scholarly journals The internal ribosome entry site of the Dengue virus mRNA is active when cap-dependent translation initiation is inhibited.

2020 ◽  
Author(s):  
Leandro Fernández-García ◽  
Jenniffer Angulo ◽  
Hade Ramos ◽  
Aldo Barrera ◽  
Karla Pino ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Dengue Virus ◽  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Mouth Disease Virus ◽  
Independent Mechanism ◽  
In Cells

Dengue virus (DENV) is an enveloped, positive-sense, single-stranded RNA virus belonging to the Flaviviridae family. Translation initiation of the DENV mRNA can occur following a cap-dependent or a cap-independent mechanism. Two non-mutually exclusive cap-independent mechanisms of translation initiation have been described for the DENV mRNA. The first corresponds to a 5′end-dependent internal ribosome entry site (IRES)-independent mechanism, while the second relies on IRES-dependent initiation. In this report, we study the recently discovered DENV IRES. Results show that the DENV IRES is functional in the rabbit reticulocyte (RRL) in vitro translation system. In accordance, the activity of DENV IRES was resistant to the cleavage of eIF4G by the Foot-and-mouth disease virus leader protease in RRL. In cells, the DENV IRES exhibited only a marginal activity under standard culture conditions. The DENV IRES showed weak activity in HEK 293T cells; however, the DENV IRES activity was significantly enhanced in HEK 293T cells expressing the Human rhinovirus 2A protease. These findings suggest that the DENV IRES enables viral protein synthesis under conditions that suppress canonical translation initiation. IMPORTANCE Dengue virus (DENV), the etiological agent of Dengue, a febrile and hemorrhagic disease, infects millions of people per year in tropical and subtropical countries. When infecting cells, DENV induces stress conditions known to inhibit canonical protein synthesis. Under these conditions, DENV mRNA thrives using non-canonical modes of translation initiation. In this study, we characterize the mechanism dependent upon an internal ribosome entry site (IRES). Herein, we describe the activity of the DENV IRES in vitro and cells. We show that in cells, DENV IRES enables the viral mRNA to translate under conditions that suppress canonical translation initiation.

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.

scholarly journals Interaction of Eukaryotic Initiation Factor eIF4B with the Internal Ribosome Entry Site of Foot-and-Mouth Disease Virus Is Independent of the Polypyrimidine Tract-Binding Protein

1999 ◽  
Vol 73 (7) ◽  
pp. 6111-6113 ◽  
Author(s):  
René C. Rust ◽  
Kerstin Ochs ◽  
Karsten Meyer ◽  
Ewald Beck ◽  
Michael Niepmann
Keyword(s):  
Disease Virus ◽  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Foot And Mouth Disease ◽  
Initiation Factor ◽  
Polypyrimidine Tract ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding Protein ◽  
Mouth Disease Virus

ABSTRACT Eukaryotic translation initiation factor 4B (eIF4B) binds directly to the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV). Mutations in all three subdomains of the IRES stem-loop 4 reduce binding of eIF4B and translation efficiency in parallel, indicating that eIF4B is functionally involved in FMDV translation initiation. In reticulocyte lysate devoid of polypyrimidine tract-binding protein (PTB), eIF4B still bound well to the wild-type IRES, even after removal of the major PTB-binding site. In conclusion, the interaction of eIF4B with the FMDV IRES is essential for IRES function but independent of PTB.

scholarly journals Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Proteinase Inhibits Viral Internal Ribosome Entry Site-Mediated Translation

2008 ◽  
Vol 82 (19) ◽  
pp. 9389-9399 ◽  
Author(s):  
Jennifer M. Bonderoff ◽  
Jennifer L. LaRey ◽  
Richard E. Lloyd
Keyword(s):  
Internal Ribosome Entry Site ◽  
Binding Protein ◽  
Virus Protein ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
Polypyrimidine Tract Binding Protein ◽  
In Cells

