scholarly journals An internal ribosome entry site located upstream of the crucifer-infecting tobamovirus coat protein (CP) gene can be used for CP synthesis in vivo

2006 ◽  
Vol 87 (9) ◽  
pp. 2693-2697 ◽  
Author(s):  
Yu. L. Dorokhov ◽  
P. A. Ivanov ◽  
T. V. Komarova ◽  
M. V. Skulachev ◽  
J. G. Atabekov
Keyword(s):  
Coat Protein ◽  
Internal Ribosome Entry Site ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Cp Gene ◽  
Green Fluorescent

It was previously shown that, unlike the type member of the genus Tobamovirus (TMV U1), a crucifer-infecting tobamovirus (crTMV) contains a 148 nt internal ribosome entry site (IRES)CP,148 CR upstream of the coat protein (CP) gene. Here, viral vectors with substitutions in the stem–loop (SL) region of CP subgenomic promoters (TMV U1-CP–GFP/SL-mut and crTMV-CP–GFP/SL-mut) were constructed and the levels of CP synthesis in agroinoculation experiments were compared. No CP–GFP (green fluorescent protein) synthesis was detected in Nicotiana benthamiana leaves inoculated with TMV U1-CP–GFP/SL-mut, whereas a small amount of CP–GFP synthesis was obtained in crTMV-CP–GFP/SL-mut-injected leaves. Northern blots proved that both promoters were inactive. It could be hypothesized that IRES-mediated early production of the CP by crTMV is needed for realization of its crucifer-infecting capacity.

scholarly journals Hepatitis C Virus Internal Ribosome Entry Site (IRES) Stem Loop IIId Contains a Phylogenetically Conserved GGG Triplet Essential for Translation and IRES Folding

2000 ◽  
Vol 74 (22) ◽  
pp. 10430-10437 ◽  
Author(s):  
Ronald Jubin ◽  
Nicole E. Vantuno ◽  
Jeffrey S. Kieft ◽  
Michael G. Murray ◽  
Jennifer A. Doudna ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Apical Loop ◽  
Hcv Ires

ABSTRACT The hepatitis C virus (HCV) internal ribosome entry site (IRES) is a highly structured RNA element that directs cap-independent translation of the viral polyprotein. Morpholino antisense oligonucleotides directed towards stem loop IIId drastically reduced HCV IRES activity. Mutagenesis studies of this region showed that the GGG triplet (nucleotides 266 through 268) of the hexanucleotide apical loop of stem loop IIId is essential for IRES activity both in vitro and in vivo. Sequence comparison showed that apical loop nucleotides (UUGGGU) were absolutely conserved across HCV genotypes and the GGG triplet was strongly conserved among related Flavivirus andPestivirus nontranslated regions. Chimeric IRES elements with IIId derived from GB virus B (GBV-B) in the context of the HCV IRES possess translational activity. Mutations within the IIId stem loop that abolish IRES activity also affect the RNA structure in RNase T1-probing studies, demonstrating the importance of correct RNA folding to IRES function.

scholarly journals Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements

2001 ◽  
Vol 21 (8) ◽  
pp. 2826-2837 ◽  
Author(s):  
Arun Venkatesan ◽  
Asim Dasgupta
Keyword(s):  
Internal Ribosome Entry Site ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Independent Manner ◽  
Internal Ribosome Entry

ABSTRACT We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.

scholarly journals Mutational Analysis of the GB Virus B Internal Ribosome Entry Site

2000 ◽  
Vol 74 (2) ◽  
pp. 773-783 ◽  
Author(s):  
René Rijnbrand ◽  
Geoffrey Abell ◽  
Stanley M. Lemon
Keyword(s):  
Internal Ribosome Entry Site ◽  
Mutational Analysis ◽  
General Structure ◽  
Point Mutations ◽  
Structural Domain ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry

ABSTRACT GB virus B (GBV-B) is a recently discovered hepatotropic flavivirus that is distantly related to hepatitis C virus (HCV). We show here that translation of its polyprotein is initiated by internal entry of ribosomes on GBV-B RNA. We analyzed the translational activity of dicistronic RNA transcripts containing wild-type or mutated 5′ nontranslated GBV-B RNA (5′NTR) segments, placed between the coding sequences of two reporter proteins, in vitro in rabbit reticulocyte lysate and in vivo in transfected BT7-H cells. We related these results to a previously proposed model of the secondary structure of the GBV-B 5′NTR (M. Honda, et al. RNA 2:955–968, 1996). We identified an internal ribosome entry site (IRES) bounded at its 5′ end by structural domain II, a location analogous to the 5′ limit of the IRES in both the HCV and pestivirus 5′NTRs. Mutational analysis confirmed the structure proposed for domain II of GBV-B RNA, and demonstrated that optimal IRES-mediated translation is dependent on each of the putative RNA hairpins in this domain, including two stem-loops not present in the HCV or pestivirus structures. IRES activity was also absolutely dependent on (i) phylogenetically conserved, adenosine-containing bulge loops in domain III and (ii) the primary nucleotide sequence of stem-loop IIIe. IRES-directed translation was inhibited by a series of point mutations predicted to stabilize stem-loop IV, which contains the initiator AUG codon in its loop segment. A reporter gene was translated most efficiently when fused directly to the initiator AUG codon, with no intervening downstream GBV-B sequence. This finding indicates that the 3′ limit of the GBV-B IRES is at the initiator AUG and that it does not require downstream polyprotein-coding sequence as suggested for the HCV IRES. These results show that the GBV-B IRES, while sharing a common general structure, differs both structurally and functionally from other flavivirus IRES elements.

