complementation also works in P. pastoris and it can be used in a variety of in vivo studies. The functional IRESes screened in this study can be used to introduce multiple genes into P. pastoris via a prokaryotic-like polycistronic manner, which provided new efficient tools for metabolic engineering and synthetic biology researches in P. pastoris.

Real-Time Temporal Dynamics of Bicistronic Expression Mediated by Internal Ribosome Entry Site and 2A Cleaving Sequence.

Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.

Design and Development of Modified mRNA Encoding Core Antigen of Hepatitis C Virus: a Possible Application in Vaccine Production

Background:Hepatitis C virus (HCV) is a blood-borne pathogen, resulting in liver cirrhosis and liver cancer. Despite of many efforts in development of treatments for HCV, no vaccine has been licensed yet. The purpose of this study was to design and prepare a specific mRNA, without 5’ cap and poly (A) tail transcribed in vitro capable of coding core protein and also to determine its functionality.Methods:Candidate mRNA was prepared by in vitro transcription of the designed construct consisting of 5’ and 3’ untranslated regions of heat shock proteins 70 (hsp70) mRNA, T7 promoter, internal ribosome entry site (IRES) sequences of eIF4G related to human dendritic cells (DCs), and the Core gene of HCV. To design the modified mRNA, the 5’ cap and poly (A) tail structures were not considered. DCs were transfected by in vitro-transcribed messenger RNA (IVT-mRNA) and the expressions of green fluorescent protein (GFP), and Core genes were determined by microscopic examination and Western blotting assay.Results:Cell transfection results showed that despite the absence of 5’ cap and poly (A) tail, the structure of the mRNA was stable. Moreover, the successful expressions of GFP and Core genes were achieved.Conclusion:Our findings indicated the effectiveness of a designed IVT-mRNA harboring the Core gene of HCV in transfecting and expressing the antigens in DCs. Considering the simple and efficient protocol for the preparation of this IVT-mRNA and its effectiveness in expressing the gene that it carries, this IVT-mRNA could be suitable for development of an RNA vaccine against HCV.

Design and Development of Modified mRNA Encoding Core Antigen of Hepatitis C Virus: a Possible Application in Vaccine Production

Hepatitis C virus (HCV) is a ‎blood-borne pathogen, resulting in liver cirrhosis and liver cancer. Despite of many efforts in development of treatments for HCV, no vaccine has been licensed yet. The purpose of this study was ‎to design and prepare a specific mRNA, without 5' cap and poly (A) tail transcribed in vitro capable of coding core protein and also to determine its functionality. Candidate mRNA was prepared by in vitro transcription of the designed construct consisting of ‎‎5ʹ and 3ʹ untranslated regions of heat shock proteins 70 (hsp70) mRNA, T7 promoter, internal ribosome entry site (IRES) sequences of eIF4G related to human dendritic cells (DCs), and the ‎Core gene of HCV. To design the modified mRNA, the ‎‎5' cap and poly (A) tail structures were not considered. DCs were transfected by in vitro-transcribed messenger RNA (IVT-mRNA) and the expressions of green fluorescent protein (GFP), and Core genes were determined by microscopic examination and Western blotting assay. Cell transfection results showed that despite the absence of ‎‎5' cap and poly (A) tail, the structure of the mRNA ‎was stable. Moreover, the successful expressions of GFP and Core genes were achieved. Our findings indicated the effectiveness of a designed IVT-mRNA harboring the Core gene of HCV in transfecting and expressing the antigens in DCs. Considering the simple and efficient protocol for the preparation of this IVT-mRNA and its effectiveness in expressing the gene that it carries, this IVT-mRNA could be suitable for development of an RNA vaccine against HCV.

In Vitro Screen to Identify Silent but Activatable (S/A) Integration Sites for a Tetracycline-Inducible Transgene in Mice.

To obtain ubiquitous or simply widespread transgene expression from a single stable integrant transgene is quite challenging because the random genomic integration sites of transgenes may create expression variation or frequent silencing. The tetracycline (Tet)-inducible system requires two reliable working transgenes, one for the tetracycline transactivators (tTA or rtTA) and one for the responder transgene driven by the tet-O promoter. Therefore, the challenge of getting this system working properly is a serious prospect. In this protocol, we describe how to identify a silent but highly activatable genomic site by taking advantage of transgenic lines reliably expressing the tetracycline transactivators from the Rosa-26 locus. These lines provide optimal Tet-inducible expression: There is minimal leakiness at the "off" state and a high level of induction in the presence of the inducer, doxycycline. The procedure requires (1) an embryonic stem (ES) cell line (germline competent) expressing rtTA from the Rosa-26 locus and (2) construction of a Tet-inducible transgene. The transgene contains the tet-O promoter followed by the gene of interest linked to a βgeo gene (a fusion between lacZ and neo) through an internal ribosomal entry site (IRES) sequence, which allows the initiation of translation in a cap-independent manner.

Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemia.

