Enhanced antiviral effect in cell culture of type 1 interferon and ribozymes targeting HCV RNA

We have recently shown that the replication of an HCV-poliovirus (PV) chimera that is dependent upon the hepatitis C virus (HCV) 5' untranslated region (UTR) can be inhibited by treatment with ribozymes targeting HCV RNA. To determine the antiviral effects of anti-HCV ribozyme treatment in combination with type 1 interferon (IFN), we analysed the replication of this HCV-PV chimera in HeLa cells treated with anti-HCV ribozyme and/or IFN-α2a, IFN-α2b, or consensus IFN. The anti-HCV ribozyme, or any of the IFNs alone have significant inhibitory effects on HCV-PV replication compared to control treatment (≥ 85%, P  < 0.01). The maximal inhibition due to IFN treatment (94%, P  < 0.01) was achieved with ≥ 50 U/ml for either IFN-α2a or IFN-α2b compared to control treatment. A similar level of inhibition in viral replication could be achieved with a 5-fold lower dose of IFN if ribozyme targeting the HCV 5' UTR was given in combination. For consensus IFN, the dose could be reduced by > 12.5-fold if ribozyme targeting the HCV 5' UTR was given in combination. Conversely, the dose of ribozyme could be reduced 3-fold if given in combination with any of the IFN preparations. Moreover, treatment with low doses (1–25 U/mL) of IFN-α2a, IFN-α2b, or consensus IFN in combination with anti-HCV ribozyme resulted in > 98% inhibition of HCV-PV replication compared to control treatment ( P  < 0.01). These results demonstrate that IFN and ribozyme each have a beneficial antiviral effect that is augmented when given in combination.     

Inhibition of HCV subgenomic replicons by siRNAs derived from plasmids with opposing U6 and H1 promoters

Hepatitis C virus (HCV) is a main cause of chronic liver disease, which may lead to the development of liver cirrhosis and hepatocellular carcinoma. Therapeutic options are still limited in a significant proportion of patients. Small interfering RNAs (siRNAs) are an efficient tool to inhibit gene expression by RNA interference. As HCV RNA replicates in the cytoplasm of liver cells without integration into the genome, RNA-directed antiviral strategies are likely to successfully block its replication cycle. In this study, a panel of siRNAs was used to target various important regions of the HCV genome [5' untranslated region (UTR), NS3, NS4A, NS4B, NS5B, 3' UTR]. Convergent opposing human H1 and U6 polymerase III promoters were used to generate siRNAs. Target genes in sense and antisense orientation were attached to a luciferase reporter system to test the inhibitory efficiency of both siRNA strands. Our data revealed effective RNA interference against the HCV(+)-strand, the HCV(-)-strand or both strands simultaneously up to 65%. Subsequently, active siRNAs were tested in HCV subgenomic replicon cells and suppression of HCV RNA and NS5B protein levels up to 75% was confirmed. Interestingly, siRNAs that were effective against the sense as well as the antisense strand revealed the greatest inhibitory effects on HCV subgenomic replicons. Additionally, combinations of siRNAs induced a greater inhibition of HCV subgenomic replication of up to 90% proving the potential of this combined antiviral approach.     

An in vitro model of hepatitis C virion production

The hepatitis C virus (HCV) is a major cause of liver disease worldwide. The understanding of the viral life cycle has been hampered by the lack of a satisfactory cell culture system. The development of the HCV replicon system has been a major advance, but the system does not produce virions. In this study, we constructed an infectious HCV genotype 1b cDNA between two ribozymes that are designed to generate the exact 5' and 3' ends of HCV. A second construct with a mutation in the active site of the viral RNA-dependent RNA polymerase (RdRp) was generated as a control. The HCV-ribozyme expression construct was transfected into Huh7 cells. Both HCV structural and nonstructural proteins were detected by immunofluorescence and Western blot. RNase protection assays showed positive- and negative-strand HCV RNA. Sequence analysis of the 5' and 3' ends provided further evidence of viral replication. Sucrose density gradient centrifugation of the culture medium revealed colocalization of HCV RNA and structural proteins in a fraction with the density of 1.16 g/ml, the putative density of HCV virions. Electron microscopy showed viral particles of approximately 50 nm in diameter. The level of HCV RNA in the culture medium was as high as 10 million copies per milliliter. The HCV-ribozyme construct with the inactivating mutation in the RdRp did not show evidence of viral replication, assembly, and release. This system supports the production and secretion of high-level HCV virions and extends the repertoire of tools available for the study of HCV biology.     

