scholarly journals Hepatitis C Virus 3′X Region Interacts with Human Ribosomal Proteins

2001 ◽  
Vol 75 (3) ◽  
pp. 1348-1358 ◽  
Author(s):  
Jonny Wood ◽  
Robert M. Frederickson ◽  
Stanley Fields ◽  
Arvind H. Patel
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fusion Protein ◽  
Ribosomal Proteins ◽  
Infectious Clone ◽  
Cdna Libraries ◽  
Entry Site ◽  
Reporter Protein ◽  
Barr Virus

ABSTRACT To identify proteins that can bind the 3′ untranslated region (UTR) of hepatitis C virus (HCV) we screened human cDNA libraries using theSaccharomyces cerevisiae three-hybrid system. Screening with an RNA sequence derived from the 3′-terminal 98 nucleotides (3′X region) of an infectious clone of HCV (H77c) yielded clones of human ribosomal proteins L22, L3, S3, and mL3, a mitochondrial homologue of L3. We performed preliminary characterization of the binding between the 3′X region and these proteins by a three-hybrid mating assay using mutant 3′X sequences. We have further characterized the interaction between 3′X and L22, since this protein is known to be associated with two small Epstein-Barr virus (EBV)-encoded RNA species (EBERs) which are abundantly produced in cells latently infected with EBV. The EBERs, which have similar predicted secondary structure to the HCV 3′X, assemble into ribonucleoprotein particles that include L22 and La protein. To confirm that L22 binds HCV 3′X we performed in vitro binding assays using recombinant L22 (expressed as a glutathioneS-transferase [GST] fusion protein) together with a 3′X riboprobe. The 3′X region binds to the GST-L22 fusion protein (but not to GST alone), and this interaction is subject to competition with unlabeled 3′X RNA. To establish the functional role played by L22 in internal ribosome entry site (IRES)-mediated translation of HCV sequences we performed translational analysis in HuH-7 cells using monocistronic and bicistronic reporter constructs. The relative amount of core-chloramphenicol acetyltransferase reporter protein translated under the control of the HCV IRES was stimulated in the presence of L22 and La when these proteins were supplied in trans.

scholarly journals Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon

2005 ◽  
Vol 86 (11) ◽  
pp. 3075-3080 ◽  
Author(s):  
Paul Targett-Adams ◽  
John McLauchlan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Osteosarcoma Cell ◽  
Dependent Manner ◽  
Entry Site ◽  
Subgenomic Replicon ◽  
Replication Assay ◽  
Cell Extracts ◽  
Genotype 1B

Dicistronic, subgenomic hepatitis C virus (HCV) replicons were constructed containing sequences from JFH1, a genotype 2a strain, that also incorporated the firefly luciferase gene under the control of the HCV internal ribosome entry site element. Luciferase activity in Huh-7 cell extracts containing in vitro-transcribed subgenomic JFH1 RNA was monitored over a 72 h period to examine early stages of HCV replication in the absence of any selective pressure. Enzyme activities produced by the replicon were almost 200-fold greater than those generated from corresponding genotype 1b replicons and correlated with an accumulation of NS5A protein and replicon RNA. Transient replication was sensitive to IFN treatment in a dose-dependent manner and, in addition to Huh-7 cells, the U2OS human osteosarcoma cell line supported efficient replication of the JFH1 replicon. Thus, this system based on JFH1 sequences offers improvements over prior genotype 1b replicons for quantitative measurement of viral RNA replication.

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 Virological characterization of the hepatitis C virus JFH-1 strain in lymphocytic cell lines

2008 ◽  
Vol 89 (7) ◽  
pp. 1587-1592 ◽  
Author(s):  
Kyoko Murakami ◽  
Toshiro Kimura ◽  
Motonao Osaki ◽  
Koji Ishii ◽  
Tatsuo Miyamura ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Culture Systems ◽  
Polyprotein Processing ◽  
Lymphocytic Cell

While hepatocytes are the major site of hepatitis C virus (HCV) infection, a number of studies have suggested that HCV can replicate in lymphocytes. However, in vitro culture systems to investigate replication of HCV in lymphocytic cells are severely limited. Robust HCV culture systems have been established using the HCV JFH-1 strain and Huh-7 cells. To gain more insights into the tissue tropism of HCV, we investigated the infection, replication, internal ribosome entry site (IRES)-dependent translation and polyprotein processing of the HCV JFH-1 strain in nine lymphocytic cell lines. HCV JFH-1 failed to infect lymphocytes and replicate, but exhibited efficient polyprotein processing and IRES-dependent translation in lymphocytes as well as in Huh-7 cells. Our results suggest that lymphocytic cells can support HCV JFH-1 translation and polyprotein processing, but may lack some host factors essential for HCV JFH-1 infection and replication.

