scholarly journals Internal Ribosome Entry Site Dramatically Reduces Transgene Expression in Hematopoietic Cells in a Position-Dependent Manner

Viruses ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 920 ◽  
Author(s):  
Qingyun Zheng ◽  
Xueyan Zhang ◽  
Hua Yang ◽  
Jinyan Xie ◽  
Yilin Xie ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Transgene Expression ◽  
Hematopoietic Cells ◽  
Inhibitory Effect ◽  
Encephalomyocarditis Virus ◽  
Transduction Efficiency ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Virus Serotype

Bicistronic transgene expression mediated by internal ribosome entry site (IRES) elements has been widely used. It co-expresses heterologous transgene products from a message RNA driven by a single promoter. Hematologic gene delivery is a promising treatment for both inherited and acquired diseases. A combined strategy was recently documented for potential genome editing in hematopoietic cells. A transduction efficiency exceeding ~90% can be achieved by capsid-optimized recombinant adeno-associated virus serotype 6 (rAAV6) vectors. In this study, to deliver an encephalomyocarditis virus (EMCV) IRES-containing rAAV6 genome into hematopoietic cells, we observed that EMCV IRES almost completely shut down the transgene expression during the process of mRNA–protein transition. In addition, position-dependent behavior was observed, in which only the EMCV IRES element located between a promoter and the transgenes had an inhibitory effect. Although further studies are warranted to evaluate the involvement of cellular translation machinery, our results propose the use of specific IRES elements or an alternative strategy, such as the 2A system, to achieve bicistronic transgene expression in hematopoietic cells.

scholarly journals Involvement of Proteasome α-Subunit PSMA7 in Hepatitis C Virus Internal Ribosome Entry Site-Mediated Translation

2001 ◽  
Vol 21 (24) ◽  
pp. 8357-8364 ◽  
Author(s):  
Martin Krüger ◽  
Carmela Beger ◽  
Peter J. Welch ◽  
Jack R. Barber ◽  
Michael P. Manns ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Inhibitory Effect ◽  
Encephalomyocarditis Virus ◽  
Luciferase Reporter ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Α Subunit ◽  
Hcv Ires

ABSTRACT Ribozymes are small catalytic RNA molecules that can be engineered to enzymatically cleave RNA transcripts in a sequence-specific fashion and thereby inhibit expression and function of the corresponding gene product. With their simple structures and site-specific cleavage activity, they have been exploited as potential therapeutic agents in a variety of human disorders, including hepatitis C virus (HCV) infection. We have designed a hairpin ribozyme (Rz3′X) targeting the HCV minus-strand replication intermediate at position 40 within the 3′X tail. Surprisingly, Rz3′X was found to induce ganciclovir (GCV)-resistant colonies in a bicistronic cellular reporter system with HCV internal ribosome entry site (IRES)-dependent translation of herpes simplex virus thymidine kinase (TK). Rz3′X-transduced GCV-resistant HeLa reporter cells showed substantially reduced IRES-mediated HCV core protein translation compared with control vector-transduced cells. Since these reporter systems do not contain the HCV 3′X tail sequences, the results indicate that Rz3′X probably exerted an inhibitory effect on HCV IRES activity fortuitously through another gene target. A novel technique of ribozyme cleavage-based target gene identification (cleavage-specific amplification of cDNA ends) (M. Krüger, C. Beger, P. J. Welch, J. R. Barber, and F. Wong-Staal, Nucleic Acids Res. 29:e94, 2001) revealed that human 20S proteasome α-subunit PSMA7 mRNA was a target RNA recognized and cleaved by Rz3′X. We then showed that additional ribozymes directed against PSMA7 RNA inhibited HCV IRES activity in two assay systems: GCV resistance in the HeLa IRES TK reporter cell system and a transient transfection assay performed with a bicistronicRenilla-HCV IRES-firefly luciferase reporter in Huh7 cells. In contrast, ribozymes were inactive against IRES of encephalomyocarditis virus and human rhinovirus. Additionally, proteasome inhibitor MG132 exerted a dose-dependent inhibitory effect on HCV IRES-mediated translation but not on cap-dependent translation. These data suggest a principal role for PSMA7 in regulating HCV IRES activity, a function essential for HCV replication.

scholarly journals Effect of NS3 and NS5B proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation

2008 ◽  
Vol 89 (4) ◽  
pp. 994-999 ◽  
Author(s):  
Ming Xiao ◽  
Yan Bai ◽  
Hui Xu ◽  
Xiaolu Geng ◽  
Jun Chen ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Classical Swine Fever ◽  
Rna Helicase ◽  
Helicase Domain ◽  
Dependent Manner ◽  
Helicase Activity ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Cellular Translation