MicroRNA-122 antagonism against hepatitis C virus genotypes 1-6 and reduced efficacy by host RNA insertion or mutations in the HCV 5' UTR

MicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5' untranslated region (5' UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo. However, virus-producing culture systems with 5' UTR of different HCV genotypes have not been available for testing 5' UTR-based treatment approaches. Using JFH1-based Core-NS2 genotype recombinants, we developed 5' UTR-NS2 recombinants of HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a with efficient growth in Huh7.5 cells. Deletion mutagenesis studies demonstrated that the 5' UTR SLI was essential for genotypes 1-6 infection. However, lack of SLI could be compensated for by insertion of other structured HCV or host RNA sequences, including U3 small nucleolar RNA. We demonstrated that SPC3649-induced miR-122 antagonism had a potent antiviral effect against HCV genotypes 1-6 5' UTR-NS2 viruses. Strikingly, HCV recombinant virus with substitution of SLI and miR-122 binding site 1 (S1) by the U3 RNA sequence was not affected by miR-122 antagonism; this was attributed to the lack of an intact S1 by reverse genetics studies. Therefore, we engineered the corresponding U3 RNA sequences into S1 and demonstrated that HCV recombinants with wild-type SLI and single or combined mutations at four of eight nucleotides of S1 were viable in Huh7.5 cells. These mutations reduced the efficacy of SPC3649 treatment, indicating that escape variants to miR-122 antagonism-based HCV therapy could potentially occur.     

丙型肝炎病毒全长基因嵌合萤光素酶重组质粒转染细胞系的建立与初步应用

目的 构建能产生较高效价重组病毒的HCV细胞感染模型,为HCV致病机制的研究和抗病毒药物的筛选提供一个有效的体外细胞培养系统.方法 利用PCR技术在HCV NS5A C端插入海肾萤光素酶( Renilla Luciferase,Rluc)报告基因,并引入能提高HCV效价的突变,酶切鉴定重组基因序列构建成功后,转染入...     

Interference of hepatitis C virus replication in cell culture by antisense peptide nucleic acids targeting the X-RNA

The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is the essential catalytic enzyme for viral genome replication. It initiates minus-strand RNA synthesis from a highly conserved 98-nt sequence, called the X-RNA, at the 3'-end of the plus-strand viral genome. In this study, we evaluated the antiviral effects of peptide nucleic acids (PNAs) targeting the X-RNA. Our in vitro RdRp assay results showed that PNAs targeting the three major stem-loop (SL) domains of X-RNA can inhibit RNA synthesis initiation. Delivery of X-RNA-targeted PNAs by fusing the PNAs to cell-penetrating peptides (CPPs) into HCV-replicating cells effectively suppressed HCV replication. Electrophoretic mobility shift assays revealed that the PNA targeting the SL3 region at the 5'-end of X-RNA dissociated the viral RdRp from the X-RNA. Furthermore, delivery of the SL3-targeted PNA into HCV-infected cells resulted in the suppression of HCV RNA replication without activation of interferon β expression. Collectively, our results indicate that the HCV X-RNA can be effectively targeted by CPP-fused PNAs to block RNA-protein and/or RNA-RNA interactions essential for viral RNA replication and identify X-RNA SL3 as an RdRp binding site crucial for HCV replication. In addition, the ability to inhibit RNA synthesis initiation by targeting HCV X-RNA using antisense PNAs suggests their promising therapeutic potential against HCV infection.     

