Selective inhibition of hepatitis B virus replication by RNA interference

Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells including genes of viral origin. To evaluate the therapeutic efficacy of siRNA against the hepatitis B virus (HBV), we studied the effect of transfection of the HBV-inducible cell lines HepAD38 and HepAD79 with siRNA specific for the core gene of the HBV genome. HepAD38 cells produce wild-type HBV, whereas HepAD79 cells produce the lamivudine resistant YMDD variant. Transfection of HepAD38 cells with either 1.6 or 4 microg/ml siRNA resulted in a profound inhibition (72% and 98%, respectively) of viral replication (as assessed by real-time quantitative PCR). The inhibitory effect was corroborated by a marked reduction of HBV core protein synthesis in induced HepAD38 cells. In HepAD79 cells, transfected with 1.6 or 4 microg/ml HBV-specific siRNA, virus production was reduced by 75% and 89%, respectively.     

Inhibition of virus replication by RNA interference

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene function by reverse genetics. Additionally, inhibition of virus replication by means of induced RNAi has now been reported for numerous viruses, including several important human pathogens such as human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, dengue virus, poliovirus and influenza virus A. In this review, we will summarize the current data on RNAi-mediated inhibition of virus replication and discuss the possibilities for the development of RNAi-based antiviral therapeutics.     

Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.     

A retrovirus-based system to stably silence hepatitis B virus genes by RNA interference

RNA interference (RNAi) might be an efficient antiviral therapy for some obstinate illness. Herein, a retrovirus-based RNAi system was developed to drive expression and delivery of Hepatitis B virus (HBV)-specific short hairpin RNA (shRNA) in HepG2 cells. The levels of HBsAg and HBeAg and that of HBV mRNA were dramatically decreased by this RNAi system in HepG2 cells transfected with Topo-HBV plasmid. Retrovirus-based RNAi thus may be useful for therapy in HBV and other viral infections and provide new clues for prophylactic vaccine development.     

Inhibition of virus production in JC virus-infected cells by postinfection RNA interference

RNA interference has been applied for the prevention of virus infections in mammalian cells but has not succeeded in eliminating infections from already infected cells. We now show that the transfection of JC virus-infected SVG-A human glial cells with small interfering RNAs that target late viral proteins, including agnoprotein and VP1, results in a marked inhibition both of viral protein expression and of virus production. RNA interference directed against JC virus genes may thus provide a basis for the development of new strategies to control infections with this polyomavirus.     

Inhibition of herpesvirus-6B RNA replication by short interference RNAs

RNA interference (RNAi) is a process of sequence-specific gene silencing, which is initiated by double-stranded RNA (dsRNA). RNAi may also serve as an antiviral system in vertebrates. This study describes the inhibition of herpesvirus-6B (HHV-6B) replication by short interference RNAs (siRNAs) that are targeted to the U38 sequence that encodes DNA polymerase. When virus-infected SupT1 cells were treated by siRNA, these cells blocked the cytopathic effect (CPE) and detected the HHV-6B antibody-negative in indirect immunofluorescence assays (IFA). Our result suggests that RNAi can efficiently block Herpesvirus-6B replication.     

Inhibition of alpha interferon signaling by hepatitis B virus

Alpha interferon (IFN-alpha) and pegylated IFN-alpha (pegIFN-alpha) are used for the treatment of chronic hepatitis B (CHB). Unfortunately, only a minority of patients can be cured. The mechanisms responsible for hepatitis B virus (HBV) resistance to pegIFN-alpha treatment are not known. pegIFN-alpha is also used to treat patients with chronic hepatitis C (CHC). As with chronic hepatitis B, many patients with chronic hepatitis C cannot be cured. In CHC, IFN-alpha signaling has been found to be inhibited by an upregulation of protein phosphatase 2A (PP2A). PP2A inhibits protein arginine methyltransferase 1 (PRMT1), the enzyme that catalyzes the methylation of the important IFN-alpha signal transducer STAT1. Hypomethylated STAT1 is less active because it is bound by its inhibitor, PIAS1. In the present work, we investigated whether similar molecular mechanisms are also responsible for the IFN-alpha resistance found in many patients with chronic hepatitis B. We analyzed the expression of PP2A, the enzymatic activity of PRMT1 (methylation assays), the phosphorylation and methylation of STAT1, the association of STAT1 with PIAS1 (via coimmunoprecipitation assays), the binding of activated STAT1 to interferon-stimulated response elements (via electrophoretic mobility shift assays), and the induction of interferon target genes (via real-time RT-PCR) in human hepatoma cells expressing HBV proteins as well as in liver biopsies from patients with chronic hepatitis B and from controls. We found an increased expression of PP2A and an inhibition of IFN-alpha signaling in cells expressing HBV proteins and in liver biopsies of patients with CHB. The molecular mechanisms involved are similar to those found in chronic hepatitis C.     

