scholarly journals IRES-mediated ribosome repositioning directs translation of a +1 overlapping ORF that enhances viral infection

2018 ◽  
Author(s):  
Craig H Kerr ◽  
Qing S Wang ◽  
Kyung-Mee Moon ◽  
Kathleen Keatings ◽  
Douglas W Allan ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Stop Codon ◽  
Open Reading Frame ◽  
Wild Type Virus ◽  
Infection Model ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Reading Frame ◽  
Coding Capacity ◽  
Paralysis Virus

AbstractRNA structures can interact with the ribosome to alter translational reading frame maintenance and promote recoding that result in alternative protein products. Here, we show that the internal ribosome entry site (IRES) from the dicistrovirus Cricket paralysis virus drives translation of the 0-frame viral polyprotein and an overlapping +1 open reading frame, called ORFx, via a novel mechanism whereby a subset of ribosomes recruited to the IRES bypasses downstream to resume translation at the +1-frame 13th non-AUG codon. A mutant of CrPV containing a stop codon in the +1 frame ORFx sequence, yet synonymous in the 0-frame, is attenuated compared to wild-type virus in a Drosophila infection model, indicating the importance of +1 ORFx expression in promoting viral pathogenesis. This work demonstrates a novel programmed IRES-mediated recoding strategy to increase viral coding capacity and impact virus infection, highlighting the diversity of RNA-driven translation initiation mechanisms in eukaryotes.Significance StatementViruses use alternate mechanisms to increase the coding capacity of their viral genomes. Here, we provide biochemical evidence that ribosomes recruited to the dicistrovirus cricket paralysis virus IRES undergo a bypass event to direct translation of a downstream +1 frame overlapping open reading frame, called ORFx. Mutations that block ORFx expression inhibit +1 frame translation and infection in fruit flies. These findings highlight the diversity of RNA-driven translation initiation mechanisms in eukaryotes.

scholarly journals Ctk1 Function Is Necessary for Full Translation Initiation Activity in Saccharomyces cerevisiae

2014 ◽  
Vol 14 (1) ◽  
pp. 86-95 ◽  
Author(s):  
Britta Coordes ◽  
Katharina M. Brünger ◽  
Kaspar Burger ◽  
Boumediene Soufi ◽  
Juliane Horenk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Elongation Factor ◽  
Entry Site ◽  
Translation Elongation ◽  
Internal Ribosome Entry ◽  
Content Type ◽  
Phosphorylation Pattern ◽  
Subunit Joining ◽  
Paralysis Virus

ABSTRACT Translation is a fundamental and highly regulated cellular process. Previously, we reported that the kinase and transcription elongation factor Ctk1 increases fidelity during translation elongation in Saccharomyces cerevisiae . Here, we show that loss of Ctk1 function also affects the initiation step of translation. Translation active extracts from Ctk1-depleted cells show impaired translation activity of capped mRNA, but not mRNA reporters containing the cricket paralysis virus (CrPV) internal ribosome entry site (IRES). Furthermore, the formation of 80S initiation complexes is decreased, which is probably due to reduced subunit joining. In addition, we determined the changes in the phosphorylation pattern of a ribosome enriched fraction after depletion of Ctk1. Thus, we provide a catalogue of phosphoproteomic changes dependent on Ctk1. Taken together, our data suggest a stimulatory function of Ctk1 in 80S formation during translation initiation.

scholarly journals Replication of Poliovirus RNA with Complete Internal Ribosome Entry Site Deletions

2004 ◽  
Vol 78 (3) ◽  
pp. 1393-1402 ◽  
Author(s):  
Kenneth E. Murray ◽  
Benjamin P. Steil ◽  
Allan W. Roberts ◽  
David J. Barton
Keyword(s):  
Internal Ribosome Entry Site ◽  
Mrna Translation ◽  
Rna Replication ◽  
Open Reading Frame ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Viral Rnas ◽  
Reading Frame ◽  
Nontranslated Region ◽  
Positive Strand Rna

ABSTRACT cis-acting RNA sequences and structures in the 5′ and 3′ nontranslated regions of poliovirus RNA interact with host translation machinery and viral replication proteins to coordinately regulate the sequential translation and replication of poliovirus RNA. The poliovirus internal ribosome entry site (IRES) in the 5′ nontranslated region (NTR) has been implicated as a cis-active RNA required for both viral mRNA translation and viral RNA replication. To evaluate the role of the IRES in poliovirus RNA replication, we exploited the advantages of cell-free translation-replication reactions and preinitiation RNA replication complexes. Genetic complementation with helper mRNAs allowed us to create preinitiation RNA replication complexes containing RNA templates with defined deletions in the viral open reading frame and the IRES. A series of deletions revealed that no RNA elements of either the viral open reading frame or the IRES were required in cis for negative-strand RNA synthesis. The IRES was dispensable for both negative- and positive-strand RNA syntheses. Intriguingly, although small viral RNAs lacking the IRES replicated efficiently, the replication of genome length viral RNAs was stimulated by the presence of the IRES. These results suggest that RNA replication is not directly dependent on a template RNA first functioning as an mRNA. These results further suggest that poliovirus RNA replication is not absolutely dependent on any protein-RNA interactions involving the IRES.

