Efficient inhibition of hepatitis B virus replication by small interfering RNAs targeted to the viral X gene in mice
Introduction
RNA interference (RNAi) is a general cellular process of gene silencing in plants and animals (Bosher and Labouesse, 2000, Hannon, 2002). Specifically, large double-stranded RNA segments are cleaved by the RNase III-like enzyme, Dicer, into 21–23 nt molecules, designated ‘small interfering RNAs’ (siRNAs) (Elbashir et al., 2001a, Elbashir et al., 2001b). And then these siRNAs are incorporated into a ribonuclease protein complex named RNA-induced silencing complex (RISC) (Nykanen et al., 2001) to mediate sequence-specific degradation of homologous target RNA (Hammond et al., 2000). Numerous trials have been performed to identify potent siRNAs and to elucidate their functions eradicating pathogenic RNA viruses, such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), and poliovirus (Gitlin et al., 2002, Novina et al., 2002, Wilson et al., 2003). Hepatitis B virus (HBV) is one of the major candidates for RNAi, as its pregenomic RNA is a key intermediate in maintaining DNA replication via reverse transcription in the viral life cycle.
HBV is a major cause of acute and chronic hepatitis in humans. Chronic infections are widespread and significantly associated with an increased risk of liver cirrhosis and hepatocellular carcinoma development (Ganem and Varmus, 1987, Lee, 1997). The major anti-HBV therapy options comprise treatment with interferon (IFN)-α or nucleoside analogues, such as lamivudine and adefovir. However, IFN-α has several limitations as a representative anti-viral drug, including low efficacy, side-effects and high costs. Lamivudine is a potent and specific inhibitor of HBV reverse transcriptase, but frequently induces viral genomic mutations resistant to drugs and reactivation of viral replication after cessation of treatment in patients. Moreover, only about 20% HBV patients respond to combination therapy with IFN-α and lamivudine (Dienstag et al., 2003, Jaboli et al., 2003, Lin and Keeffe, 2001). Therefore, there is an urgent need to develop an alternative anti-viral material for treating HBV infections.
The HBV genome is a partial double-stranded circular DNA of 3.2 kb that is transcribed into the 3.5-, 2.4-, 2.1-, and 0.7-kb viral transcripts containing four open reading frames (denoted S, C, P, and X). The HBV X (HBx) gene containing 462 nucleotides is the smallest of them, and encodes a 17 kDa protein with 154 amino acids (Fujiyama et al., 1983). This protein is an apheliotropic transactivator that stimulates HBV promoters and enhancers as well as a wide range of other viral promoters via protein–protein interactions (Nakatake et al., 1993, Spandau and Lee, 1988). In addition, it plays a critical role in inducing cell transformation and liver tumor, either through interactions with cellular transcription factors or through a signal transduction pathway (Kekule et al., 1993). A number of earlier studies reported RNAi effects of vector-derived or synthetic siRNAs targeted to the HBV genome (Giladi et al., 2003, Klein et al., 2003, McCaffrey et al., 2003, Uprichard et al., 2005). Since HBx is critically implicated in HBV-mediated HCC and its sequences expresses in all four viral mRNAs, it seems to be an attractive target for anti-HBV siRNA development.
In this investigation, we prepared an HBV replication-competent construct causing authentic infection conditions under which viral DNA replicates, viral antigens are produced, and then humoral immune responses are induced, particularly in a small animal model. Using this HBV replication system, we demonstrate that selected HBx-specific siRNAs are efficient and potent therapeutic inhibitors of viral replication in an acute and chronic HBV-infected mouse models as well as cultured cells.
Section snippets
Cell culture and transfection
The human hepatoma cell line, Huh7, was maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum. Cells were seeded at a density of 4 × 105 cells per well in six-well clustered plates. After incubation for 1 day, cells were transfected with 0.5 μg of pHBV-MBRI and 1.5 μg of pRNAiDu-control or pRNAiDu-HBx plasmid using Lipofectamine 2000 (Invitrogen). At 1, 2 and 3 days after transfection, media were collected for measuring the secreted HBsAg levels, and transfection efficiency normalized
Construction of an siRNA expression vector
To prepare vector-derived HBx-specific siRNAs, we constructed a plasmid directly expressing siRNA, similar to synthetic siRNA, under the control of two convergent opposing promoters (human U6 and H1), designated ‘pRNAiDu’ (Fig. 1A). In this siRNA expression cassette, both human promoters were modified to contain RNA polymerase III termination sequences of five thymidine nucleotides at positions −5 to −1, followed by BamHI and HindIII sites at −11 to −6 positions, respectively (Zheng et al., 2004
Discussion
In HBV-infected humans, the HBV X protein is expressed in HCC tissue, where it activates a number of viral and cellular promoters as well as enhancers essential for viral replication and HCC development. The coding region is preferentially integrated into host chromosomal DNA (Bergametti et al., 1999, Cromlish, 1996, Nakatake et al., 1993, Paterlini et al., 1995, Wang et al., 1991, Xu et al., 2002). Based on these findings, we propose that degradation of HBx RNA appeared in all HBV transcripts
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These authors contributed equally to the study.