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

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.

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Heterogeneous Nuclear Ribonucleoprotein L Interacts with the 3′ Border of the Internal Ribosomal Entry Site of Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.72.11.8782-8788.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8782-8788 ◽

Cited By ~ 102

Author(s):

Bumsuk Hahm ◽

Yoon Ki Kim ◽

Jong Heon Kim ◽

Tae Yoon Kim ◽

Sung Key Jang

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Internal Ribosomal Entry Site ◽

Hcv Rna ◽

Entry Site ◽

Coding Sequence ◽

The Core ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Hcv Ires ◽

Nuclear Ribonucleoprotein

ABSTRACT Translation initiation of hepatitis C virus (HCV) RNA occurs by internal entry of a ribosome into the 5′ nontranslated region in a cap-independent manner. The HCV RNA sequence from about nucleotide 40 up to the N terminus of the coding sequence of the core protein is required for efficient internal initiation of translation, though the precise border of the HCV internal ribosomal entry site (IRES) has yet to be determined. Several cellular proteins have been proposed to direct HCV IRES-dependent translation by binding to the HCV IRES. Here we report on a novel cellular protein that specifically interacts with the 3′ border of the HCV IRES in the core-coding sequence. This protein with an apparent molecular mass of 68 kDa turned out to be heterogeneous nuclear ribonucleoprotein L (hnRNP L). The binding of hnRNP L to the HCV IRES correlates with the translational efficiencies of corresponding mRNAs. This finding suggests that hnRNP L may play an important role in the translation of HCV mRNA through the IRES element.

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La protein is a potent regulator of replication of hepatitis C virus in patients with chronic hepatitis C through internal ribosomal entry site-directed translation

Gastroenterology ◽

10.1053/j.gastro.2004.11.064 ◽

2005 ◽

Vol 128 (2) ◽

pp. 449-462 ◽

Cited By ~ 15

Author(s):

Masao Honda ◽

Takeo Shimazaki ◽

Shuichi Kaneko

Keyword(s):

Chronic Hepatitis ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Chronic Hepatitis C ◽

Internal Ribosomal Entry Site ◽

Entry Site ◽

La Protein ◽

Ribosomal Entry

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

Journal of General Virology ◽

10.1099/vir.0.82782-0 ◽

2007 ◽

Vol 88 (7) ◽

pp. 1992-2002 ◽

Cited By ~ 8

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.

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

Journal of Virology ◽

10.1128/jvi.01405-08 ◽

2008 ◽

Vol 82 (24) ◽

pp. 12082-12093 ◽

Cited By ~ 56

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.

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Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes

Journal of General Virology ◽

10.1099/vir.0.010579-0 ◽

2009 ◽

Vol 90 (8) ◽

pp. 1812-1819 ◽

Cited By ~ 18

Author(s):

Nithya Subramanian ◽

Prashant Mani ◽

Swagata Roy ◽

Sivakumar Vadivel Gnanasundram ◽

Debi P. Sarkar ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Targeted Delivery ◽

Selective Inhibition ◽

Significant Inhibition ◽

Viral Rna ◽

Hcv Rna ◽

Entry Site ◽

Short Hairpin ◽

Hcv Ires

Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV–H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.

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Hepatitis C Virus Translation Regulation

International Journal of Molecular Sciences ◽

10.3390/ijms21072328 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2328 ◽

Cited By ~ 6

Author(s):

