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 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 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 Influence of the Theiler's Virus L∗ Protein on Macrophage Infection, Viral Persistence, and Neurovirulence

2000 ◽  
Vol 74 (19) ◽  
pp. 9071-9077 ◽  
Author(s):  
Olivier van Eyll ◽  
Thomas Michiels
Keyword(s):  
Viral Persistence ◽  
Initiation Codon ◽  
Divergent Evolution ◽  
Theiler's Virus ◽  
Macrophage Infection ◽  
Reading Frame ◽  
L Protein ◽  
Related Clone ◽  
Weak Influence

ABSTRACT The genome of picornaviruses contains a large open reading frame (ORF) translated as a precursor polypeptide that is processed to yield all the proteins necessary for the viral life cycle. In persistent but not in neurovirulent strains of Theiler's virus, an overlapping ORF encodes an additional 18-kDa protein called L∗. We confirmed previous work showing that the L∗ ORF of persistent strains facilitates the infection of macrophage cell lines, and we present evidence that this effect is due to the L∗ protein itself rather than to competition for the translation of the two overlapping ORFs. The introduction of an AUG codon to restore the L∗ ORF of the neurovirulent GDVII strain also enhanced the infection of macrophages, in spite of the divergent evolution of this protein. The presence or the absence of the L∗ AUG initiation codon had only a weak influence on the neurovirulence of the GDVII strain and on the persistence of the DA1 strain. The results obtained with DA1 in vivo contrast with the results reported previously for DAFL3, another molecular clone of the same virus strain, where the AUG-to-ACG mutation of the L∗ initiation codon totally blocked viral persistence (G. D. Ghadge, L. Ma, S. Sato, J. Kim, and R. P. Roos, J. Virol. 72:8605–8612, 1998). Thus, a factor that is critical for the persistence of a given clone of Theiler's virus is dispensable for the persistence of a closely related clone, indicating that different adjustments in the expression of persistence determinants occur in related viral strains.

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.

Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants

1986 ◽  
Vol 6 (7) ◽  
pp. 2704-2711 ◽  
Author(s):  
D S Peabody ◽  
S Subramani ◽  
P Berg
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Initiation Codon ◽  
Efficient Production ◽  
Translation Efficiency ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
Internal Translation ◽  
Late Region

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.

A Novel Mechanism in Generation of Self-Antigen Repertoire - An Unconventional Antigen Translated by a Novel Internal Ribosome Entry Site Elicits Anti-Tumor Humoral Immune Responses.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3702-3702
Author(s):  
Xiao-Feng Yang ◽  
Zeyu Xiong ◽  
Enli Liu ◽  
Yan Yan ◽  
Richard T. Silver ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Internal Ribosome Entry Site ◽  
Tumor Antigens ◽  
Genomic Structure ◽  
Myelogenous Leukemia ◽  
Human Testis ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Reading Frame ◽  
Self Antigen

Abstract Self-tumor antigens that elicit anti-tumor immune/autoimmune responses in responses to interferon-a (IFN-a) stimulation remain poorly defined. We screened a human testis cDNA library with sera from three polycythemia vera (PV) patients who responded to IFN-a and identified a novel antigen, MPD6. MPD6 belongs to the group of cryptic antigens without conventional genomic structure and is encoded by a cryptic open reading frame located in the 3′untranslated region (3′UTR) of myotrophin mRNA. MPD6 elicits IgG antibody responses in a subset of PV patients, as well as patients with chronic myelogenous leukemia and prostate cancer, suggesting that it is broadly immunogenic. The expression of myotrophin-MPD6 transcripts in tumor cells was upregulated in some tumor cells, but only slightly increased in K562 cells in response to IFN-a treatment. By using bicistronic reporter constructs, we showed that the translation of MPD6 was mediated by a novel internal ribosome entry site (IRES) upstream of the MPD6 reading frame. Furthermore, the MPD6-IRES mediated translation, but not myotrophin-MPD6 transcription, was significantly upregulated in response to IFN-a stimulation. These findings demonstrate for the first time that a novel IRES-mediated mechanism is responsible for the translation of unconventional self-antigen MPD6 in responsive to IFN-a stimulation. The eliciting anti-tumor immune response against unconventional antigen MPD6 in patients with myeloproliferative diseases suggests MPD6 as a potential target of novel immunotherapy. Our results also suggest that the enhancement of IRES-mediated translation of unconventional self-antigen(s) by IFN-a is a novel mechanism of IFN-a enhanced anti-tumor immune/autoimmune responses.