A full-length NS3 (NS3F) and a truncated NS3 protein (NS3H) with an RNA helicase domain possess RNA helicase activity. Using an in vitro system with a monocistronic reporter RNA or DNA, containing the CSFV 5′-UTR, we observed that both NS3F and NS3H enhanced internal ribosome entry site (IRES)-mediated and cellular translation in a dose-dependent manner, but NS3 protease (NS3P) that lacks a helicase domain did not. NS3F was stronger than NS3H in promoting both translations. These results showed that viral RNA helicase could promote viral and cellular translation, and higher RNA helicase activity might be more efficient. The NS5B protein, the viral replicase, did not significantly affect the IRES-directed or cellular translation alone. NS5B significantly enhanced the stimulative effect of NS3F on both IRES-mediated and cellular translation, but did not affect that of NS3H or NS3P. This suggests that NS5B and NS3 interact via the protease domain during the enhancement of translation.

Internal ribosome entry site (IRES) from Encephalomyocarditis virus (EMCV) as a tool for shuttle expression plasmids

2015 ◽  
Vol 468 (4) ◽  
pp. 548-553 ◽  
Author(s):  
Sandra Aurora Telpalo-Carpio ◽  
Francisco Diaz-Mitoma ◽  
Jorge Eugenio Moreno-Cuevas ◽  
José Manuel Aguilar-Yáñez
Keyword(s):  
Internal Ribosome Entry Site ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Expression Plasmids

scholarly journals Novel Characteristics of the Biological Properties of the YeastSaccharomyces cerevisiaeEukaryotic Initiation Factor 2A

2005 ◽  
Vol 280 (16) ◽  
pp. 15601-15611 ◽  
Author(s):  
Anton A. Komar ◽  
Stephane R. Gross ◽  
Diane Barth-Baus ◽  
Ryan Strachan ◽  
Jack O. Hensold ◽  
...  
Keyword(s):  
Translational Control ◽  
Internal Ribosome Entry Site ◽  
Biological Properties ◽  
Yeast Cells ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Entry Site ◽  
Regulation Of Expression ◽  
Internal Ribosome Entry ◽  
Expression Of Genes

Eukaryotic initiation factor 2A (eIF2A) has been shown to direct binding of the initiator methionyl-tRNA (Met-tRNAi) to 40 S ribosomal subunits in a codon-dependent manner, in contrast to eIF2, which requires GTP but not the AUG codon to bind initiator tRNA to 40 S subunits. We show here that yeast eIF2A genetically interacts with initiation factor eIF4E, suggesting that both proteins function in the same pathway. The doubleeIF2A/eIF4E-tsmutant strain displays a severe slow growth phenotype, which correlated with the accumulation of 85% of the double mutant cells arrested at the G2/M border. These cells also exhibited a disorganized actin cytoskeleton and elevated actin levels, suggesting that eIF2A might be involved in controlling the expression of genes involved in morphogenic processes. Further insights into eIF2A function were gained from the studies of eIF2A distribution in ribosomal fractions obtained from either aneIF5BΔ (fun12Δ) strain or aeIF3b-ts(prt1-1) strain. It was found that the binding of eIF2A to 40 and 80 S ribosomes was not impaired in either strain. We also found that eIF2A functions as a suppressor of Ure2p internal ribosome entry site-mediated translation in yeast cells. The regulation of expression from theURE2internal ribosome entry site appears to be through the levels of eIF2A protein, which has been found to be inherently unstable with a half-life of ∼17 min. It was hypothesized that this instability allows for translational control through the level of eIF2A protein in yeast cells.

scholarly journals Translation Mediated by the Internal Ribosome Entry Site of thecat-1mRNA Is Regulated by Glucose Availability in a PERK Kinase-dependent Manner

2002 ◽  
Vol 277 (14) ◽  
pp. 11780-11787 ◽  
Author(s):  
James Fernandez ◽  
Barry Bode ◽  
Antonis Koromilas ◽  
J. Alan Diehl ◽  
Irene Krukovets ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Glucose Availability

scholarly journals Alpha interferon inhibits translation mediated by the internal ribosome entry site of six different hepatitis C virus genotypes

2005 ◽  
Vol 86 (11) ◽  
pp. 3047-3053 ◽  
Author(s):  
Sidhartha Hazari ◽  
Asha Patil ◽  
Virendra Joshi ◽  
Deborah E. Sullivan ◽  
Cesar D. Fermin ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Transforming Growth Factor Beta ◽  
Internal Ribosome Entry Site ◽  
Transforming Growth Factor ◽  
Interleukin 1 ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Hcv Genotypes