RNA aptamer‐mediated interference of HCV replication by targeting the CRE‐5BSL3.2 domain

Summary. The RNA genome of hepatitis C virus (HCV) contains multiple conserved structural RNA domains that play key roles in essential viral processes. A conserved structural component within the 3′ end of the region coding for viral RNA‐dependent RNA polymerase (NS5B) has been characterized as a functional cis‐acting replication element (CRE). This study reports the ability of two RNA aptamers, P‐58 and P‐78, to interfere with HCV replication by targeting the essential 5BSL3.2 domain within this CRE. Structure‐probing assays showed the binding of the aptamers to the CRE results in a structural reorganization of the apical portion of the 5BSL3.2 stem‐loop domain. This interfered with the binding of the NS5B protein to the CRE and induced a significant reduction in HCV replication (≈50%) in an autonomous subgenomic HCV replication system. These results highlight the potential of this CRE as a target for the development of anti‐HCV therapies and underscore the potential of antiviral agents based on RNA aptamer molecules.     

短发夹状双链RNA抑制丙型肝炎病毒IRES介导的基因表达

目的研究载体表达的短发夹状双链RNA（shRNA）对丙型肝炎病毒（HCV）IRES介导的基因表达的特异性抑制作用。方法构建HCV IRES调控的绿色荧光蛋白表达载体（pIRES—GFP）和虫荧光索酶表达载体（p5′ UTR—Luc），以及针对HCV IRES的shRNA表达载体（pshRNA-HCV）。共转染HepG2细胞，于转染后24、48、72h观察绿色荧光的强弱，用Western blot检测绿色荧光蛋白的表达，半定量逆转录聚合酶链反应法检测GFP的mRNA水平。双荧光索酶系统检测虫荧光索酶活性。结果pshRNA-HCV作用组绿色荧光强度明显弱于未干扰组，GFP蛋白表达量及虫荧光素酶活性降低60％～70％，半定量逆转录聚合酶链反应显示pshRNA-HCV导致了GFP基因mRNA水平的降低。结论针对HCV IRES的shRNA能够显著和特异地抑制该区域调控的蛋白表达水平及mRNA水平，该研究结果为利用RNA干扰技术治疗HCV感染进行了初步探索。     

抑制性RNA在细胞内对丙型肝炎病毒5′非编码区翻译启动的抑制作用

目的：研究核糖体内部进入位点（IRES）特异性抑制性RNA（IRNA）在细胞内对HCV 5′非编码区（NCR）IRES翻译启动功能的抑制作用。方法：通过脂质体介导的基因转染方法,转染IRNA真核表达体与带荧光素（luc)的靶基因真核表达载体于人肝癌细胞（HHCC）,应用发光仪检测luc报告基因的表达活性。结果：IRNA能有效地抑制由HCV 5′NCR IRES介导的luc基因表达,抑制率最高达90％,最低50％,而IRNA突变体（mIRNA)无此活性。结论：IRES特异性IRNA能有效地抑制HCV 5′NCR介导的蛋白翻译启动作用。     

Stabilization of hepatitis C virus RNA by an Ago2-miR-122 complex

MicroRNAs (miRNAs) are small noncoding RNAs that regulate eukaryotic gene expression by binding to regions of imperfect complementarity in mRNAs, typically in the 3' UTR, recruiting an Argonaute (Ago) protein complex that usually results in translational repression or destabilization of the target RNA. The translation and decay of mRNAs are closely linked, competing processes, and whether the miRNA-induced silencing complex (RISC) acts primarily to reduce translation or stability of the mRNA remains controversial. miR-122 is an abundant, liver-specific miRNA that is an unusual host factor for hepatitis C virus (HCV), an important cause of liver disease in humans. Prior studies show that it binds the 5' UTR of the messenger-sense HCV RNA genome, stimulating translation and promoting genome replication by an unknown mechanism. Here we show that miR-122 binds HCV RNA in association with Ago2 and that this slows decay of the viral genome in infected cells. The stabilizing action of miR-122 does not require the viral RNA to be translationally active nor engaged in replication, and can be functionally substituted by a nonmethylated 5' cap. Our data demonstrate that a RISC-like complex mediates the stability of HCV RNA and suggest that Ago2 and miR-122 act coordinately to protect the viral genome from 5' exonuclease activity of the host mRNA decay machinery. miR-122 thus acts in an unconventional fashion to stabilize HCV RNA and slow its decay, expanding the repertoire of mechanisms by which miRNAs modulate gene expression.     