scholarly journals The Majority of Infiltrating CD8+ T Cells in the Central Nervous System of Susceptible SJL/J Mice Infected with Theiler's Virus Are Virus Specific and Fully Functional

2002 ◽  
Vol 76 (13) ◽  
pp. 6577-6585 ◽  
Author(s):  
Bong-Su Kang ◽  
Michael A. Lyman ◽  
Byung S. Kim
Keyword(s):  
Central Nervous System ◽  
T Cells ◽  
Nervous System ◽  
T Cell ◽  
Demyelinating Disease ◽  
Class I ◽  
Theiler's Virus ◽  
Ctl Response ◽  
The Central Nervous System ◽  
Ifn Γ

ABSTRACT Theiler's virus infection of the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains, such as SJL/J, and serves as a relevant infectious model for human multiple sclerosis. It has been previously suggested that susceptible SJL/J mice do not mount an efficient cytotoxic T-lymphocyte (CTL) response to the virus. In addition, genetic studies have shown that resistance to Theiler's virus-induced demyelinating disease is linked to the H-2D major histocompatibility complex class I locus, suggesting that a compromised CTL response may contribute to the susceptibility of SJL/J mice. Here we show that SJL/J mice do, in fact, generate a CD8+ T-cell response in the CNS that is directed against one dominant (VP3159-166) and two subdominant (VP111-20 and VP3173-181) capsid protein epitopes. These virus-specific CD8+ T cells produce gamma interferon (IFN-γ) and lyse target cells in the presence of the epitope peptides, indicating that these CNS-infiltrating CD8+ T cells are fully functional effector cells. Intracellular IFN-γ staining analysis indicates that greater than 50% of CNS-infiltrating CD8+ T cells are specific for these viral epitopes at 7 days postinfection. Therefore, the susceptibility of SJL/J mice is not due to the lack of an early functional Theiler's murine encephalomyelitis virus-specific CTL response. Interestingly, T-cell responses to all three epitopes are restricted by the H-2Ks molecule, and this skewed class I restriction may be associated with susceptibility to demyelinating disease.

scholarly journals Translation Initiation at the CUU Codon Is Mediated by the Internal Ribosome Entry Site of an Insect Picorna-Like Virus In Vitro

1999 ◽  
Vol 73 (2) ◽  
pp. 1219-1226 ◽  
Author(s):  
Jun Sasaki ◽  
Nobuhiko Nakashima
Keyword(s):  
Capsid Protein ◽  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Protein Gene ◽  
Initiation Codon ◽  
Coding Region ◽  
Entry Site ◽  
Capsid Protein Gene ◽  
Internal Ribosome Entry

ABSTRACT AUG-unrelated translation initiation was found in an insect picorna-like virus, Plautia stali intestine virus (PSIV). The positive-strand RNA genome of the virus contains two nonoverlapping open reading frames (ORFs). The capsid protein gene is located in the 3′-proximal ORF and lacks an AUG initiation codon. We examined the translation mechanism and the initiation codon of the capsid protein gene by using various dicistronic and monocistronic RNAs in vitro. The capsid protein gene was translated cap independently in the presence of the upstream cistron, indicating that the gene is translated by internal ribosome entry. Deletion analysis showed that the internal ribosome entry site (IRES) consisted of approximately 250 bases and that its 3′ boundary extended slightly into the capsid-coding region. The initiation codon for the IRES-mediated translation was identified as the CUU codon, which is located just upstream of the 5′ terminus of the capsid-coding region by site-directed mutagenesis. In vitro translation assays of monocistronic RNAs lacking the 5′ part of the IRES showed that this CUU codon was not recognized by scanning ribosomes. This suggests that the PSIV IRES can effectively direct translation initiation without stable codon-anticodon pairing between the initiation codon and the initiator methionyl-tRNA.

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