scholarly journals N6-methyladenosine modification of HCV RNA genome regulates cap-independent IRES-mediated translation via YTHDC2 recognition

2021 ◽  
Vol 118 (10) ◽  
pp. e2022024118 ◽  
Author(s):  
Geon-Woo Kim ◽  
Aleem Siddiqui
Keyword(s):  
Translation Initiation ◽  
Mutational Analysis ◽  
Rna Helicase ◽  
Hcv Rna ◽  
Helicase Domain ◽  
Entry Site ◽  
Ires Element ◽  
Rna Genome ◽  
Hcv Ires

Hepatitis C virus (HCV) infections are associated with the risk of progression to fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV RNA genome is translated by an internal ribosome entry site (IRES)-dependent mechanism. The structure and function of the HCV IRES have been investigated by both biological and biophysical criteria. Recently, the role of N6-methyladenosine (m6A) in cellular RNA and viral transcripts has been intensely investigated. The HCV RNA genome is m6A-methylated, and this modification regulates the viral life cycle. In this study, we investigated the role of m6A modification of the HCV genome in the IRES-dependent translation function by mutating m6A consensus motifs (DRACH) within the IRES element in stem–loop III and IV regions and studied their effect on translation initiation. There are several DRACH motifs within the IRES element. Of these, the DRACH motif at nucleotide (nt) 329-333, located about 7 nt upstream of initiator AUG (iAUG) codon, regulates IRES-mediated translation initiation. Mutational analysis showed that m6A methylation of the adenosine at nt 331 is essential for the IRES-dependent translation. m6A reader protein YTHDC2, containing the RNA helicase domain, recognizes m6A-methylated adenosine at nt 331 and, in concert with the cellular La antigen, supports HCV IRES-dependent translation. The RNA helicase dead YTHDC2 (E332Q) mutant failed to stimulate HCV translation initiation. This report highlights the functional roles of m6A modification and YTHDC2 in the HCV IRES-dependent translation initiation, thus offering alternative therapeutic avenues to interfere with the infectious process.

scholarly journals Lower concentration of La protein required for internal ribosome entry on hepatitis C virus RNA than on poliovirus RNA

1999 ◽  
Vol 80 (9) ◽  
pp. 2319-2327 ◽  
Author(s):  
Takeshi Isoyama ◽  
Nobuhiko Kamoshita ◽  
Kotaro Yasui ◽  
Atsushi Iwai ◽  
Kazuko Shiroki ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Translation Initiation ◽  
Internal Ribosomal Entry Site ◽  
Translation System ◽  
Hcv Rna ◽  
Entry Site ◽  
La Protein ◽  
Reticulocyte Lysates ◽  
Hcv Ires

Translation initiation of poliovirus and hepatitis C virus (HCV) RNA occurs by entry of ribosomes to the internal RNA sequence, called the internal ribosomal entry site (IRES). Both IRES bind to the La protein and are thought to require the protein for their translation initiation activity, although they are greatly different in both the primary and predicted secondary structures. To compare the La protein requirement for these IRES, we took advantage of I-RNA from the yeast Saccharomyces cerevisiae, which has been reported to bind to La protein and block poliovirus IRES-mediated translation initiation. In a cell-free translation system prepared from HeLa cells, yeast I-RNA inhibited translation initiation on poliovirus RNA as expected, but did not significantly inhibit translation initiation on HCV RNA. However, the translation initiation directed by either IRES was apparently inhibited by I-RNA in rabbit reticulocyte lysates, in which La protein is limiting. I-RNA-mediated inhibition of HCV IRES-dependent translation in rabbit reticulocyte lysates was reversed by exogenous addition of purified recombinant La protein of smaller amounts than necessary to reverse poliovirus IRES-dependent translation. These results suggest that HCV IRES requires lower concentrations of La protein for its function than does poliovirus IRES. Immunofluorescence studies showed that HCV infection appeared not to affect the subcellular localization of La protein, which exists mainly in the nucleus, although La protein redistributed to the cytoplasm after poliovirus infection. The data are compatible with the low requirement of La protein for HCV IRES activity.

scholarly journals Differential Effects on the Hepatitis C Virus (HCV) Internal Ribosome Entry Site by Vitamin B12 and the HCV Core Protein

2004 ◽  
Vol 78 (21) ◽  
pp. 12075-12081 ◽  
Author(s):  
Dongsheng Li ◽  
William B. Lott ◽  
John Martyn ◽  
Gholamreza Haqshenas ◽  
Eric J. Gowans
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Core Protein ◽  
Vitamin B ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Hcv Core Protein ◽  
Hcv Ires

ABSTRACT To investigate the role of the hepatitis C virus internal ribosome entry site (HCV IRES) domain IV in translation initiation and regulation, two chimeric IRES elements were constructed to contain the reciprocal domain IV in the otherwise HCV and classical swine fever virus IRES elements. This permitted an examination of the role of domain IV in the control of HCV translation. A specific inhibitor of the HCV IRES, vitamin B12, was shown to inhibit translation directed by all IRES elements which contained domain IV from the HCV and the GB virus B IRES elements, whereas the HCV core protein could only suppress translation from the wild-type HCV IRES. Thus, the mechanisms of translation inhibition by vitamin B12 and the core protein differ, and they target different regions of the IRES.