Amiodarone inhibits the entry and assembly steps of hepatitis C virus life cycle

2013 ◽  
Vol 125 (9) ◽  
pp. 439-448 ◽  
Author(s):  
Yuan-Lung Cheng ◽  
Keng-Hsueh Lan ◽  
Wei-Ping Lee ◽  
Szu-Han Tseng ◽  
Li-Rong Hung ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Inhibitory Effect ◽  
Hcv Infection ◽  
Receptor Expression ◽  
Current Standard ◽  
Protein Activity ◽  
Entry Site

HCV (hepatitis C virus) infection affects an estimated 180 million people in the world's population. Adverse effects occur frequently with current standard treatment of interferon and ribavirin, while resistance of new direct anti-viral agents, NS3 protease inhibitors, is a major concern because of their single anti-HCV mechanism against the viral factor. New anti-viral agents are needed to resolve the problems. Amiodarone, an anti-arrhythmic drug, has recently been shown to inhibit HCV infection in vitro. The detailed mechanism has yet to be clarified. The aim of the present study was to elucidate the molecular mechanism of the inhibitory effect of amiodarone on HCV life cycle. The effect of amiodarone on HCV life cycle was investigated in Huh-7.5.1 cells with HCVcc (cell culture-derived HCV), HCVpp (HCV pseudoviral particles), sub-genomic replicons, IRES (internal ribosomal entry site)-mediated translation assay, and intracellular and extracellular infectivity assays. The administration of amiodarone appeared to inhibit HCV entry independent of genotypes, which was attributed to the down-regulation of CD81 receptor expression. The inhibitory effect of amiodarone also manifested in the HCV assembly step, via the suppression of MTP (microsomal triacylglycerol transfer protein) activity. Amiodarone revealed no effects on HCV replication and translation. With the host factor-targeting characteristics, amiodarone may be an attractive agent for the treatment of HCV infection.

scholarly journals The 5′ untranslated region of GB virus B shows functional similarity to the internal ribosome entry site of hepatitis C virus

1999 ◽  
Vol 80 (9) ◽  
pp. 2337-2341 ◽  
Author(s):  
Ken Grace ◽  
Margaret Gartland ◽  
Peter Karayiannis ◽  
Michael J. McGarvey ◽  
Berwyn Clarke
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Secondary Structure ◽  
Internal Ribosome Entry Site ◽  
Mutational Analysis ◽  
Sequence Similarity ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Close Phylogenetic Relationship

Since its characterization in 1995, there has been increasing interest in the significance of GB virus B (GBV-B) due to its close phylogenetic relationship to hepatitis C virus (HCV). The genome of GBV-B is similar in length and organization to that of HCV and the two viruses share sequence similarity in their 5′ untranslated regions (5′UTR). A secondary structure model of the GBV-B 5′UTR has been proposed by comparative sequence analysis with HCV. The highly conserved secondary structure, present in HCV and the pestiviruses, is also present in the 5′UTR of GBV-B. Translation of the HCV polyprotein initiates via an internal ribosome entry site (IRES) and it is proposed that the GBV-B UTR may function in a similar manner. Dicistronic reporter constructs were made to investigate the function of the GBV-B 5′UTR. Mutational analysis and in vitro translation experiments demonstrate that GBV-B initiates translation via an IRES.

scholarly journals Transcriptional slippage prompts recoding in alternate reading frames in the hepatitis C virus (HCV) core sequence from strain HCV-1

2008 ◽  
Vol 89 (7) ◽  
pp. 1569-1578 ◽  
Author(s):  
Maxime Ratinier ◽  
Steeve Boulant ◽  
Christophe Combet ◽  
Paul Targett-Adams ◽  
John McLauchlan ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Infectious Clone ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Subgenomic Replicon ◽  
Luciferase Reporter System ◽  
Transcriptional Slippage