Michael Niepmann ◽

Gesche K. Gerresheim

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Translation Initiation ◽

Core Protein ◽

Mrna Translation ◽

Start Codon ◽

Translation Regulation ◽

Hcv Rna ◽

Entry Site ◽

Initiation Factors

Translation of the hepatitis C virus (HCV) RNA genome is regulated by the internal ribosome entry site (IRES), located in the 5’-untranslated region (5′UTR) and part of the core protein coding sequence, and by the 3′UTR. The 5′UTR has some highly conserved structural regions, while others can assume different conformations. The IRES can bind to the ribosomal 40S subunit with high affinity without any other factors. Nevertheless, IRES activity is modulated by additional cis sequences in the viral genome, including the 3′UTR and the cis-acting replication element (CRE). Canonical translation initiation factors (eIFs) are involved in HCV translation initiation, including eIF3, eIF2, eIF1A, eIF5, and eIF5B. Alternatively, under stress conditions and limited eIF2-Met-tRNAiMet availability, alternative initiation factors such as eIF2D, eIF2A, and eIF5B can substitute for eIF2 to allow HCV translation even when cellular mRNA translation is downregulated. In addition, several IRES trans-acting factors (ITAFs) modulate IRES activity by building large networks of RNA-protein and protein–protein interactions, also connecting 5′- and 3′-ends of the viral RNA. Moreover, some ITAFs can act as RNA chaperones that help to position the viral AUG start codon in the ribosomal 40S subunit entry channel. Finally, the liver-specific microRNA-122 (miR-122) stimulates HCV IRES-dependent translation, most likely by stabilizing a certain structure of the IRES that is required for initiation.

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Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

Journal of Virology ◽

10.1128/jvi.75.24.12047-12057.2001 ◽

2001 ◽

Vol 75 (24) ◽

pp. 12047-12057 ◽

Cited By ~ 236

Author(s):

Peter Friebe ◽

Volker Lohmann ◽

Nicole Krieger ◽

Ralf Bartenschlager

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Virus ◽

Rna Replication ◽

Hcv Rna ◽

Entry Site ◽

Internal Ribosome Entry ◽

Nontranslated Region ◽

High Level ◽

Hcv Ires

ABSTRACT Sequences in the 5′ and 3′ termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5′ end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5′ NTR required for RNA replication. We show that deletions introduced into the 5′ terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5′ NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5′ NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5′ NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5′ NTR of HCV RNA essential for replication of this virus.

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Genetic Analysis of a Poliovirus/Hepatitis C Virus Chimera: New Structure for Domain II of the Internal Ribosomal Entry Site of Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.75.8.3719-3730.2001 ◽

2001 ◽

Vol 75 (8) ◽

pp. 3719-3730 ◽

Cited By ~ 35

Author(s):

Wei Dong Zhao ◽

Eckard Wimmer

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Internal Ribosomal Entry Site ◽

Genome Replication ◽

Entry Site ◽

Internal Ribosomal Entry Sites ◽

Ribosomal Entry ◽

Hcv Ires

ABSTRACT Internal ribosomal entry sites (IRESs) of certain plus-strand RNA viruses direct cap-independent initiation of protein synthesis both in vitro and in vivo, as can be shown with artificial dicistronic mRNAs or with chimeric viral genomes in which IRES elements were exchanged from one virus to another. Whereas IRESs of picornaviruses can be readily analyzed in the context of their cognate genome by genetics, the IRES of hepatitis C virus (HCV), a Hepacivirus belonging toFlaviviridae, cannot as yet be subjected to such analyses because of difficulties in propagating HCV in tissue culture or in experimental animals. This enigma has been overcome by constructing a poliovirus (PV) whose translation is controled by the HCV IRES. Within the PV/HCV chimera, the HCV IRES has been subjected to systematic 5′ deletion analyses to yield a virus (P/H710-d40) whose replication kinetics match that of the parental poliovirus type 1 (Mahoney). Genetic analyses of the HCV IRES in P/H710-d40 have confirmed that the 5′ border maps to domain II, thereby supporting the validity of the experimental approach applied here. Additional genetic experiments have provided evidence for a novel structural region within domain II. Arguments that the phenotypes observed with the mutant chimera relate solely to impaired genome replication rather than deficiencies in translation have been dispelled by constructing novel dicistronic poliovirus replicons with the gene order [PV]cloverleaf-[HCV]IRES-Δcore-R-Luc-[PV]IRES-F-Luc-P2,3-3′NTR, which have allowed the measurement of HCV IRES-dependent translation independently from the replication of the replicon RNA.

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