丁型肝炎病毒核酶在细胞内抑制丙型肝炎病毒RNA活性的研究

目的 探讨丁型肝炎病毒(HDV)核酶在细胞内抑制丙型肝炎病毒(HCV)RNA的可能性。方法 针对HCV-5′NCR-C RNA的不同靶位构建了pC1-RzC1(107～113nt)、pC1-RzC2(268～274nt)、pC1-RzC3(345～351nt)3个HDV核酶重组真核表达载体,采用脂质体介导将它们转染至HCV阳性胎肝细胞中,通过荧光定量聚合酶链反应(PCR)检测细胞和培养上清液中的HCV RNA,初步探讨HDV核酶对HCV RNA的抑制活性。结果 (1)重组表达载体通过PCR、酶切和碱基序列测定证实了3组HDV核酶定向插入到真核表达载体。(2)免疫组织化学观察到HCV阳性胎肝细胞中HCV NS3、NS5抗原及逆转录-PCR法检测胎肝细胞及上清液中的HCV RNA正、负链,同时利用Light Cycler荧光PCR定量检测HCV RNA的含量,证明HCV感染胎肝细胞成功。(3)HCV阳性胎肝细胞中HDV核酶对全长HCV RNA的抑制活性,当剂量为0.5μmol/L时,pC1-RzC1、pC1-RzC2、pC1-RzC3抑制活性分别为53.2%、50.5%、10.6%(t=5.25,P<0.01)。pC1-RzC1连续1周作用于HCV阳性的胎肝细胞,结果显示第2～7天的抑制活性分别是60.7%、64.2%、68.4%、71.9%、78.8%、83.1%(t=15.34,P<0.01)。结论 pC1-RzC1、pC1-RzC2在HCV阳性胎肝细胞中对HCV RNA的抑制活性明显高于pC1-RzC3。     

Masking the 5' terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex

Hepatitis C virus subverts liver-specific microRNA, miR-122, to upregulate viral RNA abundance in both infected cultured cells and in the liver of infected chimpanzees. These findings have identified miR-122 as an attractive antiviral target. Thus, it is imperative to know whether a distinct functional complex exists between miR-122 and the viral RNA versus its normal cellular target mRNAs. Toward this goal, effects on viral RNA abundance of mutated miR-122 duplex molecules, bound at each of the two target sites in the viral genome, were compared to effects on microRNA- or siRNA-mediated regulation of reporter target mRNAs. It was found that miR-122 formed an unusual microRNA complex with the viral RNA that is distinct from miR-122 complexes with reporter mRNAs. Notably, miR-122 forms an oligomeric complex in which one miR-122 molecule binds to the 5' terminus of the hepatitis C virus (HCV) RNA with 3' overhanging nucleotides, masking the 5' terminal sequences of the HCV genome. Furthermore, specific internal nucleotides as well as the 3' terminal nucleotides in miR-122 were absolutely required for maintaining HCV RNA abundance but not for microRNA function. Both miR-122 molecules utilize similar internal nucleotides to interact with the viral genome, creating a bulge and tail in the miR-122 molecules, revealing tandemly oriented oligomeric RNA complexes. These findings suggest that miR-122 protects the 5' terminal viral sequences from nucleolytic degradation or from inducing innate immune responses to the RNA terminus. Finally, this remarkable microRNA-mRNA complex could be targeted with compounds that inactivate miR-122 or interfere with this unique RNA structure.     

Inhibition of hepatitis C virus-directed gene expression by a DNA ribonuclease.