scholarly journals Regulating Intracellular Antiviral Defense and Permissiveness to Hepatitis C Virus RNA Replication through a Cellular RNA Helicase, RIG-I

2005 ◽  
Vol 79 (5) ◽  
pp. 2689-2699 ◽  
Author(s):  
Rhea Sumpter ◽  
Yueh-Ming Loo ◽  
Eileen Foy ◽  
Kui Li ◽  
Mitsutoshi Yoneyama ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Infections ◽  
Cultured Cells ◽  
Rna Replication ◽  
Rna Helicase ◽  
Hcv Rna ◽  
Helicase Domain ◽  
Replication Studies ◽  
Virus Rna

ABSTRACT Virus-responsive signaling pathways that induce alpha/beta interferon production and engage intracellular immune defenses influence the outcome of many viral infections. The processes that trigger these defenses and their effect upon host permissiveness for specific viral pathogens are not well understood. We show that structured hepatitis C virus (HCV) genomic RNA activates interferon regulatory factor 3 (IRF3), thereby inducing interferon in cultured cells. This response is absent in cells selected for permissiveness for HCV RNA replication. Studies including genetic complementation revealed that permissiveness is due to mutational inactivation of RIG-I, an interferon-inducible cellular DExD/H box RNA helicase. Its helicase domain binds HCV RNA and transduces the activation signal for IRF3 by its caspase recruiting domain homolog. RIG-I is thus a pathogen receptor that regulates cellular permissiveness to HCV replication and, as an interferon-responsive gene, may play a key role in interferon-based therapies for the treatment of HCV infection.

scholarly journals Unlike for cellular mRNAs and other viral internal ribosome entry sites (IRESs), the eIF3 subunit e is not required for the translational activity of the HCV IRES

2020 ◽  
Vol 295 (7) ◽  
pp. 1843-1856
Author(s):  
Baptiste Panthu ◽  
Solène Denolly ◽  
Cendrine Faivre-Moskalenko ◽  
Théophile Ohlmann ◽  
François-Loïc Cosset ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Hcv Rna ◽  
Internal Ribosome Entry ◽  
Internal Ribosome Entry Sites ◽  
Subunit E ◽  
Cellular Mrnas ◽  
Hcv Ires

Viruses depend on the host cell translation machinery for their replication, and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors. The hepatitis C virus (HCV) IRES binds eukaryotic translation initiation factor 3 (eIF3), but the exact functional role of the eIF3 complex and of its subunits remains to be precisely defined. Toward this goal, here we focused on eIF3 subunit e. We used an in vitro assay combining a ribosome-depleted rabbit reticulocyte lysate and ribosomes prepared from HeLa or Huh-7.5 cells transfected with either control or eIF3e siRNAs. eIF3e silencing reduced translation mediated by the 5′UTR of various cellular genes and HCV-like IRESs. However, this effect was not observed with the bona fide HCV IRES. Silencing of eIF3e reduced the intracellular levels of the c, d, and l subunits of eIF3 and their association with the eIF3 core subunit a. A pulldown analysis of eIF3 subunits associated with the HCV IRES disclosed similar effects and that the a subunit is critical for binding to the HCV IRES. Carrying out HCV infections of control and eIF3e-silenced Huh-7.5 cells, we found that in agreement with the in vitro findings, eIF3e silencing does not reduce HCV replication and viral protein expression. We conclude that unlike for host cellular mRNAs, the entire eIF3 is not required for HCV RNA translation, favoring viral expression under conditions of low eIF3e levels.

scholarly journals Functional interaction of translation initiation factor eIF4G with the foot-and-mouth disease virus internal ribosome entry site

2001 ◽  
Vol 82 (4) ◽  
pp. 757-763 ◽  
Author(s):  
Lanja Saleh ◽  
René C. Rust ◽  
Ralf Füllkrug ◽  
Ewald Beck ◽  
Gergis Bassili ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Foot And Mouth Disease ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
Mouth Disease Virus ◽  
Ires Element

In the life-cycle of picornaviruses, the synthesis of the viral polyprotein is initiated cap-independently at the internal ribosome entry site (IRES) far downstream from the 5′ end of the viral plus-strand RNA. The cis-acting IRES RNA elements serve as binding sites for translation initiation factors that guide the ribosomes to an internal site of the viral RNA. In this study, we show that the eukaryotic translation initiation factor eIF4G interacts directly with the IRES of foot-and-mouth disease virus (FMDV). eIF4G binds mainly to the large Y-shaped stem–loop 4 RNA structure in the 3′ region of the FMDV IRES element, whereas stem–loop 5 contributes only slightly to eIF4G binding. Two subdomains of stem–loop 4 are absolutely essential for eIF4G binding, whereas another subdomain contributes to a lesser extent to binding of eIF4G. At the functional level, the translational activity of stem–loop 4 subdomain mutants correlates with the efficiency of binding of eIF4G in the UV cross-link assay. This indicates that the interaction of eIF4G with the IRES is crucial for the initiation of FMDV translation. A model for the interaction of initiation factors with the IRES element is discussed.

