scholarly journals The Rhopalosiphum padi virus 5′ internal ribosome entry site is functional in Spodoptera frugiperda 21 cells and in their cell-free lysates: implications for the baculovirus expression system

2004 ◽  
Vol 85 (6) ◽  
pp. 1565-1569 ◽  
Author(s):  
Elizabeth Royall ◽  
Kathryn E. Woolaway ◽  
Jens Schacherl ◽  
Stefan Kubick ◽  
Graham J. Belsham ◽  
...  
Keyword(s):  
Spodoptera Frugiperda ◽  
Internal Ribosome Entry Site ◽  
Expression System ◽  
Rhopalosiphum Padi ◽  
Baculovirus Expression System ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Baculovirus Expression ◽  
Initiation Of Translation

Cap-independent internal initiation of translation occurs on a number of viral and cellular mRNAs and is directed by internal ribosome entry site (IRES) elements. Rhopalosiphum padi virus (RhPV) is a member of the Dicistroviridae. These viruses have single-stranded, positive-sense RNA genomes that contain two open reading frames, both preceded by IRES elements. Previously, the activity of the RhPV 5′ UTR IRES has been demonstrated in mammalian, Drosophila and wheat germ in vitro translation systems. It is now shown that this IRES also functions within Spodoptera frugiperda (Sf21) cells which are widely used in the baculovirus expression system, and in a novel Sf21 cell-based lysate system. Inclusion of the RhPV IRES in a dicistronic reporter mRNA transcript increased translation of the second cistron 23-fold within Sf21 cells. In contrast, the encephalomyocarditis virus IRES was inactive in both systems. The RhPV IRES therefore has the potential to be utilized in insect cell expression systems.

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.

scholarly journals Regulation of the cell-cycle-dependent internal ribosome entry site of the PITSLRE protein kinase: roles of Unr (upstream of N-ras) protein and phosphorylated translation initiation factor eIF-2α

2004 ◽  
Vol 385 (1) ◽  
pp. 155-163 ◽  
Author(s):  
Sandrine A. TINTON ◽  
Bert SCHEPENS ◽  
Yanik BRUYNOOGHE ◽  
Rudi BEYAERT ◽  
Sigrid CORNELIS
Keyword(s):  
Cell Cycle ◽  
Internal Ribosome Entry Site ◽  
Encephalomyocarditis Virus ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Α Subunit ◽  
Initiation Of Translation ◽  
Cycle Dependent

The PITSLRE kinases belong to the large family of cyclin-dependent protein kinases. Their function has been related to cell-cycle regulation, splicing and apoptosis. We have previously shown that the open reading frame of the p110PITSLRE transcript contains an IRES (internal ribosome entry site) that allows the expression of a smaller p58PITSLRE isoform during the G2/M stage of the cell cycle. In the present study we investigated further the role of cis- and trans-acting factors in the regulation of the PITSLRE IRES. Progressive deletion analysis showed that both a purine-rich sequence and a Unr (upstream of N-ras) consensus binding site are essential for PITSLRE IRES activity. In line with these observations, we demonstrate that the PITSLRE IRES interacts with the Unr protein, which is more prominently expressed at the G2/M stage of the cell cycle. We also show that phosphorylation of the α-subunit of the canonical initiation factor eIF-2 is increased at G2/M. Interestingly, phosphorylation of eIF-2α has a permissive effect on the efficiency of both the PITSLRE IRES and the ornithine decarboxylase IRES, two cell cycle-dependent IRESs, in mediating internal initiation of translation, whereas this was not observed with the viral EMCV (encephalomyocarditis virus) and HRV (human rhinovirus) IRESs.

scholarly journals Presence of an Encephalomyocarditis Virus Internal Ribosome Entry Site Sequence in Avian Infectious Bronchitis Virus Defective RNAs Abolishes Rescue by Helper Virus

2004 ◽  
Vol 78 (6) ◽  
pp. 2711-2721 ◽  
Author(s):  
Brian Dove ◽  
David Cavanagh ◽  
Paul Britton
Keyword(s):  
Internal Ribosome Entry Site ◽  
Expression System ◽  
Serial Passage ◽  
Helper Virus ◽  
Encephalomyocarditis Virus ◽  
Avian Infectious Bronchitis Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Infectious Bronchitis ◽  
Defective Rnas

