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.

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 Cell Type-Specific Enhancement of Hepatitis C Virus Internal Ribosome Entry Site-Directed Translation due to 5′ Nontranslated Region Substitutions Selected during Passage of Virus in Lymphoblastoid Cells

2000 ◽  
Vol 74 (15) ◽  
pp. 7024-7031 ◽  
Author(s):  
Hervé Lerat ◽  
Yoko K. Shimizu ◽  
Stanley M. Lemon
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Internal Ribosome Entry Site ◽  
Nucleotide Substitutions ◽  
Entry Site ◽  
Lymphoblastoid Cells ◽  
Internal Ribosome Entry ◽  
Translational Activity ◽  
Nontranslated Region

ABSTRACT Low-level replication of hepatitis C virus (HCV) in cultured lymphoblastoid cells inoculated with H77 serum inoculum led to the appearance of new virus variants containing identical substitutions at three sites within the viral 5′ nontranslated RNA (5′NTR): G107→A, C204→A, and G243→A (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325–3329, 1996). These results suggest that virus with this 5′NTR sequence may have a greater capacity for replication in such cells, possibly due to more efficient cap-independent translation, since these nucleotide substitutions reside within the viral internal ribosome entry site (IRES). To test this hypothesis, we examined the translation of dicistronic RNAs containing upstream and downstream reporter sequences (Renilla and firefly luciferases, respectively) separated by IRES sequences containing different combinations of these substitutions. The activity of the IRES was assessed by determining the relative firefly and Renillaluciferase activities expressed in transfected cells. Compared with the IRES present in the dominant H77 quasispecies, an IRES containing all three nucleotide substitutions had significantly greater translational activity in three of five human lymphoblastoid cell lines (Raji, Bjab, and Molt4 but not Jurkat or HPBMa10-2 cells). In contrast, these substitutions did not enhance IRES activity in cell lines derived from monocytes or granulocytes (HL-60, KG-1, or THP-1) or hepatocytes (Huh-7) or in cell-free translation assays carried out with rabbit reticulocyte lysates. Each of the three substitutions was required for maximally increased translational activity in the lymphoblastoid cells. The 2- to 2.5-fold increase in translation observed with the modified IRES sequence may facilitate the replication of HCV, possibly accounting for differences in quasispecies variants recovered from liver tissue and peripheral blood mononuclear cells of the same patient.

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 The Hepatitis C Virus RNA 3′-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site

2006 ◽  
Vol 80 (23) ◽  
pp. 11579-11588 ◽  
Author(s):  
Yutong Song ◽  
Peter Friebe ◽  
Eleni Tzima ◽  
Christiane Jünemann ◽  
Ralf Bartenschlager ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Internal Ribosome Entry Site ◽  
Hepatoma Cell ◽  
Rna Replication ◽  
Viral Rna ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Positive Strand Rna

ABSTRACT The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5′- and 3′-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5′-UTR, and subsequent viral RNA replication requires sequences in the 3′-UTR and in the 5′-UTR. Addressing previous conflicting reports on a possible function of the 3′-UTR for RNA translation in this study, we found that reporter construct design is an important parameter in experiments testing 3′-UTR function. A translation enhancer function of the HCV 3′-UTR was detected only after transfection of monocistronic reporter RNAs or complete RNA genomes having a 3′-UTR with a precise 3′ terminus. The 3′-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. The variable region, the poly(U · C) tract, and the most 3′ terminal stem-loop 1 of the highly conserved 3′ X region contribute significantly to translation enhancement, whereas stem-loops 2 and 3 of the 3′ X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minus-strand synthesis in the HCV 3′-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3′-5′ end interaction and a switch between translation and RNA replication.