多种小分子干扰RNA联合抑制乙型肝炎病毒的体外研究

小分子干扰RNA(siRNA)能够在哺乳动物细胞中引起包括病毒基因在内的基因沉默。为了研究多种siRNA联合应用在体外抑制乙型肝炎病毒(HBV)复制中的效果,本研究设计了12种针对不同HBV靶点的siRNA,转染可稳定分泌HBV颗粒的HepG22．2．15细胞,并采用了酶联免疫法检测上清液中HBsAg和HBeAg的含量,实时定量PCR法检测细胞中HBV的DNA含量。结果发现这12种分子均能在不同程度上抑制病毒复制。进一步研究表明它们对HBV的抑制作用在一定程度上存在剂量效应和协同作用,单分子siRNA在80nmol／L处对HBsAg和HBeAg的抑制率分别可达到约80％和60％,而多分子siRNA组合在20nmol／L处对HBsAg就能达到90％的抑制率,但对HBeAg表达的抑制率提高不明显。单分子siRNA在80nmol／L处对HBVDNA复制的抑制率可达到90％以上,而多分子siRNA组合在20nmol／L处对DNA含量就能达到约90％的抑制率。本研究的结果为进一步开发新的联合应用多种siRNA治疗HBV的途径打下了基础。     

Inhibition of retroviral pathogenesis by RNA interference

BACKGROUND: RNA interference (RNAi) is a newly discovered cellular defense system that is known to suppress replication of genomic parasites in model organisms. It has been widely conjectured that RNAi may also serve as an antiviral system in vertebrates. RESULTS: Retroviral infection could be initiated by electroporation of cloned Rous sarcoma virus (RSV) proviral DNA into the developing chick neural tube. Coelectroporation of proviral DNA and short double-stranded RNAs matching sequences of avain retroviruses, which were designed to induce RNAi against RSV, inhibited viral replication. Replication of RSV after electroporation resulted in disruption of embryonic development and early death, but this, too, could be suppressed by RNAi against the RSV genome. RNAi could also inhibit the growth of RSV and HIV in cell culture. Analysis of the step of the retroviral life cycle that is inhibited by RNAi revealed that it primarily prevented accumulation of the viral RNAs synthesized late during infection. RNA genomes introduced in viral particles early during infection were less sensitive. CONCLUSIONS: RNAi can block retroviral infection in vertebrates. The tissue electroporation method described here should allow RNAi to be used widely to study gene function and control of infection in vertebrate animals.     

Inhibition of gammaherpesvirus replication by RNA interference

RNA interference (RNAi) is a conserved mechanism in which double-stranded, small interfering RNAs (siRNAs) trigger a sequence-specific gene-silencing process. Here we describe the inhibition of murine herpesvirus 68 replication by siRNAs targeted to sequences encoding Rta, an immediate-early protein known as an initiator of the lytic viral gene expression program, and open reading frame 45 (ORF 45), a conserved viral protein. Our results suggest that RNAi can block gammaherpesvirus replication and ORF 45 is required for efficient viral production.     

Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.     

Inhibition of replication of human hepatitis B virus

Several nucleoside 5'-triphosphate analogs were investigated as inhibitors of human hepatitis B virus replication. Different analogs inhibited DNA synthesis differently, 3'-azido-2',3'-dideoxythymidine 5'triphosphate being the most active compound. This inhibitor blocked DNA synthesis by 50% at inhibitor: substrate molar ratio 1:8, and by 80% - at 1:1. The hypothesis is formulated that 3'-azido-2',3'-dideoxythymidine 5'-triphosphate inhibits RNA directed viral DNA replication due to incorporation of this compound into 3'-termini of newly synthesized DNA chains. The phenomenon observed opens new possibilities for chemotherapy of acute and chronic human hepatitis B.     

Stable inhibition of hepatitis B virus expression and replication in HepG2.2.15 cells by RNA interference based on retrovirus delivery

RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral strategy. In order to suppress hepatitis B virus (HBV) expression and replication, a retrovirus-based RNAi system was developed, which utilized the U6-RNA polymerase III (Pol III) promoter to drive efficient expression and deliver the HBV-specific short hairpin RNAs (shRNAs) in HepG2.2.15 (2215) cells. In this system, the retrovirus vector with a puromycin selection marker was integrated into the host cell genome and allowed stable expression of shRNAs. In Puro-resistant 2215 cells, the levels of both HBV protein and mRNA were dramatically reduced by over 88% and HBV replication was suppressed. The results demonstrated that retrovirus-based RNAi technology will have foreseeable applications both in experimental biology and molecular medicine.     

Inhibition of hepatitis B virus replication by interferon requires proteasome activity

Hepatitis B virus (HBV) replication is inhibited in a noncytopathic manner by alpha/beta interferon (IFN-alpha/beta) and IFN-gamma. We demonstrate here that inhibitors of cellular proteasome activity can block this antiviral effect. These results suggest that a critical component of the IFN-induced antiviral response may be the proteasome-dependent degradation of viral or cellular proteins that are required for HBV replication.