Quick identification of effective small interfering RNAs that inhibit the replication of coxsackievirus A16

Coxsackievirus A16 (CA16) is a major causative agent of hand, foot, and mouth disease (HFMD). It can cause myocarditis, pericarditis and fatal shock. There is no effective therapy against CA16. RNA interference (RNAi) is a powerful tool to silence gene expression. The small interfering RNA (siRNA) that induces RNA degradation has recently been used as an anti-virus agent to inhibit virus replication. In this study, we established the complete nucleotide sequence of CA16 strain Shzh05-1, and then compared the nucleotide sequences of Shzh05-1 with sequences of other CA16 strains in GenBank. We chose conserved regions between Shzh05-1 and the two other CA16 strains to design 30 siRNAs and construct siRNA-encoding plasmids. Thirteen siRNAs targeting conserved regions of the virus could effectively block replication of CA16 in cultured cells. Combination transfection of these 13 effective siRNAs could also produce a high inhibitory effect. These strategies and results suggest that RNAi has potential therapeutic use for suppression of CA16 infection.     

Effective small interfering RNAs targeting matrix and nucleocapsid protein gene inhibit influenza A virus replication in cells and mice

RNA interference (RNAi) is a powerful tool to silence gene expression. Small interfering RNA (siRNA)-induced RNA degradation has been recently used as an antivirus agent to inhibit specific virus replication. Here, we showed that several siRNAs specific for conserved regions of influenza virus matrix (M2) and nucleocapsid protein (NP) genes could effectively inhibit expression of the corresponding viral protein. We also evaluated the antiviral potential of these siRNAs targeting M2 and NP of H5N1 avian influenza virus (AIV), which are essential to viral replication. We investigated the inhibitory effect of M2-specific siRNAs and NP-specific siRNAs on influenza A virus (H5N1, H1N1 and H9N2) replication in Madin-Darby canine kidney (MDCK) cells and BALB/c mice. The results showed that treatment with these siRNAs could specifically inhibit influenza A virus replication in MDCK cells (0.51-1.63 TCID(50) reduction in virus titers), and delivery of pS-M48 and pS-NP1383 significantly reduced lung virus titers in the infected mice (16-50-fold reduction in lung virus titers) and partially protected the mice from lethal influenza virus challenge (a survival rate of 4/8 for H1N1 virus-infected mice and 2/8 for H5N1 virus infected mice). Moreover, the treatment of pS-M48 and pS-NP1383 could suppress replication of different subtypes of influenza A viruses, including a H5N1 highly pathogenic avian isolate strain. The results provided a basis for further development of siRNA for prophylaxis and therapy of influenza virus infection in humans and animals.     

Inhibition of multiple strains of Venezuelan equine encephalitis virus by a pool of four short interfering RNAs

RNA interference, mediated by short interfering RNAs (siRNAs), has been shown to have activity against a wide range of viruses and is a promising new antiviral therapy. Using multiple siRNAs that target conserved areas of the genome allows for increased chances of antiviral activity against different viral strains and also helps to prevent the emergence of escape mutants. In this study, four siRNAs were designed to target areas of conserved sequence between divergent strains of Venezuelan equine encephalitis virus (VEEV). A pool of these siRNAs inhibited the replication of all six strains of VEEV tested. A single nucleotide mismatch at the extreme 3' end of one of the siRNA sense strands did not affect antiviral activity but other mutations were not tolerated. Two strains of VEEV were tested for their abilities to overcome the inhibitory effects of RNA interference following 10 consecutive incubations in the presence of siRNAs. One strain remained susceptible throughout the course of the experiment but the other strain became resistant to the activity of siRNAs. Sequence analysis of the siRNA target sites in this strain showed that no mutations had been generated, indicating that the virus may had become resistant in some other manner. In the absence of effective antiviral drugs and vaccines to combat VEEV infection, these siRNAs offer a potential new therapeutic approach but, as with all antimicrobial agents, caution needs to be exercised with respect to the generation of resistance.     