ABSTRACT Avian infectious bronchitis virus (IBV) defective RNAs (D-RNAs) have been used for the expression of heterologous genes in a helper-virus-dependent expression system. The heterologous genes were expressed under the control of an IBV transcription-associated sequence (TAS) derived from gene 5 of IBV Beaudette. However, coronavirus D-RNA expression vectors display an inherent instability following serial passage with helper virus, resulting in the eventual loss of the heterologous genes. The use of the picornavirus encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence to initiate gene translation was investigated as an alternative method to the coronavirus-mediated TAS-controlled heterologous gene expression system. IBV D-RNAs containing the chloramphenicol acetyltransferase (CAT) reporter gene, under EMCV IRES control, were assessed for IRES-mediated CAT protein translation. CAT protein was detected from T7-derived IBV D-RNA transcripts in a cell-free protein synthesis system and in situ in avian chick kidney (CK) cells following T7-derived D-RNA synthesis from a recombinant fowlpox virus expressing the bacteriophage T7 DNA-dependent RNA polymerase. However, CAT protein was not detected in CK cells from IRES-containing IBV D-RNAs, in which the IRES-CAT construct was inserted at two different positions within the D-RNA, in the presence of helper IBV. Northern blot analysis demonstrated that the IRES-containing D-RNAs were not rescued on serial passage with helper virus, indicating that the EMCV IRES sequence had a detrimental effect on IBV D-RNA rescue.

scholarly journals The 5′ Untranslated Region of Rhopalosiphum padi Virus Contains an Internal Ribosome Entry Site Which Functions Efficiently in Mammalian, Plant, and Insect Translation Systems

2001 ◽  
Vol 75 (21) ◽  
pp. 10244-10249 ◽  
Author(s):  
Kathryn E. Woolaway ◽  
Konstantinos Lazaridis ◽  
Graham J. Belsham ◽  
Michael J. Carter ◽  
Lisa O. Roberts
Keyword(s):  
Internal Ribosome Entry Site ◽  
Rhopalosiphum Padi ◽  
Encephalomyocarditis Virus ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ires Element ◽  
Translation Systems

ABSTRACT Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5′ UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5′ UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, andDrosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5′ IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.

scholarly journals Internal initiation of translation from the human rhinovirus-2 internal ribosome entry site requires the binding of Unr to two distinct sites on the 5′ untranslated region

2007 ◽  
Vol 88 (11) ◽  
pp. 3043-3052 ◽  
Author(s):  
Emma C. Anderson ◽  
Sarah L. Hunt ◽  
Richard J. Jackson
Keyword(s):  
Internal Ribosome Entry Site ◽  
Binding Sites ◽  
Cold Shock ◽  
Human Rhinovirus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding Protein ◽  
Initiation Of Translation ◽  
Cold Shock Domain ◽  
Internal Initiation

Internal initiation of translation from the human rhinovirus-2 (HRV-2) internal ribosome entry site (IRES) is dependent upon host cell trans-acting factors. The multiple cold shock domain protein Unr and the polypyrimidine tract-binding protein have been identified as synergistic activators of HRV-2 IRES-driven translation. In order to investigate the mechanism by which Unr acts in this process, we have mapped the binding sites of Unr to two distinct secondary structure domains of the HRV-2 IRES, and have identified specific nucleotides that are involved in the binding of Unr to the IRES. The data suggest that Unr acts as an RNA chaperone to maintain a complex tertiary IRES structure required for translational competency.

scholarly journals A Peptide Derived from RNA Recognition Motif 2 of Human La Protein Binds to Hepatitis C Virus Internal Ribosome Entry Site, Prevents Ribosomal Assembly, and Inhibits Internal Initiation of Translation

2005 ◽  
Vol 79 (15) ◽  
pp. 9842-9853 ◽  
Author(s):  
Renuka Pudi ◽  
Sudhamani S. Ramamurthy ◽  
Saumitra Das
Keyword(s):  
Internal Ribosome Entry Site ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Recognition Motif ◽  
La Protein ◽  
Initiation Of Translation ◽  
Internal Initiation ◽  
Hcv Ires

ABSTRACT Human La protein is known to interact with hepatitis C virus (HCV) internal ribosome entry site (IRES) and stimulate translation. Previously, we demonstrated that mutations within HCV SL IV lead to reduced binding to La-RNA recognition motif 2 (RRM2) and drastically affect HCV IRES-mediated translation. Also, the binding of La protein to SL IV of HCV IRES was shown to impart conformational alterations within the RNA so as to facilitate the formation of functional initiation complex. Here, we report that a synthetic peptide, LaR2C, derived from the C terminus of La-RRM2 competes with the binding of cellular La protein to the HCV IRES and acts as a dominant negative inhibitor of internal initiation of translation of HCV RNA. The peptide binds to the HCV IRES and inhibits the functional initiation complex formation. An Huh7 cell line constitutively expressing a bicistronic RNA in which both cap-dependent and HCV IRES-mediated translation can be easily assayed has been developed. The addition of purified TAT-LaR2C recombinant polypeptide that allows direct delivery of the peptide into the cells showed reduced expression of HCV IRES activity in this cell line. The study reveals valuable insights into the role of La protein in ribosome assembly at the HCV IRES and also provides the basis for targeting ribosome-HCV IRES interaction to design potent antiviral therapy.

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 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.

Sign in / Sign up

Export Citation Format

Share Document