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

2009 ◽  
Vol 90 (8) ◽  
pp. 1812-1819 ◽  
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.

scholarly journals Sequence-specific cleavage of hepatitis C virus RNA by DNAzymes: inhibition of viral RNA translation and replication

2008 ◽  
Vol 89 (7) ◽  
pp. 1579-1586 ◽  
Author(s):  
Swagata Roy ◽  
Nidhi Gupta ◽  
Nithya Subramanian ◽  
Tanmoy Mondal ◽  
Akhil Chandra Banerjea ◽  
...  
Keyword(s):  
Hepatitis C ◽  
Rna Replication ◽  
Significant Inhibition ◽  
Viral Rna ◽  
Hcv Rna ◽  
Hepatitis C Virus Rna ◽  
Rna Translation ◽  
Virus Rna ◽  
Huh7 Cells ◽  
Hcv Ires

DNAzyme (Dz) molecules have been shown to be highly efficient inhibitors of virus replication. Hepatitis C virus RNA translation is mediated by an internal ribosome entry site (IRES) element located mostly in the 5′ untranslated region (UTR), the mechanism of which is fundamentally different from cap-dependent translation of cellular mRNAs, and thus an attractive target for designing antiviral drugs. Inhibition of HCV IRES-mediated translation has drastic consequences for the replication of viral RNA as well. We have designed several Dzs, targeting different regions of HCV IRES specific for 1b and also sequences conserved across genotypes. The RNA cleavage and translation inhibitory activities of these molecules were tested in a cell-free system and in cell culture using transient transfections. The majority of Dzs efficiently inhibited HCV IRES-mediated translation. However, these Dz molecules did not show significant inhibition of coxsackievirus B3 IRES-mediated translation or cap-dependent translation of reporter gene, showing high level of specificity towards target RNA. Also, Northern blot hybridization analysis showed significant cleavage of HCV IRES by the Dz molecules in Huh7 cells transiently transfected with the HCV–FLuc monocistronic construct. Interestingly, one of the Dzs was more effective against genotype1b, whereas the other showed significant inhibition of viral RNA replication in Huh7 cells harbouring a HCV 2a monocistronic replicon. As expected, mutant-Dz failed to cleave RNA and inhibit HCV RNA translation, showing the specificity of inhibition. Taken together, these findings suggest that the Dz molecule can be used as selective and effective inhibitor of HCV RNA replication, which can be explored further for development of a potent therapeutic agent against HCV infection.

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.

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

2001 ◽  
Vol 75 (24) ◽  
pp. 12047-12057 ◽  
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.

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

1998 ◽  
Vol 72 (11) ◽  
pp. 8782-8788 ◽  
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.

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 Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition

2016 ◽  
Vol 113 (27) ◽  
pp. 7620-7625 ◽  
Author(s):  
Qisheng Li ◽  
Catherine Sodroski ◽  
Brianna Lowey ◽  
Cameron J. Schweitzer ◽  
Helen Cha ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Surface ◽  
Viral Entry ◽  
Epithelial To Mesenchymal Transition ◽  
Hcv Infection ◽  
Hcv Rna ◽  
Entry Site ◽  
Mesenchymal Transition ◽  
E Cadherin

Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry.

scholarly journals Characterization of Occult Hepatitis C Virus (Hcv) Infection among Hemodialysis Patient

2017 ◽  
Vol 16 (1) ◽  
Author(s):  
Siti Nurul Fazlin Abdul Rahman ◽  
Hairul Aini binti Hamzah ◽  
Mohammed Imad Mustafa ◽  
Mohamed Hadzri Hasmoni
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hemodialysis Patients ◽  
Hcv Infection ◽  
Viral Rna ◽  
Cell Counting ◽  
Hcv Rna ◽  
Viral Rnas ◽  
Occult Hepatitis ◽  
Occult Hepatitis C

Introduction: The existence of new entity called occult hepatitis C virus (HCV) has become a raising and escalating concern among healthcare professionals worldwide. It is defined by the presence of viral RNA in liver and/or peripheral blood mononuclear cells (PBMCs) within non HCV-infected patients. Previous study had shown the occult HCV is infectious and capable of transmitting the virus to another host. Till today, HCV infection remains common among hemodialysis patients despite having the best preventive plans. Because of this, there is a significant concern about the source of viral transmission. The aim of the study was to identify and characterize occult HCV infection in PBMC sample of hemodialysis patients. This was an observational and cross sectional study. Materials and method: PBMCs were isolated from the whole blood using Ficoll-gradient centrifugation technique. The PBMCs were then subjected for cell counting and stored in -70O C until further used. HCV RNA were extracted from these cells and viral RNA were subjected for molecular assays, immune cells analysis and cells culture. Results: PBMCs were isolated from eleven (11) study patients and five (5) anti-HCV positive (control) patients. By using automated flow cytometry, PBMCs of each sample were counted and the average number of cells obtained range from 2x104 to 5x106 cells/ ml. Viral RNAs were extracted and quantitatively measured by using NanoDrop Spectrophotometers. The viral RNAs concentration obtained were between 24.7 and 258.9 ng/ml. The RNAs would be subjected for purification (ethanol precipitation) and further assays. Conclusion: The final findings might contribute to the clinical management of dialysis patients.

scholarly journals mTORC1 Restricts Hepatitis C Virus Replication Through ULK1-mediated Suppression of miR-122 and Facilitates Post-replication Events

2018 ◽  
Author(s):  
Manish Kumar Johri ◽  
Hiren Vasantrai Lashkari ◽  
Dhiviya Vedagiri ◽  
Divya Gupta ◽  
Krishnan Harinivas Harshan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Infection ◽  
Virus Replication ◽  
Viral Rna ◽  
Hcv Rna ◽  
Significant Drop ◽  
Mechanistic Target Of Rapamycin ◽  
Mtorc1 Activation ◽  
Rna Levels

ABSTRACTMechanistic target of rapamycin (mTOR) is an important kinase that assimilates several upstream signals including viral infection and facilitates appropriate response by the cell through two unique complexes mTORC1 and mTORC2. Here, we demonstrate that mTORC1 is activated early during HCV infection as antiviral response. Pharmacological inhibition of mTORC1 promoted HCV replication as suggested by elevated levels of HCV (+) and (-) RNA strands. This was accompanied by significant drop in extracellular HCV RNA levels indicating defective post-replication stages. The increase in viral RNA levels failed to augment intracellular infectious virion levels, suggesting that mTORC1 inhibition is detrimental to post-replication steps. Lower infectivity of the supernatant confirmed this observation. Depletion of Raptor and ULK1 accurately reproduced these results suggesting that mTORC1 imparted these effects on HCV through mTORC1-ULK1 arm. Interestingly, ULK1 depletion resulted in increased levels of miR-122, a critical host factor for HCV replication, thus revealing a new mechanism of regulation by ULK1. The binary effect of mTORC1 on HCV replication and egress suggests that mTORC1-ULK1 could be critical in replication: egress balance. Interestingly we discover that ULK1 depletion did not interfere with autophagy in Huh7.5 cells and hence the effects on HCV replication and post-replication events are not resultant of involvement of autophagy. Our studies demonstrate an overall ULK1 mediated anti-HCV function of mTORC1 and identifies an ULK1-independent autophagy that allows HCV replication in spite of mTORC1 activation.

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