Exploiting cis -Acting Replication Elements To Direct Hepatitis C Virus-Dependent Transgene Expression

Abstract

We describe here a novel targeting gene therapy strategy to direct gene expression responsive to hepatitis C virus (HCV). The goal was approached by engineering a construct containing the antisense sequence of the transgene and internal ribosome entry site of encephalomyocarditis virus flanked by 5-and 3-end sequences of HCV cDNA that contain cis-acting replication elements. Thus, expression of the transgene is only promoted when the minus-strand RNA has been synthesized by the functional replication machinery present in infected cells. Reporter assay and strand-specific reverse transcription-PCR showed selective transgene expression in Huh-7 cells harboring an autonomously replicating HCV subgenome but remaining silent in uninfected cells. Furthermore, using the cytosine deaminase suicide gene as a transgene coupled with recombinant adenovirus delivery, we demonstrated that cytosine deaminase was specifically expressed in replicon cells, resulting in marked chemosensitization of replicon cells to the cytotoxic effects of flucytosine. This new targeting strategy could be extended to other single-stranded RNA viruses encoding the unique RNA-dependent RNA polymerase that has no parallel in mammalian cells.Chronic hepatitis C virus (HCV) infection, which frequently leads to liver cirrhosis and hepatocellular carcinoma (3, 34), remains a major public health problem worldwide. It has been estimated that more than 3% of the world population is infected with HCV. The plus-strand HCV RNA genome is approximately 9,600 nucleotides in length and encodes a polyprotein precursor of about 3,010 amino acids, which is cleaved co-and posttranslationally by cellular and viral proteases to produce structural and nonstructural (NS) proteins (8,13,15). One of the NS proteins, NS5B, is an RNA-dependent RNA polymerase (RdRp) that catalyzes the replication of HCV (5, 30).Current treatment modalities available for HCV infection, including alpha interferon, has limited effectiveness. Only 20 to 30% of alpha interferon-treated patients develop a sustained remission, and increased or prolonged systemic administration is often associated with severe side effects or viral resistance. The combination of ribavirin and interferon is known to be significantly more effective than interferon monotherapy in naïve and relapser patients, but it induces a sustained response only in 41% of patients and in less than 30% of patients infected with genotype 1 (18). The therapeutic potential of small inhibitory molecules that target serine protease, helicase, or RdRp has proved to be promising (4, 6, 10), however, one unavoidable problem of this approach is selection of resistant mutants conferred by single or multiple mutations due to the error-prone nature of RdRp (24,26,29). Thus, an alternative approach for treating HCV patients that results in the death of infected cells, thereby limiting or eliminating virus production, while leaving uninfected cells unharmed would have an advantage over the HCV therapies now available.Here we describe a targeting gen...

Helper virus-independent trans-replication of hepatitis C virus-derived minigenome

“…If the minigenome could be accepted as a template by the replication complex provided in trans, the luciferase gene, which is encoded by synthesized minusstrand RNA, would express in HCV-infected cells. Fully consistent with this hypothesis, luciferase activity was selectively detected in Huh-7 cells harboring an autonomously replicating HCV subgenome (Huh-NNRZ) [13].…”

“…Plasmids. HCV 1b-derived minigenome p1b-1b was previously referred to as pT7cRLNS5B1 [13]. For construction of chimeric minigenome p2a-1b, the first 376 nucelotides of HCV 2a cDNA with the T7 promoter directly coupled at the 5 0 -end were amplified by PCR with primers 5 0 -tataa gcttTAATACGACTCACTATAACCTGCCCCTAATAGGGGC-3 0 and 5 0 -tgcgcatgcTTTGGTTTTTCTTTGAGGTT-3 0 .…”

“…The resulting PCR products were digested with HindIII-SphI and SphI-XbaI, respectively, and inserted into the HindIII/XbaI sites of p1b-1b. The 3 0 -part of the NS5B coding region fused 3 0 UTR of HCV 2a cDNA was amplified by PCR using primers 5 0 -ataggatccCCTCAGAA AACTTGGGG-3 0 and 5 0 -ataggcgccagcgaggaggctgggaccatgccggccACAT GATCTGCAGAGAGACC-3 0 , digested with BamHI and NarI, and cloned, along with the annealed oligonucleotides containing partial sequence of the HDV ribozyme [13], into BamHI/EcoRI-cut p1b-1b or p2a-1b, creating p1b-2a and p2a-2a, respectively.…”

“…Synthetic minigenomes have been described in a number of minus-and plus-stranded RNA viruses, which has contributed greatly to the analysis of cis-acting sequences and trans-acting proteins required for viral replication, maturation, and packaging [11,12]. We previously established a helper virus-dependent expression system utilizing HCVderived minigenome, and Huh-7 cells harboring autonomously replicating HCV subgenome [13]. In this study, we further investigated whether the minigenome replication system could be reconstituted by transfection of naïve Huh-7 cells with plasmid expressing NS proteins.…”

Ultrasensitive monitoring of ribozyme cleavage product using molecular-beacon-ligation system

“…Targeted destruction of viral RNA or cellular mRNA eliminates the formation of a protein that is deleterious in a disease state, which has potential therapeutic utility [2][3][4][5] . Ribozyme cleaves RNA substrate, resulting in a 2′,3′-cyclic phosphate product and a 5′-hydroxyl terminus product [6] .…”