Small Interfering RNA-Mediated Inhibition of Hepatitis C Virus Replication in the Human Hepatoma Cell Line Huh-7

Seo, Mi-Young; Abrignani, Sergio; Houghton, Michael; Han, Jang H.

doi:10.1128/jvi.77.1.810-812.2003

Cited by 120 publications

(81 citation statements)

References 18 publications

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“…1). The sequence of 5'UTR shRNA is identical to the one previously reported (44). The NS5B shRNA covers the same region as reported previously (29) but differs from the reported sequence by three residues due to the difference in the HCV replicons used in the studies.…”

Section: Hcv Replicon-silencing Effects Of Pavu6ϩ27 Plasmid-mediated supporting

confidence: 54%

“…was reported that siRNAs and/or shRNAs against 5'-untranslated region (5'UTR) (44), and nonstructural regions 3 (NS3) (19,49) and NS5B (29) of the HCV genome could effectively suppress the replication of HCV replicons without induction of IFN. In their approaches, however, siRNAs or plasmid vectors expressing shRNAs should be introduced to the target cell by using a transfection reagent or electroporation, that cannot be applied to human use.…”

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confidence: 99%

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Suppression of Hepatitis C Virus Replicon by RNA Interference Directed against the NS3 and NS5B Regions of the Viral Genome

Takigawa

Nagano-Fujii

Deng

et al. 2004

Microbiology and Immunology

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RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence‐specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5′‐untranslated region (5′UTR) (nt 286 to 304), Core (nt 371 to 389), NS3–1 (nt 2052 to 2060), NS3–2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV‐1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27‐derived expression cassette. Although 5′UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5′UTR. In both plasmid‐ and lentivirus‐mediated expression systems, shRNAs against NS3–1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3–1 also inhibited replication of the HCV replicon in a dose‐dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3–1 would be a useful tool to inhibit HCV‐1b infection.

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Section: Hcv Replicon-silencing Effects Of Pavu6ϩ27 Plasmid-mediated supporting

confidence: 54%

mentioning

confidence: 99%

Suppression of Hepatitis C Virus Replicon by RNA Interference Directed against the NS3 and NS5B Regions of the Viral Genome

Takigawa

Nagano-Fujii

Deng

et al. 2004

Microbiology and Immunology

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“…They exist primarily in a circular conformation but also in a linear conformation; these two conformations can be resolved using appropriate conditions of gel electrophoresis (2). The third RNA species consists of relatively lower amounts of an 800-nt polyadenylated RNA (of the same polarity as the antigenome), which is translated to produce a 195-amino-acid protein, known as the delta antigen (␦Ag-S), and is essential for HDV genome replication (14).An increasing number of reports have shown that small interfering RNAs (siRNA) can be exogenously provided to cells undergoing animal virus replication and achieve inhibition (6,8,9,12,13,17,23). For the following reasons, we were specifically interested in the possible susceptibility to siRNA of HDV RNAs.…”

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confidence: 99%

Susceptibility of Human Hepatitis Delta Virus RNAs to Small Interfering RNA Action

Chang

Taylor

2003

J Virol

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In animal cells, small interfering RNAs (siRNA), when exogenously provided, have been reported to be capable of inhibiting replication of several different viruses. In preliminary studies, siRNA species were designed and tested for their ability to act on the protein expressed in Huh7 cells transfected with DNAdirected mRNA constructs containing hepatitis delta virus (HDV) target sequences. The aim was to achieve siRNA specific for each of the three RNAs of HDV replication: (i) the 1,679-nucleotide circular RNA genome, (ii) its exact complement, the antigenome, and (iii) the less abundant polyadenylated mRNA for the small delta protein.Many of the 16 siRNA tested gave >80% inhibition in this assay. Next, these three classes of siRNA were tested for their ability to act during HDV genome replication. It was found that only siRNA targeted against HDV mRNA sequences could interfere with HDV genome replication. In contrast, siRNA targeted against genomic and antigenomic RNA sequences had no detectable effect on the accumulation of these RNAs. Reconstruction experiments with nonreplicating HDV RNA sequences support the interpretation that neither the potential for intramolecular rod-like RNA folding nor the presence of the delta protein conferred resistance to siRNA. In terms of replicating HDV RNAs, it is considered more likely that the genomic and antigenomic RNAs are resistant because their location within the nucleus makes them inaccessible to siRNA-mediated degradation.Human hepatitis delta virus (HDV) has a 1,679-nucleotide (nt) single-stranded circular RNA genome that is replicated by RNA-directed RNA synthesis, most probably involving host RNA polymerase II (25). During this replication, three RNA species accumulate (5), as represented in Fig. 1. The genome and its exact complement, the antigenome, are considered unit length. They exist primarily in a circular conformation but also in a linear conformation; these two conformations can be resolved using appropriate conditions of gel electrophoresis (2). The third RNA species consists of relatively lower amounts of an 800-nt polyadenylated RNA (of the same polarity as the antigenome), which is translated to produce a 195-amino-acid protein, known as the delta antigen (␦Ag-S), and is essential for HDV genome replication (14).An increasing number of reports have shown that small interfering RNAs (siRNA) can be exogenously provided to cells undergoing animal virus replication and achieve inhibition (6,8,9,12,13,17,23). For the following reasons, we were specifically interested in the possible susceptibility to siRNA of HDV RNAs. (i) The HDV genomic and antigenomic RNAs can fold into an unbranched rod-like structure with 74% of the bases paired (15), and this folding might interfere with siRNA action. (ii) The delta protein has the ability to bind doublestranded RNA (4) and thus might also interfere. (iii) While several reports indicate that HDV genomic and antigenomic RNAs are predominantly located in the cell nucleus (7,21,24), two recent studies using cell fract...

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“…They are short enough to bypass general dsRNA-induced nonspecific interferon response (28,34) and thus provided a powerful reverse genetic approach to develop siRNA in gene functional study and antiviral drug development. To date, several laboratories have demonstrated that siRNA can be used as powerful antiviral agents for different viral infection, such as poliovirus (18), influenza A virus (17,33), respiratory syncytial virus (3), hepatitis B (21, 50), C (27,37,41,46,51), and D virus (6), human immunodeficiency virus type 1 (HIV-1) (4,7,9,24,25,29,35,36), and West Nile virus (33). Therefore, there has been considerable interest in the development of siRNA as a possible treatment for CVB3-induced heart diseases.…”

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confidence: 99%

Inhibition of Coxsackievirus B3 Replication by Small Interfering RNAs Requires Perfect Sequence Match in the Central Region of the Viral Positive Strand

Cheung

Zhang

Chau

et al. 2005

J Virol

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Small Interfering RNA-Mediated Inhibition of Hepatitis C Virus Replication in the Human Hepatoma Cell Line Huh-7

Cited by 120 publications

References 18 publications

Suppression of Hepatitis C Virus Replicon by RNA Interference Directed against the NS3 and NS5B Regions of the Viral Genome

Suppression of Hepatitis C Virus Replicon by RNA Interference Directed against the NS3 and NS5B Regions of the Viral Genome

Susceptibility of Human Hepatitis Delta Virus RNAs to Small Interfering RNA Action

Inhibition of Coxsackievirus B3 Replication by Small Interfering RNAs Requires Perfect Sequence Match in the Central Region of the Viral Positive Strand

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