The hepatitis C virus internal ribosome-entry site: a new target for antiviral research

2002 ◽  
Vol 30 (2) ◽  
pp. 140-145 ◽  
Author(s):  
J. Gallego ◽  
G. Varani
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Entry Site ◽  
Viral Protein Synthesis ◽  
Initiation Mechanism ◽  
Internal Ribosome Entry

The hepatitis C virus (HCV) is the main causative agent of non-A, non-B hepatitis in humans and a major cause of mortality and morbidity in the world. Currently there is no effective treatment available for the infection caused by this virus, whose replication depends on an unusual translation-initiation mechanism. The viral RNA contains an internal ribosome-entry site (IRES) that is recognized specifically by the small ribosomal subunit and by eukaryotic initiation factor 3, and these interactions allow cap (7-methylguanine nucleotide)-independent initiation of viral protein synthesis. In this article, we review the structure and mechanism of translation initiation of the HCV IRES, and its potential as a target for novel antivirals.

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.

scholarly journals Hepatitis C virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast

2007 ◽  
Vol 88 (7) ◽  
pp. 1992-2002 ◽  
Author(s):  
Tomas Masek ◽  
Vaclav Vopalensky ◽  
Ondrej Horvath ◽  
Lucie Vortelova ◽  
Zuzana Feketova ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Yeast Cells ◽  
Initiation Codon ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Broad Dynamic Range ◽  
Hcv Ires

Hepatitis C virus (HCV) is an important pathogen causing both acute and chronic infections in humans. The HCV polyprotein is synthesized by cap-independent translation initiation after ribosome binding to the highly structured internal ribosome entry site (IRES). The HCV IRES has been shown to have a low requirement for translation initiation factors and the ability to bind directly to the 40S ribosomal subunit. A novel yeast bicistronic reporter system, suitable for sensitive and accurate analysis of IRES activity, has been developed. It employs signal amplification based on the Gal4p transcription factor-mediated activation of a variety of secondary reporter genes. The system has a broad dynamic range and, depending on the nature of the particular secondary reporter, can be used both for precise measurements of IRES activity and for selection and screening for novel IRES variants and IRES trans-acting factors. By using this novel bicistronic system, it was shown that the HCV IRES is functional in yeast cells. Mutational analysis of the IRES loop IV and the adjacent region revealed that, in yeast, as in mammalian cells, translation initiates preferentially at the authentic 342AUG codon and that disruption of the HCV IRES loop IV abrogates its function, whilst minor positional changes or substitutions of the initiation codon within loop IV are largely tolerated. These findings bring more general insights to translation initiation, but also open the door for utilization of yeast and its sophisticated genetics for searching for new antiviral drugs and HCV IRES trans-acting proteins.

scholarly journals Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site

2014 ◽  
Vol 112 (2) ◽  
pp. 319-325 ◽  
Author(s):  
Gabriele Fuchs ◽  
Alexey N. Petrov ◽  
Caleb D. Marceau ◽  
Lauren M. Popov ◽  
Jin Chen ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Single Molecule ◽  
Internal Ribosome Entry Site ◽  
Large Scale ◽  
Ribosomal Subunit ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ribosomal Subunits ◽  
Hcv Ires

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

scholarly journals Cytoplasmic Expression of mRNAs Containing the Internal Ribosome Entry Site and 3′ Noncoding Region of Hepatitis C Virus: Effects of the 3′ Leader on mRNA Translation and mRNA Stability

2002 ◽  
Vol 76 (24) ◽  
pp. 12457-12462 ◽  
Author(s):  
Li Kuo Kong ◽  
Peter Sarnow
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Binding Protein ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Noncoding Regions ◽  
End Sequences ◽  
End To End

ABSTRACT Translation initiation in many eukaryotic mRNAs is modulated by an interaction between the cap binding protein complex, bound to the 5′ end of the mRNA, and the polyadenosine binding protein, bound to the 3′-terminal polyadenosine sequences. A few cellular and viral mRNAs, such as the hepatitis C virus (HCV) mRNA genome, lack 3′-terminal polyadenosine sequences. For such mRNAs, the question of whether their 3′-end sequences also regulate the initiation phase of protein synthesis via an interaction with their 5′ ends has received intense scrutiny. For HCV mRNA, various experimental designs have led to conflicting interpretations, that the 3′ end of the RNA can modulate translation initiation either in a positive or in a negative fashion. To examine the possibility of end-to-end communication in HCV in detail, mRNAs containing the HCV internal ribosome entry site linked to a luciferase coding region, followed by different 3′ noncoding regions, were expressed in the cytoplasm of cultured cells by T7 RNA polymerase. The intracellular translation efficiencies, steady-state levels, stabilities, and 3′-end sequences of these chimeric RNAs were examined. It was found that the HCV 3′ noncoding region modulates neither the translation nor the stability of the mRNAs. Thus, there is no detectable end-to-end communication in cytoplasmically expressed chimeric mRNAs containing the HCV noncoding regions. However, it remains an open question whether end-to-end communication occurs in full-length HCV mRNAs in the infected liver.

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