scholarly journals La Autoantigen Is Necessary for Optimal Function of the Poliovirus and Hepatitis C Virus Internal Ribosome Entry Site In Vivo and In Vitro

2004 ◽  
Vol 24 (15) ◽  
pp. 6861-6870 ◽  
Author(s):  
Mauro Costa-Mattioli ◽  
Yuri Svitkin ◽  
Nahum Sonenberg
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Ribosomal Subunit ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5′ untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.

Splicing signals and factors in plant intron removal

2002 ◽  
Vol 30 (2) ◽  
pp. 146-149 ◽  
Author(s):  
J. W. S. Brown ◽  
C. G. Simpson ◽  
G. Thow ◽  
G. P. Clark ◽  
S. N. Jennings ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Viral Vectors ◽  
Binding Proteins ◽  
Fluorescent Protein ◽  
Splicing Factors ◽  
Small Nuclear Rna ◽  
Intron Removal ◽  
Arabidopsis Proteins ◽  
Green Fluorescent

Constitutive splicing of the potato invertase miniexon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5′ splice site of the adjacent intron and a U11 element found just downstream of the branchpoint in the upstream intron [Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422–433]. The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences. Plant introns differ from their vertebrate and yeast couterparts in being UA- or U-rich (up to 85% UA). One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins. We are adopting three approaches to studying the RNA-protein interactions in plant splicing. First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants. Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts. Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened.

scholarly journals Sequence variability at the internal ribosome entry site of the HCV genome in relation to therapy outcome

2009 ◽  
Vol 61 (2) ◽  
pp. 205-212
Author(s):  
Snezana Jovanovic-Cupic ◽  
Jasmina Simonovic-Babic ◽  
Jelena Blagojevic ◽  
Milena Bozic ◽  
Rada Jesic ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Response To Therapy ◽  
Therapy Outcome ◽  
Sequence Variability ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Different Types ◽  
Genotype 3A

Different types of interferon are widely used to treat hepatitis C virus (HCV) infection. Results obtained in vitro suggest that interferon inhibits internal ribosome entry site (IRES)-mediated translation of the HCV genome. To elucidate the possible effect of the nucleotide sequence of IRES on therapy outcome, we compared HCV isolates from patients with sustained response and non-response to interferon/ribavirin combination therapy. In 56 analyzed HCV isolates, nucleotide changes appeared strictly in the stem-loop IIIb region, the stem part from 243 nt to 248 nt, and the polypyrimidine-II region. The natural sequence variability of IRES in isolates of genotype 3a was significantly higher than in isolates of genotype 1b (p < 0.05). The average number of nucleotide changes in genotype 3a correlated with response to therapy (p < 0.05).

Long-range RNA–RNA interactions between distant regions of the hepatitis C virus internal ribosome entry site element

2002 ◽  
Vol 83 (5) ◽  
pp. 1113-1121 ◽  
Author(s):  
Esther Lafuente ◽  
Ricardo Ramos ◽  
Encarnación Martínez-Salas
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Long Range ◽  
Internal Ribosome Entry Site ◽  
Translation Efficiency ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Hcv Ires ◽  
Rna Complexes

Efficient internal initiation of translation from the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires sequences of domain II, but the precise role of these sequences is still unknown. In this study, the formation of RNA–RNA complexes in the HCV IRES was evaluated. Using transcripts that contain the sequences of the structural HCV IRES domains II, IIIabcd, IIIabc, IV and IIIef-IV, specific long-range interactions between domains II and IV, as well as domains II and IIIabcd, have been found. These interactions were readily detected in a gel mobility-shift assay and required the presence of magnesium ions. A high concentration of nonspecific competitors, an 80 nt fragment of 18S rRNA or poly(I:C), did not interfere with the formation of RNA complexes. Interestingly, an RNA oligonucleotide bearing the sequence of stem–loop IIId interacted with domain II but not with domain IV or IIIef-IV, strongly suggesting that the interaction between domains II and IIIabcd was mediated by the IIId hairpin. Interaction between domains IIIabcd and IV was barely detected, consistent with the result that the apical part of domain III folds independently of the rest of the IRES. Moreover, the addition of stem–loop IIIef sequences to domain IV significantly reduced its ability to interact, which is in agreement with the formation of a compact RNA structure of domain IV with IIIef. The interactions observed in the absence of proteins between domains II and IV as well as stem–loop IIId and domain II may be transient, having a regulatory role in the translation efficiency of the HCV IRES.

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