Small Interfering RNA Targeted to Hepatitis C Virus 5′ NontranslatedRegion Exerts Potent Antiviral Effect

Kanda, Tatsuo; Steele, Robert; Ray, Ranjit

doi:10.1128/jvi.01496-06

Cited by 78 publications

(54 citation statements)

References 64 publications

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“…Untreated viruses were used similarly for comparison. Cells were washed following incubation, and infectivity was determined as described previously (17,18,24).…”

Section: Methodsmentioning

confidence: 99%

Sulfated Homologues of Heparin Inhibit Hepatitis C Virus Entry into Mammalian Cells

Basu¹,

Kanda²,

Beyene³

et al. 2007

J Virol

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The mechanism of entry of hepatitis C virus (HCV) through interactions between the envelope glycoproteins and specific cell surface receptors remains unclear at this time. We have previously shown with the vesicular stomatitis virus (VSV)/HCV pseudotype model that the hypervariable region 1 of the HCV E2 envelope glycoprotein helps in binding with glycosaminoglycans present on the cell surface. In this study, we have examined the binding of HCV envelope glycoproteins with chemically modified derivatives of heparin. Furthermore, we have determined the functional relevance of the interaction of heparin derivatives with HCV envelope glycoproteins for infectivity by using a human immunodeficiency virus (HIV)/HCV pseudotype, a VSV/HCV pseudotype, and cell culture-grown HCV genotype 1a. Taken together, our results suggest that the HCV envelope glycoproteins rely upon O-sulfated esters of a heparin homologue to facilitate entry into mammalian cells.We first reported the generation of a vesicular stomatitis virus (VSV)/hepatitis C virus (HCV) pseudotype to understand the role of the ectodomains of HCV envelope glycoproteins in relation to the initiation of virus infection (20). Since VSV particles assemble at the plasma membrane (1), we expressed chimeric HCV envelope glycoproteins (E1G and E2G) by appending the transmembrane domain and cytoplasmic tail of VSV glycoprotein at their C termini to provide membrane anchor signals. This modification allowed for the localization of the glycoproteins to the cell surface for efficient incorporation onto VSV pseudotype particles. Pseudotypes generated from either E1G or E2G which had been expressed individually displayed a low level of infectivity, while the presence of both envelope glycoproteins significantly increased the infectious titer of the pseudotype (26). Interestingly, each HCV envelope protein is suggested to have class I and/or class II fusion peptide motifs (10,11,12,31), which may contribute to the infectivity of the parent virus. Murine leukemia virus (MuLV)-and human immunodeficiency virus (HIV)-derived pseudotypes were subsequently generated from human embryonic kidney epithelial cells (293T) by the expression of an unmodified HCV envelope genomic region (5, 15). Although there are apparent differences, the overall observations from MuLV-and HIV-derived pseudotypes were similar to observations from the VSV/HCV pseudotype (8, 26). We have previously observed that the E2 glycoprotein binds to cell surface glycosaminoglycans (GAGs) (7,24). Further studies suggested that the hypervariable region 1 (HVR1) of E2 binds with heparin, and the infectivity of the VSV/HCV pseudotype is diminished in the presence of soluble GAGs or decreased after the enzymatic removal of heparinlike GAGs from the cell surface (7). A different group of investigators have suggested that HCV particles can be efficiently purified from patient sera by using heparin, and GAG binding sites are located within E2 (27, 32).The binding of E2 with a heparin-like molecule appears to facilitate VSV-deri...

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“…Untreated viruses were used similarly for comparison. Cells were washed following incubation, and infectivity was determined as described previously (17,18,24).…”

Section: Methodsmentioning

confidence: 99%

Sulfated Homologues of Heparin Inhibit Hepatitis C Virus Entry into Mammalian Cells

Basu¹,

Kanda²,

Beyene³

et al. 2007

J Virol

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“…5'UTR bölgesi İnternal Ribozomal Entry Site (IRES) adı verilen ve replikasyonun başlangıcın-da görev alan translasyonu başlatan bir bölge içerir (17). Yeni tedavi alternatifleri geliştirme çalışmalarında 5' UTR bölgesi ve IRES kısmını inhibe eden RNA molekülleri geliştirilmiş ve bu bölgenin inhibisyonunun potent antiviral etkisi gösterilmiştir (18). Laporte ve ark.…”

