scholarly journals Mechanism of Initiation Site Selection Promoted by the Human Rhinovirus 2 Internal Ribosome Entry Site

2010 ◽  
Vol 84 (13) ◽  
pp. 6578-6589 ◽  
Author(s):  
Ann Kaminski ◽  
Tuija A. A. Pöyry ◽  
Peter J. Skene ◽  
Richard J. Jackson
Keyword(s):  
Internal Ribosome Entry Site ◽  
Point Mutations ◽  
Apical Part ◽  
Cell Extract ◽  
Initiation Site ◽  
Human Rhinovirus ◽  
Translation Initiation Site ◽  
Base Pairing ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT Translation initiation site usage on the human rhinovirus 2 internal ribosome entry site (IRES) has been examined in a mixed reticulocyte lysate/HeLa cell extract system. There are two relevant AUG triplets, both in a base-paired hairpin structure (domain VI), with one on the 5′ side at nucleotide (nt) 576, base paired with the other at nt 611, which is the initiation site for polyprotein synthesis. A single residue was inserted in the apical loop to put AUG-576 in frame with AUG-611, and in addition another in-frame AUG was introduced at nt 593. When most of the IRES was deleted to generate a monocistronic mRNA, the use of these AUGs conformed to the scanning ribosome model: improving the AUG-576 context increased initiation at this site and decreased initiation at downstream sites, whereas the converse was seen when AUG-576 was mutated to GUA; and AUG-593, when present, took complete precedence over AUG-611. Under IRES-dependent conditions, by contrast, much less initiation occurred at AUG-576 than in a monocistronic mRNA with the same AUG-576 context, mutation of AUG-576 decreased initiation at downstream sites by ∼70%, and introduction of AUG-593 did not completely abrogate initiation at AUG-611, unless the apical base pairing in domain VI was destroyed by point mutations. These results indicate that ribosomes first bind at the AUG-576 site, but instead of initiating there, most of them are transferred to AUG-611, the majority by strictly linear scanning and a substantial minority by direct transfer, which is possibly facilitated by the occasional persistence of base pairing in the apical part of the domain VI stem.

scholarly journals The Triticum Mosaic Virus Internal Ribosome Entry Site Relies on a Picornavirus-Like YX-AUG Motif To Designate the Preferred Translation Initiation Site and To Likely Target the 18S rRNA

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Helena Jaramillo-Mesa ◽  
Megan Gannon ◽  
Elijah Holshbach ◽  
Jincan Zhang ◽  
Robyn Roberts ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
18S Rrna ◽  
Initiation Site ◽  
Upstream Region ◽  
Base Pairing ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
First Report

ABSTRACTSeveral viruses encode an internal ribosome entry site (IRES) at the 5′ end of their RNA, which, unlike most cellular mRNAs, initiates translation in the absence of a 5′ m7GpppG cap. Here, we report a uniquely regulated translation enhancer found in the 739-nucelotide (nt) sequence of the Triticum mosaic virus (TriMV) leader sequence that distinguishes the preferred initiation site from a plethora of IRES-encoded AUG triplets. Through deletion mutations of the TriMV 5′ untranslated region (UTR), we show that the TriMV 5′ UTR encodes acis-acting picornaviral Y16-X11-AUG-like motif with a 16-nt polypyrimidine CU-tract (Y16), at a precise, 11-nt distance (X11) from the preferred 13th AUG. Phylogenetic analyses indicate that this motif is conserved among potyviral leader sequences with multiple AUGs. Consistent with a broadly conserved mechanism, the motif could be functionally replaced with known picornavirus YX-AUG motifs and is predicted to function as target sites for the 18S rRNA by direct base pairing. Accordingly, mutations that disrupted overall complementarity to the 18S rRNA markedly reduced TriMV IRES activity, as did the delivery of antisense oligonucleotides designed to block YX-AUG accessibility. To our knowledge, this is the first report of a plant viral IRES YX-AUG motif, and our findings suggest that a conserved mechanism regulates translation for multiple economically important plant and animal positive single-stranded RNA viruses.IMPORTANCEUncapped viral RNAs often rely on their 5′ leader sequences to initiate translation, and the Triticum mosaic virus (TriMV) devotes an astonishing 7% of its genome to directing ribosomes to the correct AUG. Here we uncover a novel mechanism by which a TriMVcis-regulatory element controls cap-independent translation. The upstream region of the functional AUG contains a 16-nt polypyrimidine tract located 11 nt from the initiation site. Based on functional redundancy with similar motifs derived from human picornaviruses, the motif is likely to operate by directing ribosome targeting through base pairing with 18S rRNA. Our results provide the first report of a broad-spectrum mechanism regulating translation initiation for both plant- and animal-hosted picornaviruses.

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

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Yu-rong Chai ◽  
Meng-meng Ge ◽  
Ting-ting Wei ◽  
Yan-long Jia ◽  
Xiao Guo ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Transgene Expression ◽  
Human Rhinovirus ◽  
Entry Site ◽  
Internal Ribosome Entry

scholarly journals Quasispecies Changes with Distinctive Point Mutations in the Hepatitis C Virus Internal Ribosome Entry Site (IRES) Derived from PBMCs and Plasma

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Luca Mercuri ◽  
Emma C. Thomson ◽  
Joseph Hughes ◽  
Peter Karayiannis
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Rna Structure ◽  
Mononuclear Cells ◽  
Point Mutations ◽  
Pcr Cloning ◽  
Entry Site ◽  
Peripheral Blood Mononuclear ◽  
Internal Ribosome Entry

The 5’ untranslated region (UTR) of the hepatitis C virus (HCV) genome contains the internal ribosome entry site (IRES), a highly conserved RNA structure essential for cap-independent translation of the viral polyprotein. HCV, apart from the liver, is thought to be associated with lymphocyte subpopulations of peripheral blood mononuclear cells (PBMCs), in lymph nodes and brain tissue. In this study, RT-PCR, cloning, and sequence analysis were employed to investigate the quasispecies nature of the 5’UTR following extraction of viral RNA from PBMCs and plasma of HCV infected individuals. The nucleotide variation between IRES-derived sequences from PBMCs and plasma indicated the existence of polymorphic sites within the IRES. HCV isolates had divergent variants with unique mutations particularly at positions 107, 204, and 243 of the IRES. Most of the PBMC-derived sequences contained an A-A-A variant at these positions. The mutations associated with the IRESes suggested the presence of unique quasispecies populations in PBMCs compared with plasma.

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.

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