BACKGROUND/AIMS: The aim of this study was to determine whether DNA analogs of ribozymes could be prepared to inhibit hepatitis C virus (HCV) gene expression. METHODS: Two DNA ribonucleases, Dz2 and Dz4, were designed with varying arm lengths, to cleave at the 5'-noncoding region (NCR) just upstream from the translation start site, and core region of HCV genome, respectively. A reporter vector was prepared to contain target HCV regulatory sequences controlling a downstream luciferase gene. DNA ribonucleases with normal phosphodiester, as well as with terminal phosphorothioate linkages, were administered to Huh7 cells, and luciferase activity was measured. RESULTS: DNA ribonucleases were highly active in cleaving HCV RNA targets. Enzymes with longer arms had consistently higher cleavage activity compared to enzymes with shorter arms under cell-free conditions. Furthermore, in Huh7 cells, terminal phosphorothioate derivatives, Dz2 and Dz4, significantly suppressed HCV-luciferase fusion gene expression up to 45% and 67% of controls, respectively. Interestingly, phosphorothioate-modified DNA ribonucleases had greater inhibitory effects on target gene expression than their unmodified counterparts. In contrast, DNA ribonucleases with point mutations in the catalytic domain had significantly lower inhibitory effects compared to wild-type DNA ribonucleases. However, activity was not eliminated, suggesting that some antisense contribution was present. CONCLUSIONS: DNA ribonucleases directed against the HCV genome can specifically cleave target HCV RNA. Modifications of the extreme 3'- and 5'-termini protect against nuclease degradation without appreciable reduction in inhibitory activity against viral gene expression under intracellular conditions.     

A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice

Hepatitis C virus (HCV) is an RNA virus infecting one in every 40 people worldwide. Current treatments are ineffective and HCV is the leading cause of liver failure leading to transplantation in the United States and Europe. Translational control of HCV is a prime therapeutic target. We assessed the inhibitory potential of morpholino phosphoramidate antisense oligonucleotides (morpholinos) on HCV translation by codelivering them with reporter plasmids expressing firefly luciferase under the translational control of the HCV internal ribosome entry site (IRES) into the livers of mice. Real-time imaging of HCV IRES luciferase reporter messenger RNA (mRNA) translation in living mice showed that a 20-mer complementary to nucleotides 345-365 of the IRES inhibited translation by greater than 95% for at least 6 days and showed mismatch specificity. No significant nonspecific inhibition of a cap-dependent luciferase or encephalomyocarditis virus (EMCV) IRES luciferase reporter translation was observed. Inhibition by the 20-mer morpholino was dose dependent, with 1 nmol/mouse giving the highest inhibition. In conclusion, morpholino antisense oligonucleotides are potent inhibitors of HCV IRES translation in a preclinical mouse model; morpholinos have potential as molecular therapeutics for treating HCV and other viral infections. The in vivo model described is a broadly applicable, straightforward, and rapid readout for inhibitor efficacy. As such, it will greatly facilitate the development of novel therapeutic strategies for viral hepatitis. Notably, the level of antisense inhibition observed in this in vivo model is similar to the maximal inhibition we have obtained previously with RNA interference in mice.     

丙型肝炎病毒5′非翻译区 DNA 序列在 HepG2细胞中的启动子活性

目的 研究丙型肝炎病毒(HCV)基因组5′非翻译区(5′UTR)DNA 序列在 HepG2细胞中 的启动子活性，以了解 HCV 的复制调控机制。方法 分别构建 HCV 基因组5′UTR DNA 正反向序列驱动 虫荧光素酶基因表达的质粒5′UTR-Luc(+)/(-)和5′UTR DNA 序列驱动绿色荧光蛋白基因表达的质粒5′ UTR-EGFP(+)/(-)，分别转染 HepG2细胞，用双荧光素酶检测系统检测虫荧光素酶的表达水平，逆转录 聚合酶链反应检测虫荧光素酶基因 m R N A 水平，荧光显微镜观察绿色荧光蛋白基因的表达水平，并与相应 对照作比较，来证实 HCV 基因组5′UTR DNA 序列的启动子活性。结果 5′UTR-Luc(+)有明显的虫 荧光素酶表达，但比 pGL3 control 表达水平低(Luc/R为0．690±0．086，Luc/RL 为4．210±0．340)，而5 ′UTR-Luc(-)和 pGL3 enhancer 无明显虫荧光素酶表达(Luc/RL 分别为0．095±0．008和0．044±0． 005)；逆转录聚合酶链反应结果与之相符，5′UTR-Luc(+)检测到虫荧光素酶基因 mRNA，而5′UTR- Luc(-)则未检测到。5′UTR-EGFP(+)观察到较强绿色荧光，而5′UTR-EGFP(-)无荧光表达。 结论 HCV 基因组5′U TR DNA 序列具有明显的启动子活性，能启动下游基因的表达，在 HCV 基因组复 制过程中有重要作用。