Section: Gereç Ve Yöntemunclassified

Genotype Distribution And 5' Utr Nucleotide Changes In Hepatitis C Virus

Karslığil¹,

Savaş²,

Savaş³

2010

TUTFD

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ÖZETAmaç: HCV, tek zincirli 9500 nükleotid uzunluğunda bir RNA virusdür. Genomun 3' ve 5' uçlarında oldukça korunmuş ve farklı HCV tipleri arasında %92 oranında homoloji gösteren iki adet translasyona uğramayan bölge (untranslated region=UTR) bulunmaktadır. 5'UTR bölgesi genotip saptanmasında kullanılmaktadır. Çalışmada genotip ve bu bölgedeki nük-leik asid değişiklikleri araştırılmıştır. ABSTRACT Objective: HCV is a single-stranded RNA virus which has 9500 nucleotide. On the 3' and 5' ends of the genome, there are two untranslated regions (UTR) which are highly protected and which have 92% homology among various HCV types. 5' UTR is used for the genotype detection. In this study, genotype and nucleic acid changes in this region were analyzed. Material and Methods:In 51 patients diagnosed with HCV, HCV-RNA was isolated and purified from serum samples. The 341 nucleotide-long UTR region at the 5'end of the genome was sequenced and genotypes were detected. Nucleotide changes were analyzed with on-line BLAST program.Results: In 45 (88.2%) of 51 patients, genotype-1 (78.4% of all genotypes were 1b, 9.8% were 1a) was detected. In 41 (80.4%) of the 51 sequences, nucleic acid changes were detected. These changes generally occurred as an insertion in codon 84 ; deletion in codon 43 and codon 46; transversion in codon 15, 17 and 18, and transition in codon 62. No significant relationship was found between viral load and nucleic acid changes. Conclusion:Although the 5'UTR region is a protected region, mutation can be observed, and the mutations may affect the genotype, viral load and treatment response. Therefore, further investigation is required in a large series.Key Words: HCV, Genotyping, 5' UTR nucleotid changes : 30.03.2010: 30.03. Accepted: 21.07.2010 Giriş Hepatit C Virüs (HCV) enfeksiyonu, kronik hepatit, karaciğer sirozu ve karaciğer kanserine yol açabilen önemli bir sağlık sorunudur. Dünyada 130-170 milyon kişinin hepatit C virusu (HCV) ile infekte olduğu ve prevalansın yaklaşık %2.2-3 arasında olduğu tahmin edilmektedir (1). Ülkemiz dünya haritasında prevalansı %1-1.9 arasında olan ülkeler içinde yer almaktadır (2). ReceivedHepatit C Virüsü, Hepacivirüsler genusunda Flaviviridae ailesinde yaklaşık 40-50 nm çapında, dışta lipid zarf taşıyan bir virüstür (3, 4). HCV'nin genomu tek zincirli pozitif sens bir RNA molekülüdür. Yaklaşık 9500 baz uzunluğundadır ve tek bir protein kodlayıcı bölge, open reading frame (ORF) içerir. ORF genomun büyük bir kısmını kapsamaktadır ve yaklaşık 3010 aminoasit uzunluğunda bir poliproteini kodlar. HCV genomunun her iki ucunda (5' ve 3') ise ürüne dönüşmeyen (untranslated region, 5'UTR, 3'UTR) bölgeler bulunmaktadır (3, 5).Hepatit C viruslarında görülen yüksek mutasyon potansiyeli sayesinde fenotip kısa sürede değişebilir. Bunun biyolojik etkileri, konak savunmasından kaçan mutasyonların oluşması, hücre tropizminde ve virulansta değişiklik ve antiviral ajanlara direnç şeklinde olabilir. Virus-konak ilişkilerinde etkili olabilecek genotipler ve mutasyonlar hakkında daha detaylı bi...