scholarly journals Regulation of Internal Ribosome Entry Site-mediated Translation by Eukaryotic Initiation Factor-2α Phosphorylation and Translation of a Small Upstream Open Reading Frame

2001 ◽  
Vol 277 (3) ◽  
pp. 2050-2058 ◽  
Author(s):  
James Fernandez ◽  
Ibrahim Yaman ◽  
William C. Merrick ◽  
Antonis Koromilas ◽  
Ronald C. Wek ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Open Reading Frame ◽  
Upstream Open Reading Frame ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Reading Frame

scholarly journals Alternative Translation Initiation of Theiler’s Murine Encephalomyelitis Virus

1999 ◽  
Vol 73 (10) ◽  
pp. 8519-8526 ◽  
Author(s):  
Kenji Yamasaki ◽  
Conrad C. Weihl ◽  
Raymond P. Roos
Keyword(s):  
Translation Initiation ◽  
Demyelinating Disease ◽  
Stop Codon ◽  
Initiation Codon ◽  
Site Mutation ◽  
Entry Site ◽  
Theiler's Murine Encephalomyelitis Virus ◽  
Reading Frame ◽  
Theiler’S Murine Encephalomyelitis Virus ◽  
Encephalomyelitis Virus

ABSTRACT DA strain and other members of the TO subgroup of Theiler’s murine encephalomyelitis virus (TMEV) produce a chronic demyelinating disease in which the virus persists but has a restricted expression. We previously reported that TO subgroup strains, in addition to synthesizing the picornaviral polyprotein, use an alternative initiation codon just downstream from the polyprotein’s AUG to translate an 18-kDa protein called L* that is out of frame with the polyprotein (H. H. Chen et al., Nat. Med. 1:927–931, 1995; W. P. Kong and R. P. Roos, J. Virol. 65:3395–3399, 1991). L* is critically important for virus persistence and the induction of the demyelinating disease (Chen et al., 1995; G. D. Ghadge et al. J. Virol. 72:8605–8612, 1998). We have proposed that variations in the amount of translation initiation from the L* AUG versus the polyprotein AUG may occur in different cell types and therefore affect the degree of expression of viral capsid proteins. We now demonstrate that ribosomal translation initiation at the polyprotein’s initiation codon affects initiation at the L* AUG, suggesting that ribosomes land at the polyprotein’s initiation codon before scanning downstream and initiating at the L* AUG. We also find that the viral 5′ untranslated region affects utilization of the L* AUG. Surprisingly, mutant DA cDNAs were found to be infectious despite the presence of mutations of the polyprotein initiation codon or placement of a stop codon upstream of the L* AUG in the polyprotein’s reading frame. Sequencing studies showed that these viruses had a second site mutation, converting the reading frame of L* into the polyprotein’s reading frame; the results suggest that translation of the polyprotein during infection of these mutant viruses can be initiated at the L* AUG. These data are important in our understanding of translation initiation of TMEV and other RNAs that contain an internal ribosome entry site.

scholarly journals Non-AUG-Initiated Internal Translation of the L* Protein of Theiler's Virus and Importance of This Protein for Viral Persistence

2002 ◽  
Vol 76 (21) ◽  
pp. 10665-10673 ◽  
Author(s):  
Olivier van Eyll ◽  
Thomas Michiels
Keyword(s):  
Demyelinating Disease ◽  
Open Reading Frame ◽  
Initiation Codon ◽  
Theiler's Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Reading Frame ◽  
L Protein ◽  
The Central Nervous System ◽  
Internal Translation

ABSTRACT Theiler's virus is a neurotropic murine picornavirus which, depending on the strain, causes either acute encephalitis or persistent demyelinating disease. Persistent strains of Theiler's virus (such as DA) produce an 18-kDa protein called L* from an open reading frame overlapping that encoding the viral polyprotein. Neurovirulent strains (such as GDVII) are thought not to produce the L* protein, as the alternative open reading frame of these strains starts with an ACG codon instead of an AUG codon. However, we observed that both persistent and neurovirulent strain derivatives can produce two forms of the L* protein through unusual type II internal ribosome entry site-mediated translation. A full-length 18-kDa protein can be expressed from an ACG or an AUG initiation codon, whereas an N-terminally truncated 15-kDa product can be translated from a downstream AUG initiation codon. The expression of the 18-kDa form is required for efficient persistence of DA virus derivatives in the central nervous system.