ABSTRACT The two enteroviral proteinases, 2A proteinase (2Apro) and 3C proteinase (3Cpro), induce host cell translation shutoff in enterovirus-infected cells by cleaving canonical translation initiation factors. Cleavage of poly(A)-binding protein (PABP) by 3Cpro has been shown to be a necessary component for host translation shutoff. Here we show that 3Cpro inhibits cap-independent translation mediated by the poliovirus internal ribosome entry site (IRES) in a dose-dependent manner in HeLa translation extracts displaying cap-poly(A) synergy. This effect is independent of the stimulatory effect of 2Apro on IRES translation, and 3Cpro-induced translation inhibition can be partially rescued by addition of recombinant PABP in vitro. 3Cpro inhibits IRES translation on transcripts containing or lacking poly(A) tails, suggesting that cleavage of PABP and IRES trans-activating factors polypyrimidine tract-binding protein and poly r(C)-binding protein 2 may also be important for inhibition. Expression of 3Cpro cleavage-resistant PABP in cells increased translation of nonreplicating viral minigenome reporter RNAs during infection and also delayed and reduced virus protein synthesis from replicating RNA. Further, expression of cleavage-resistant PABP in cells reduced the accumulation of viral RNA and the output of infectious virus. These results suggest that cleavage of PABP contributes to viral translation shutoff that is required for the switch from translation to RNA replication.

scholarly journals Translation Initiation Factor eIF4B Interacts with a Picornavirus Internal Ribosome Entry Site in both 48S and 80S Initiation Complexes Independently of Initiator AUG Location

1999 ◽  
Vol 73 (9) ◽  
pp. 7505-7514 ◽  
Author(s):  
Kerstin Ochs ◽  
RenéC. Rust ◽  
Michael Niepmann
Keyword(s):  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Entry Site ◽  
Small Ribosomal Subunit ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
Mouth Disease Virus ◽  
Energy Dependent

ABSTRACT Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation.

scholarly journals Unique Characteristics of a Picornavirus Internal Ribosome Entry Site from the Porcine Teschovirus-1 Talfan

2002 ◽  
Vol 76 (22) ◽  
pp. 11721-11728 ◽  
Author(s):  
Yoshihiro Kaku ◽  
Louisa S. Chard ◽  
Toru Inoue ◽  
Graham J. Belsham
Keyword(s):  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Mammalian Cells ◽  
Polypyrimidine Tract ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
New Class ◽  
Porcine Teschovirus ◽  
Ires Element

ABSTRACT The teschoviruses constitute a recently defined picornavirus genus. Most of the genome sequence of the porcine teschovirus-1 (PTV) Talfan and several other strains is known. We now demonstrate that initiation of protein synthesis occurs at nucleotide (nt) 412 on the PTV Talfan RNA and that nt 1 to 405 contains an internal ribosome entry site (IRES) that functions efficiently in vitro and within mammalian cells. In comparison with other picornavirus IRES elements, the PTV IRES is relatively short and lacks a significant polypyrimidine tract near the 3′ end. Expression of an enterovirus 2A protease, which induces cleavage of eIF4G within the translation initiation complex eIF4F, has little effect on the PTV IRES activity within BHK cells. The PTV IRES has a unique set of properties and represents a new class of picornavirus IRES element.

scholarly journals In vivo footprint of a picornavirus internal ribosome entry site reveals differences in accessibility to specific RNA structural elements

2007 ◽  
Vol 88 (11) ◽  
pp. 3053-3062 ◽  
Author(s):  
Olga Fernández-Miragall ◽  
Encarnación Martínez-Salas
Keyword(s):  
Internal Ribosome Entry Site ◽  
Living Cells ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Essential Information ◽  
Mouth Disease Virus ◽  
Ires Element

Internal ribosome entry site (IRES) elements were described in picornaviruses as an essential region of the viral RNA. Understanding of IRES function requires a detailed knowledge of each step involved in the internal initiation process, from RNA folding and IRES–protein interaction to ribosome recruitment. Thus, deciphering IRES accessibility to external agents due to RNA structural features, as well as RNA–protein protection within living cells, is of primary importance. In this study, two chemical reagents, dimethylsulfate (DMS) and aminomethylpsoralen, have been used to footprint the entire IRES of foot-and-mouth disease virus (FMDV) in living cells; these reagents enter the cell membrane and interact with nucleic acids in a structure-dependent manner. For FMDV, as in other picornaviruses, viral infection is dependent on the correct function of the IRES; therefore, the IRES region itself constitutes a useful target of antiviral drugs. Here, the in vivo footprint of a picornavirus IRES element in the context of a biologically active mRNA is shown for the first time. The accessibility of unpaired adenosine and cytosine nucleotides in the entire FMDV IRES was first obtained in vitro by DMS probing; subsequently, this information was used to interpret the footprint data obtained in vivo for the mRNA encompassing the IRES element in the intercistronic space. The results of DMS accessibility and UV–psoralen cross-linking studies in the competitive cellular environment provided evidence for differences in RNA structure from data obtained in vitro, and provided essential information to identify appropriate targets within the FMDV IRES aimed at combating this important pathogen.

Sign in / Sign up

Export Citation Format

Share Document