Certain genotypes of hepatitis C virus (HCV) respond less often than others to treatment with interferon (IFN). The mechanisms for this differential response are not known. In this report antiviral effects of IFN-α2b on translation were examined in a hepatic cell line using chimeric clones of internal ribosome entry site (IRES) sequences from six different HCV genotypes and the green fluorescence protein (GFP) gene. As a control, IFN action at the level of the IRES was examined in the presence of different cytokines. It was determined that IFN-α2b specifically inhibited the translation of GFP mediated by IRES sequences from six major HCV genotypes in a concentration-dependent manner. Other cytokines including tumour necrosis factor alpha, transforming growth factor beta 1, interleukin 1 and interleukin 6 have no inhibitory effect. The inhibition of translation in these experiments was not due to extensive intracellular degradation of IRES-GFP mRNA. These results suggest that the antiviral action of IFN-α2b blocks IRES-mediated translation and this effect is the same among HCVs of other genotypes.

scholarly journals Cap-dependent and hepatitis C virus internal ribosome entry site-mediated translation are modulated by phosphorylation of eIF2α under oxidative stress

2006 ◽  
Vol 87 (11) ◽  
pp. 3251-3262 ◽  
Author(s):  
Paul R. MacCallum ◽  
Samantha C. Jack ◽  
Philip A. Egan ◽  
Benjamin T. McDermott ◽  
Richard M. Elliott ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Hepg2 Cells ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Huh7 Cells ◽  
Hcv Ires

Chronic hepatitis C is often associated with oxidative stress. Hepatitis C virus (HCV) utilizes an internal ribosome entry site (IRES) element for translation, in contrast to cap-dependent translation of the majority of cellular proteins. To understand how virus translation is modulated under oxidative stress, HCV IRES-mediated translation was compared with cap-dependent translation using a bicistronic reporter construct and hydrogen peroxide (H2O2) as a stress inducer. In H2O2-sensitive HeLa cells, H2O2 repressed translation in a time- and dose-dependent manner, concomitant with the kinetics of eIF2α phosphorylation. A phosphomimetic of eIF2α, which mimics the structure of the phosphorylated eIF2α, was sufficient to repress translation in the absence of H2O2. In H2O2-resistant HepG2 cells, H2O2 activated both HCV IRES-mediated and cap-dependent translation, associated with an increased level of phospho-eIF2α. It was postulated that H2O2 might stimulate translation in HepG2 cells via an eIF2α-independent mechanism, whereas the simultaneous phosphorylation of eIF2α repressed part of the translational activities. Indeed, the translational repression was released in the presence of a non-phosphorylatable mutant, eIF2α-SA, resulting in further enhancement of both translational activities after exposure to H2O2. In HuH7 cells, which exhibited an intermediate level of sensitivity towards H2O2, both HCV IRES-mediated and cap-dependent translational activities were upregulated after treatment with various doses of H2O2, but the highest level of induction was achieved with a low level of H2O2, which may represent the physiological level of H2O2. At this level, the HCV IRES-mediated translation was preferentially upregulated compared with cap-dependent translation.

scholarly journals Conserved Nucleotides within the J Domain of the Encephalomyocarditis Virus Internal Ribosome Entry Site Are Required for Activity and for Interaction with eIF4G

2003 ◽  
Vol 77 (23) ◽  
pp. 12441-12449 ◽  
Author(s):  
Angela T. Clark ◽  
Morwenna E. M. Robertson ◽  
Graeme L. Conn ◽  
Graham J. Belsham
Keyword(s):  
Internal Ribosome Entry Site ◽  
Single Point ◽  
Encephalomyocarditis Virus ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Of Translation ◽  
Mouth Disease Virus ◽  
J Domain

ABSTRACT The internal ribosome entry site (IRES) elements of cardioviruses (e.g., encephalomyocarditis virus [EMCV] and foot-and-mouth disease virus) are predicted to have very similar secondary structures. Among these complex RNA structures there is only rather limited complete sequence conservation. Within the J domain of the EMCV IRES there are four highly conserved nucleotides (A704, C705, G723, and A724)., which are predicted to be unpaired and have been targeted for mutagenesis. Using an IRES-dependent cell selection system, we have isolated functional IRES elements from a pool of up to 256 mutants. All changes to these conserved nucleotides resulted in IRES elements that were less efficient at directing internal initiation of translation than the wild-type element, and even some of the single point mutants were highly defective. Each of the mutations adversely affected the ability of the RNAs to interact with the translation initiation factor eIF4G.

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