scholarly journals Vaccinia Virus Encodes I5, a Small Hydrophobic Virion Membrane Protein That Enhances Replication and Virulence in Mice

2008 ◽  
Vol 82 (20) ◽  
pp. 10071-10078 ◽  
Author(s):  
Cindy L. Sood ◽  
Jerrold M. Ward ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Stop Codon ◽  
Open Reading Frame ◽  
Wild Type Virus ◽  
Epidermal Keratinocytes ◽  
In Vitro Cultivation ◽  
Human Epidermal Keratinocytes ◽  
Reading Frame ◽  
Control Virus

ABSTRACT The vaccinia virus I5L open reading frame encodes a 79-amino-acid protein, with two predicted transmembrane domains, that is conserved among all sequenced members of the chordopoxvirus subfamily. No nonpoxvirus homologs or functional motifs have been recognized, and the role of the I5 protein remains unknown. We found that synthesis of I5 was dependent on viral DNA replication and occurred exclusively at late times, consistent with a consensus late promoter motif adjacent to the start of the open reading frame. I5 was present in preparations of purified virions and could be extracted with nonionic detergent, suggesting membrane insertion. Transmission electron microscopy of immunogold-labeled thawed cryosections of infected cells revealed the association of an epitope-tagged I5 with the membranes of immature and mature virions. Viable I5L deletion and frameshift mutants were constructed and found to replicate like wild-type virus in a variety of cell lines and primary human epidermal keratinocytes, indicating that the protein was dispensable for in vitro cultivation. However, mouse intranasal challenge experiments indicated that a mutant virus with a frameshift resulting in a stop codon near the N terminus of I5 was attenuated compared to control virus. The attenuation was correlated with clearance of mutant viruses from the respiratory tract and with less progression and earlier resolution of pathological changes. We suggest that I5 is involved in an aspect of host defense that is evolutionarily conserved although a role in cell tropism should also be considered.

scholarly journals Translation of a minigene in the 5′ leader sequence of the enterohaemorrhagic Escherichia coli LEE1 transcription unit affects expression of the neighbouring downstream gene

2011 ◽  
Vol 441 (1) ◽  
pp. 247-253 ◽  
Author(s):  
Md. Shahidul Islam ◽  
Robert K. Shaw ◽  
Gad Frankel ◽  
Mark J. Pallen ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Translation Initiation ◽  
Stop Codon ◽  
Leader Sequence ◽  
Open Reading Frame ◽  
Start Codon ◽  
Target Cells ◽  
Reading Frame ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Translation Start

The 5′ end of the major RNA transcript of the LEE1 operon of enterohaemorrhagic Escherichia coli contains ~170 bases before the AUG translation start codon of the first recognized gene, ler. This unusually long leader sequence carries three potential alternative AUG start codons. Using a lac fusion expression vector, we confirmed that the ler gene AUG is functional for translation initiation, and we checked for translation initiation at the three alternative AUG codons. Whereas two of the alternative AUG codons appear incompetent for translation initiation, we detected strong initiation at the third AUG, which is followed by one AAA codon and a UAG stop codon. The location of this very short two-codon open reading frame with respect to the ler translation start appears to be critical. Hence mutations that destroy the UAG stop codon, or short deletions between the UAG stop codon and the ler translation initiation region, result in big effects on ler expression. In the context of the full-length LEE1 operon leader sequence, translation of this very short two-codon open reading frame is necessary for optimal expression of the ler gene and for the subsequent interactions of enterohaemorrhagic Escherichia coli with host target cells.

scholarly journals Polyamine-Responsive Ribosomal Arrest at the Stop Codon of an Upstream Open Reading Frame of the AdoMetDC1 Gene Triggers Nonsense-Mediated mRNA Decay in Arabidopsis thaliana

2014 ◽  
Vol 55 (9) ◽  
pp. 1556-1567 ◽  
Author(s):  
Naoko Uchiyama-Kadokura ◽  
Karin Murakami ◽  
Mariko Takemoto ◽  
Naoto Koyanagi ◽  
Katsunori Murota ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Mrna Decay ◽  
Stop Codon ◽  
Open Reading Frame ◽  
Upstream Open Reading Frame ◽  
Reading Frame ◽  
Nonsense Mediated Mrna Decay

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.

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