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 Inhibitory Effect of 2′-Substituted Nucleosides on Hepatitis C Virus Replication Correlates with Metabolic Properties in Replicon Cells

2005 ◽  
Vol 49 (5) ◽  
pp. 2050-2058 ◽  
Author(s):  
Joanne E. Tomassini ◽  
Krista Getty ◽  
Mark W. Stahlhut ◽  
Sung Shim ◽  
Balkrishen Bhat ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Growth Period ◽  
Inhibitory Effect ◽  
Chemotherapeutic Agents ◽  
Metabolic Properties ◽  
Ns5b Polymerase

ABSTRACT Nucleosides have been widely used in the treatment of viral diseases, but relatively few have been identified as inhibitors of hepatitis C virus (HCV). The modified ribonucleosides, 2′-C-methyl-adenosine and 2′-O-methyl-cytidine, are potent inhibitors of HCV replication which specifically target the NS5B polymerase. Herein, a more extensive characterization of the effect of these compounds upon HCV replication in subgenomic replicons is reported. A highly selective antireplicative effect induced by the nucleosides in replicon-containing cell lines was maintained during an exponential growth period with potencies which paralleled the reduction of both positive- and negative-strand RNA replication. Moreover, the inhibitory effect closely correlated with the intrinsic metabolic properties of differing replicon clonal lines. Interestingly, while 2′-C-methyl-adenosine elicited similar inhibitory potencies in different cell lines, 2′-O-methyl-cytidine was found to be inactive in one replicon cell line tested, although the corresponding triphosphates comparably inhibited the in vitro activity of replication complexes isolated from these cells and the activity of NS5B polymerase using synthetic templates. The lack of antireplicative effect, attributed to poor intracellular conversion of the 2′-O-methyl-cytidine nucleoside to the active 5′-triphosphate, was reversed using a monophosphate prodrug. Thus, although replicon cells are useful for evaluating the effect of inhibitors upon HCV replication, these findings have important implications for their use in the identification and characterization of nucleosides and other chemotherapeutic agents requiring cellular metabolism.

scholarly journals Optimized High-Throughput Screen for Hepatitis C Virus Translation Inhibitors

2011 ◽  
Vol 16 (2) ◽  
pp. 211-220 ◽  
Author(s):  
Katherine E. Berry ◽  
Betty Peng ◽  
David Koditek ◽  
Douglas Beeman ◽  
Nikos Pagratis ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
Large Fraction ◽  
In Vitro Translation ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Hcv Ires ◽  
General Translation

Hepatitis C virus (HCV) is a considerable global health problem for which new classes of therapeutics are needed. The authors developed a high-throughput assay to identify compounds that selectively block translation initiation from the HCV internal ribosome entry site (HCV IRES). Rabbit reticulocyte lysate conditions were optimized to faithfully report on authentic HCV IRES-dependent translation relative to a 5′ capped mRNA control. The authors screened a library of ~430,000 small molecules for IRES inhibition, leading to ~1700 initial hits. After secondary counterscreening, the vast majority of hits proved to be luciferase and general translation inhibitors. Despite well-optimized in vitro translation conditions, in the end, the authors found no selective HCV IRES inhibitors but did discover a new scaffold of general translation inhibitor. The analysis of these molecules, as well we the finding that a large fraction of false positives resulted from off-target effects, highlights the challenges inherent in screens for RNA-specific inhibitors.

scholarly journals A Small Yeast RNA Blocks Hepatitis C Virus Internal Ribosome Entry Site (HCV IRES)-Mediated Translation and Inhibits Replication of a Chimeric Poliovirus under Translational Control of the HCV IRES Element

1998 ◽  
Vol 72 (7) ◽  
pp. 5638-5647 ◽  
Author(s):  
Saumitra Das ◽  
Michael Ott ◽  
Akemi Yamane ◽  
Weimin Tsai ◽  
Matthias Gromeier ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Internal Ribosome Entry Site ◽  
Hcv Infection ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ires Element ◽  
Hcv Ires

ABSTRACT Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis and cirrhosis of the liver and has been linked to development of hepatocellular carcinoma. We previously identified a small yeast RNA (IRNA) capable of specifically inhibiting poliovirus (PV) internal ribosome entry site (IRES)-mediated translation. Here we report that IRNA specifically inhibits HCV IRES-mediated translation both in vivo and in vitro. A number of human hepatoma (Huh-7) cell lines expressing IRNA were prepared and characterized. Constitutive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell lines. Additionally, Huh-7 cells constitutively expressing IRNA became refractory to infection by a PV-HCV chimera in which the PV IRES is replaced by the HCV IRES. In contrast, replication of a PV-encephalomyocarditis virus (EMCV) chimera containing the EMCV IRES element was not affected significantly in the IRNA-producing cell line. Finally, the binding of the La autoantigen to the HCV IRES element was specifically and efficiently competed by IRNA. These results provide a basis for development of novel drugs effective against HCV infection.

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