Internal ribosome entry site (IRES) sequences have become a valuable tool in the construction of gene transfer and therapeutic vectors for multi-cistronic gene expression from a single mRNA transcript. The optimal conditions for effective use of this sequence to construct a functional expression vector are not precisely defined but it is generally assumed that the internal ribosome entry site dependent expression of the second gene in such as cassette is less efficient than the cap-dependent expression of the first gene. Mainly tailoring inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector further in construction of optimised cassette for gene therapy of familial hypercholesterolemia. We tailored the size of the inter-cistronic spacer sequence at the 5′ region of the internal ribosome entry site sequence using sequential deletions and demonstrated that the expression of the 3′ gene can be significantly increased to similar levels as the cap-dependent expression of the 5’ gene. Maximum expression efficiency of the downstream gene was obtained when the spacer is composed of 18–141 base pairs. In this case a single mRNA transcriptional unit containing both the first and the second Cistron was detected. Whilst constructs with spacer sequences of 216 bp or longer generate a single transcriptional unit containing only the first Cistron. This suggests that long spacers may affect transcription termination. When the spacer is 188 bp, both transcripts were produced simultaneously in most transfected cells, while a fraction of them expressed only the first but not the second gene. Expression analyses of vectors containing optimised cassettes clearly confirm that efficiency of gene transfer and biological activity of the expressed transgenic proteins in the transduced cells can be achieved. Furthermore, Computational analysis was carried out by molecular dynamics (MD) simulation to determine the most emerges as viable containing specific binding site and bridging of 5′ and 3′ ends involving direct RNA-RNA contacts and RNA-protein interactions. These results provide a mechanistic basis for translation stimulation and RNA resembling for the synergistic stimulation of cap-dependent translation.

Mutational Analysis of the Bovine Hepacivirus Internal Ribosome Entry Site.

In recent years, hepatitis C virus (HCV)-related viruses were identified in several species, including dogs, horses, bats, and rodents. In addition, a novel virus of the genus Hepacivirus has been discovered in bovine samples and was termed bovine hepacivirus (BovHepV). Prediction of the BovHepV internal ribosome entry site (IRES) structure revealed strong similarities to the HCV IRES structure comprising domains II, IIIabcde, pseudoknot IIIf, and IV with the initiation codon AUG. Unlike HCV, only one microRNA-122 (miR-122) binding site could be identified in the BovHepV 5' nontranslated region. In this study, we analyzed the necessity of BovHepV IRES domains to initiate translation and investigated possible interactions between the IRES and core coding sequences by using a dual luciferase reporter assay. Our results suggest that such long-range interactions within the viral genome can affect IRES-driven translation. Moreover, the significance of a possible miR-122 binding to the BovHepV IRES was investigated. When analyzing translation in human Huh-7 cells with large amounts of endogenous miR-122, introduction of point mutations to the miR-122 binding site resulted in reduced translation efficiency. Similar results were observed in HeLa cells after substitution of miR-122. Nevertheless, the absence of pronounced effects in a bovine hepatocyte cell line expressing hardly any miR-122 as well suggests additional functions of this host factor in virus replication.IMPORTANCE Several members of the family Flaviviridae, including HCV, have adapted cap-independent translation strategies to overcome canonical eukaryotic translation pathways and use cis-acting RNA-elements, designated viral internal ribosome entry sites (IRES), to initiate translation. Although novel hepaciviruses have been identified in different animal species, only limited information is available on their biology on molecular level. Therefore, our aim was a fundamental analysis of BovHepV IRES functions. The findings which show that functional IRES elements are also crucial for BovHepV translation expand our knowledge on molecular mechanism of hepacivirus propagation. We also studied the possible effects of one major host factor implicated in HCV pathogenesis, miR-122. The results of mutational analyses suggested that miR-122 enhances virus translation mediated by BovHepV IRES.

Human rhinovirus internal ribosome entry site element enhances transgene expression in transfected CHO-S cells

Chinese hamster ovary (CHO) cells are mainly used for recombinant protein production. However, the unstable transgene expression and lower transgene copy numbers are the major issues need to be resolved. Here, eleven internal ribosome entry site (IRES) elements from viral and cellular IRES were evaluated for foreign gene expression in CHO-S cells. We constructed eleven fusing plasmids containing different IRES sequences downstream of the enhanced green fluorescent protein (EGFP) gene. EGFP expression was detected by flow cytometry and the transgene copy number was evaluated by quantitative PCR. The erythropoietin (EPO) protein was also used to assess the stronger IRES. The results showed that IRES from human rhinovirus (HRV) exhibited the highest EGFP expression level under transient and stable transfections. The EGFP expression level of vector with IRES from HRV was related to the gene copy number in stably transfected CHO-S cells. Moreover, IRES from HRV induced higher expression level of EPO compared with one mutant IRES from EMCV in transfected cells. In conclusion, IRES from HRV can function as a strong IRES element for stable expression in CHO-S cells, which could potentially guide more effective foreign gene expression in CHO-S cells.

Polyplex System Versus Nucleofection for Human Skin Cell Transfection and Effect of Internal Ribosome Entry Site Sequence.

Nonviral transfection has important implications on gene therapy because of its safety. In particular, polyfection and nucleofection are two widely used systems for nonviral gene delivery. Their potential depends on the transfection efficiency achieved, which is influenced in turn by the type of cells transfected and by the plasmid that carries the gene of interest. The efficiency of transfection by polyfection or nucleofection in human fibroblasts and keratinocytes was evaluated in this study. Transfections were performed with plasmids containing a gene of interest (human cathelicidin antimicrobial peptide) and two reporter genes (red or green fluorescent protein) that included or not an internal ribosome entry site (IRES). The efficiency was measured by flow cytometry in terms of percentage of cells expressing the reporter gene; viability of transfected cells was also evaluated. It was found that nucleofection was more efficient than polyplexes for transfecting fibroblasts, while no significant differences were found between both systems of transfection when applied to keratinocytes. Regarding the viability of fibroblasts after transfection, values were high in both systems. In contrast, keratinocytes were more sensitive to nucleofection. It was also noted that both types of cells decreased reporter gene expression when IRES sequence was located upstream of the reporter gene, suggesting a negative effect on the expression of this gene. These results confirm that the transfection efficiency depends on the type of cells and the system used.