Since the first report of frameshifting in HCV-1, its sequence has been the paradigm for examining the mechanism that directs alternative translation of the hepatitis C virus (HCV) genome. The region encoding the core protein from this strain contains a cluster of 10 adenines at codons 8–11, which is thought to direct programmed ribosomal frameshifting (PRF), but formal evidence for this process has not been established unequivocally. To identify the mechanisms of frameshifting, this study used a bicistronic dual luciferase reporter system in a coupled transcription/translation in vitro assay. This approach revealed +1 as well as –1 frameshifting, whereas point mutations, selectively introduced between codons 8 and 11, demonstrated that PRF did not readily account for frameshifting in strain HCV-1. Sequence analysis of cDNAs derived from RNA transcribed by T7 RNA polymerase in the dual luciferase reporter system, as well as in both a subgenomic replicon and an infectious clone derived from strain JFH1, identified additions and deletions of adenines between codons 8 and 11 due to transcriptional slippage (TS). Moreover, RNA isolated from cells infected with virus generated by JFH1 containing the A-rich tract also contained heterogeneity in the adenine sequence, strongly suggesting TS by the NS5B viral polymerase. These findings have important implications for insight into frameshifting events in HCV-1 and demonstrate for the first time the involvement of transcriptional slippage in this recoding event.

scholarly journals Hepatitis C Virus Internal Ribosome Entry Site (IRES) Stem Loop IIId Contains a Phylogenetically Conserved GGG Triplet Essential for Translation and IRES Folding

2000 ◽  
Vol 74 (22) ◽  
pp. 10430-10437 ◽  
Author(s):  
Ronald Jubin ◽  
Nicole E. Vantuno ◽  
Jeffrey S. Kieft ◽  
Michael G. Murray ◽  
Jennifer A. Doudna ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Apical Loop ◽  
Hcv Ires

ABSTRACT The hepatitis C virus (HCV) internal ribosome entry site (IRES) is a highly structured RNA element that directs cap-independent translation of the viral polyprotein. Morpholino antisense oligonucleotides directed towards stem loop IIId drastically reduced HCV IRES activity. Mutagenesis studies of this region showed that the GGG triplet (nucleotides 266 through 268) of the hexanucleotide apical loop of stem loop IIId is essential for IRES activity both in vitro and in vivo. Sequence comparison showed that apical loop nucleotides (UUGGGU) were absolutely conserved across HCV genotypes and the GGG triplet was strongly conserved among related Flavivirus andPestivirus nontranslated regions. Chimeric IRES elements with IIId derived from GB virus B (GBV-B) in the context of the HCV IRES possess translational activity. Mutations within the IIId stem loop that abolish IRES activity also affect the RNA structure in RNase T1-probing studies, demonstrating the importance of correct RNA folding to IRES function.

scholarly journals Comparative Analysis of Translation Efficiencies of Hepatitis C Virus 5′ Untranslated Regions among Intraindividual Quasispecies Present in Chronic Infection: Opposite Behaviors Depending on Cell Type

2000 ◽  
Vol 74 (22) ◽  
pp. 10827-10833 ◽  
Author(s):  
Julien Laporte ◽  
Isabelle Malet ◽  
Thibault Andrieu ◽  
Vincent Thibault ◽  
Jean-Jacques Toulme ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Expression System ◽  
Luciferase Expression ◽  
Hcv Rna ◽  
Translation Efficiency ◽  
Cell Type ◽  
Entry Site ◽  
Conserved Sequence

ABSTRACT Hepatitis C virus (HCV) RNA translation initiation is dependent on the presence of an internal ribosome entry site (IRES) that is found mostly in its 5′ untranslated region (5′ UTR). While exhibiting the most highly conserved sequence within the genome, the 5′ UTR accumulates small differences, which may be of biological and clinical importance. In this study, using a bicistronic dual luciferase expression system, we have examined the sequence of 5′ UTRs from quasispecies characterized in the serum of a patient chronically infected with HCV genotype 1a and its corresponding translational activity. Sequence heterogeneity between IRES elements led to important changes in their translation efficiency both in vitro and in different cell cultures lines, implying that interactions of RNA with related transacting factors may vary according to cell type. These data suggest that variants occasionally carried by the serum prior to reinfection could be selected toward different compartments of the same infected organism, thus favoring the hypothesis of HCV multiple tropism.

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