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“…To verify the specificity of siRNA gene silencing on HCV RNA replication, we established a robust control for comparative purposes. From previous reports, HCV replication is repressed by siRNA-targeting of regions within NS3 (Kapadia et al, 2003;Prabhu et al, 2005), NS5B (Kapadia et al, 2003;Ng et al, 2007;Prabhu et al, 2005) or the 59 untranslated region (UTR) (Chevalier et al, 2007;Kanda et al, 2007;Kronke et al, 2004;Ng et al, 2007;Seo et al, 2003;Yokota et al, 2003). The 59 UTR is the most conserved segment of the HCV genome and contains a viral IRES for cap-independent translation of the viral polyprotein (Tsukiyama-Kohara et al, 1992;Wang et al, 1993).…”

Section: Isolation Of Cell Lines Supporting Autonomous Replication Ofmentioning

confidence: 99%

Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication

Jones¹,

Domingues²,

Targett-Adams³

et al. 2010

Journal of General Virology

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Host cell factors are critical to all stages of the hepatitis C virus (HCV) life cycle. While many cellular proteins that regulate HCV genome synthesis have been identified, the mechanisms engaged in this process are incompletely understood. To identify novel cellular proteins involved in HCV RNA replication, we screened a library of small interfering RNAs (siRNAs) targeting 299 cellular factors, which principally function in RNA interactions. For the screen, a robust system was established using two cell lines (derived from Huh-7 and U2OS cells) that replicated tricistronic subgenomic replicons (SGRs). We found that the U2OS cell line gave lower levels of intracellular HCV RNA replication compared with Huh-7 cells and was more readily transfected by siRNAs. Consequently, increased gene silencing and greater effects on HCV replication were observed in the U2OS cell line. Thus, U2OS cells provided a suitable and more sensitive alternative to Huh-7 cells for siRNA studies on HCV RNA replication. From the screen, several cellular proteins that enhanced and suppressed HCV RNA replication were identified. One of the genes found to downregulate viral RNA synthesis, ISG15, is expressed in response to alpha interferon and may therefore partly contribute to the clearance of virus from infected individuals. A second gene that inhibited HCV RNA levels was the 59-39 exoRNase XRN1, which suggested a role for cellular RNA degradation pathways in modulating the abundance of viral genomes. Therefore, this study provides an important framework for future detailed analyses of these and other cellular proteins. INTRODUCTIONHepatitis C virus (HCV) possesses a single-stranded (ss), positive-sense RNA genome of approximately 9.6 kb . The RNA is translated to yield a polyprotein that is cleaved by cellular (signal peptidase and signal peptide peptidase) and virus-encoded proteases (the NS2-3 autoprotease and NS3-4A serine protease), thereby generating the mature HCV proteins. The nonstructural (NS) proteins NS3, NS4A, NS4B, NS5A and NS5B are located at punctate structures on the endoplasmic reticulum (ER) membrane, which represent viral replication complexes (RCs) (Egger et al., 2002;Gosert et al., 2003;Targett-Adams et al., 2008). Although the NS3-NS5B proteins are essential for HCV RNA synthesis, replication is dependent on host cell factors. A number of cellular proteins that participate in HCV RNA replication have been identified (see below for references). However, characterization of the complete complement of cellular factors required for HCV replication remains undefined.Small inhibitory RNA (siRNA) technology is a powerful approach to identify host cell factors that contribute to viral replication and as such, several groups have screened siRNA libraries targeting cellular genes. For example, screening with libraries that target protein kinases (Supekova et al., 2008), transporter proteins and transcription factors (Ng et al., 2007), previously documented HCV-interacting proteins (Randall et al., 2007) and proteins implicated...

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Small Interfering RNA Targeted to Hepatitis C Virus 5′ NontranslatedRegion Exerts Potent Antiviral Effect

Cited by 78 publications

References 64 publications

Sulfated Homologues of Heparin Inhibit Hepatitis C Virus Entry into Mammalian Cells

Sulfated Homologues of Heparin Inhibit Hepatitis C Virus Entry into Mammalian Cells

Genotype Distribution And 5' Utr Nucleotide Changes In Hepatitis C Virus

Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication

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