scholarly journals Effect of NS3 and NS5B proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation

2008 ◽  
Vol 89 (4) ◽  
pp. 994-999 ◽  
Author(s):  
Ming Xiao ◽  
Yan Bai ◽  
Hui Xu ◽  
Xiaolu Geng ◽  
Jun Chen ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Classical Swine Fever ◽  
Rna Helicase ◽  
Helicase Domain ◽  
Dependent Manner ◽  
Helicase Activity ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Cellular Translation

A full-length NS3 (NS3F) and a truncated NS3 protein (NS3H) with an RNA helicase domain possess RNA helicase activity. Using an in vitro system with a monocistronic reporter RNA or DNA, containing the CSFV 5′-UTR, we observed that both NS3F and NS3H enhanced internal ribosome entry site (IRES)-mediated and cellular translation in a dose-dependent manner, but NS3 protease (NS3P) that lacks a helicase domain did not. NS3F was stronger than NS3H in promoting both translations. These results showed that viral RNA helicase could promote viral and cellular translation, and higher RNA helicase activity might be more efficient. The NS5B protein, the viral replicase, did not significantly affect the IRES-directed or cellular translation alone. NS5B significantly enhanced the stimulative effect of NS3F on both IRES-mediated and cellular translation, but did not affect that of NS3H or NS3P. This suggests that NS5B and NS3 interact via the protease domain during the enhancement of translation.

The role of IRES trans-acting factors in regulating translation initiation

2010 ◽  
Vol 38 (6) ◽  
pp. 1581-1586 ◽  
Author(s):  
Helen A. King ◽  
Laura C. Cobbold ◽  
Anne E. Willis
Keyword(s):  
Translation Initiation ◽  
Alternative Form ◽  
Structural Element ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Cis Acting ◽  
Initiation Factors ◽  
Eukaryotic Initiation Factors ◽  
Cellular Pathways

The majority of mRNAs in eukaryotic cells are translated via a method that is dependent upon the recognition of, and binding to, the methylguanosine cap at the 5' end of the mRNA, by a set of protein factors termed eIFs (eukaryotic initiation factors). However, many of the eIFs involved in this process are modified and become less active under a number of pathophysiological stress conditions, including amino acid starvation, heat shock, hypoxia and apoptosis. During these conditions, the continued synthesis of proteins essential to recovery from stress or maintenance of a cellular programme is mediated via an alternative form of translation initiation termed IRES (internal ribosome entry site)-mediated translation. This relies on the mRNA containing a complex cis-acting structural element in its 5'-UTR (untranslated region) that is able to recruit the ribosome independently of the cap, and is often dependent upon additional factors termed ITAFs (IRES trans-acting factors). A limited number of ITAFs have been identified to date, particularly for cellular IRESs, and it is not yet fully understood how they exert their control and which cellular pathways are involved in their regulation.

scholarly journals Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

2001 ◽  
Vol 75 (17) ◽  
pp. 8289-8297 ◽  
Author(s):  
Chun-Ling Tai ◽  
Wen-Ching Pan ◽  
Shwu-Huey Liaw ◽  
Ueng-Cheng Yang ◽  
Lih-Hwa Hwang ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Atpase Activity ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Rna Helicase ◽  
Helicase Domain ◽  
Helicase Activity ◽  
Conserved Sequence

ABSTRACT The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3.

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 RNA-Binding Protein hnRNP D Modulates Internal Ribosome Entry Site-Dependent Translation of Hepatitis C Virus RNA

2008 ◽  
Vol 82 (24) ◽  
pp. 12082-12093 ◽  
Author(s):  
Ki Young Paek ◽  
Chon Saeng Kim ◽  
Sung Mi Park ◽  
Jong Heon Kim ◽  
Sung Key Jang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Ribosome Profiling ◽  
Hcv Rna ◽  
Entry Site ◽  
Nontranslated Region ◽  
Hnrnp D ◽  
Hcv Ires

ABSTRACT Hepatitis C virus (HCV) is one of the major causative agents of virus-related hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. Translation of the HCV polyprotein is mediated by an internal ribosomal entry site (IRES) in the 5′ nontranslated region of the genome. Here, we report that a cellular protein, hnRNP D, interacts with the 5′ border of HCV IRES (stem-loop II) and promotes translation of HCV mRNA. Overexpression of hnRNP D in mammalian cells enhances HCV IRES-dependent translation, whereas knockdown of hnRNP D with small interfering RNAs (siRNAs) inhibits translation. In addition, sequestration of hnRNP D with an interacting DNA oligomer inhibits the translation of HCV mRNA in an in vitro system. Ribosome profiling experiments reveal that HCV RNA is redistributed from heavy to light polysome fractions upon suppression of the hnRNP D level using specific siRNA. These results collectively suggest that hnRNP D plays an important role in the translation of HCV mRNA through interactions with the IRES. Moreover, knockdown of hnRNP D with siRNA significantly hampers infection by HCV. A potential role of hnRNP D in HCV proliferation is discussed.

Sign in / Sign up

Export Citation Format

Share Document