RNA interference-based gene silencing in mice: the development of a novel therapeutical strategy

RNAi (RNA interference) was originally detected in Caenorhabditis elegans as biological response to exogenous double-stranded RNA (dsRNA), which induces very effective sequence-specific silencing of gene expression. Further investigations revealed that RNAi can occur in many eukaryotic species. Increasing understanding of the biochemical components of RNAi indicates the existence of a conserved machinery for dsRNA-induced gene silencing that acts in two steps. In the first step, an RNase III family nuclease called Dicer processes the dsRNA to small interfering RNAs (siRNAs) 21-23 nt in length. These siRNAs enter a multimeric nuclease complex that identifies target mRNAs through their homology to siRNAs and induce destruction of the corresponding mRNAs. Since RNAi has become an excellent strategy for gene silencing, it is tempting to apply this technology to 'knock-down' gene expression in living animals. The generation of transgenic mice from embryonic stem cells expressing small hairpin RNAs (shRNAs) has provided evidence for in vivo application of RNAi. Furthermore, different experimental strategies have been developed to analyze the influence of chemically synthesized siRNAs and of vector-based shRNAs on the expression of different transgenes and endogenous genes in vivo. Recent studies describe the in vivo delivery of siRNAs to inhibit transgene expression in certain organs of adult mice, predominately murine liver. Strategies for the inhibition of cellular proliferation by systemic treatment of tumor-bearing animals with siRNAs are beginning to emerge. They are of utmost interest for systemic diseases such as cancer. In addition, several groups have shown that RNAi can also be used to block the infectivity or suppress the replication of different RNA viruses relevant to human diseases including human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV). In summary, multiple lines of evidence indicate that RNAi seems to become a powerful tool for the fight against undesirable gene expression in human diseases.     

Inhibition of measles virus and subacute sclerosing panencephalitis virus by RNA interference

Subacute sclerosing panencephalitis (SSPE) is a rare, but fatal outcome of measles virus (MeV) infection. SSPE develops after prolonged persistence of mutated MeV called SSPE virus. Although a combination therapy using interferon and inosiplex or ribavirin appears to prolong survival time to some extent, there is currently no effective treatment to completely cure SSPE and a new treatment strategy is greatly needed. In this study, we adopted RNA interference (RNAi) strategy and examined whether small interfering RNAs (siRNAs) can be used to inhibit replication of MeV and SSPE virus. We report here that siRNAs targeted against L mRNA of MeV, either synthetic siRNAs or those generated by pcPUR + U6i-based expression plasmids, effectively and specifically inhibited replication of both MeV and SSPE virus without exhibiting any cytotoxic effect. The L protein of MeV is a major component of RNA-dependent RNA polymerase that is essential for viral RNA replication, and yet it is least abundant among all the MeV proteins expressed. Therefore, mRNA encoding the L protein would be a good target for RNAi strategy. The present results imply the possibility that our siRNAs against MeV L mRNA are among the potential candidates to be used to treat patients with SSPE.     

Evaluation of synthetic oligonucleotides as inhibitors of West Nile virus replication

A series of synthetic oligonucleotide phosphorothioate 15-mers were generated against specific sequences in the West Nile virus RNA genome. These antisense oligonucleotides targeted (1) conserved features of the West Nile virus RNA genome that may be expected to lead to inhibition of virus replication since such features play essential roles in the virus lifecycle; (2) G-quartet oligonucleotides with potential facilitated uptake properties and that also targeted conserved sequences among a range of West Nile virus strains. Several formulations with significant in vitro antiviral activity were found. Among the active oligonucleotides were examples that targeted both C-rich RNA sequences of the West Nile RNA genome as well as recognized conserved sequences key to West Nile virus replication. Since the antiviral activity of the latter oligonucleotides diminished upon 2'-O-methyl substitution, it is likely that their activity involves RNase H-catalyzed RNA degradation. One G-rich oligonucleotide that did not target a West Nile virus RNA sequence also was found. These results suggest the potential of antisense strategies for the control of West Nile virus replication if the attendant problem of oligonucleotide delivery can be adequately addressed.     

RNA干扰技术抗肠道病毒71型复制

目的肠道病毒71型(Enterovirus 71,EV 71)是手足口病(hand,foot and mouth disease,HFMD)主要的病原体之一。研究RNA干扰技术抑制肠道病毒71型复制。方法以RNA干扰技术(RNAi)为干预手段,抑制病毒EV 71在人横纹肌肉瘤(rhabdomyosarcoma,RD)细胞中的复制。通过Western blot、Real-time PCR和病毒滴度3种方法验证,其抑制效果体现在感染细胞内部病毒RNA,病毒蛋白质的表达水平和培养基上清中子代病毒颗粒的数量上。结果靶向病毒基因组5'UTR和VP2的siRNAs具有明显的病毒抑制效应。结论此研究证实RNAi方法具有特异性抑制病毒复制的潜能和可行性。     

Potential applications of siRNA in hepatitis C virus therapy

Small interfering RNAs (siRNAs) are short RNA duplexes approximately 21 nucleotides long. When introduced into mammalian cells, siRNA can silence specific gene expression. Hepatitis C virus (HCV) replicates in the cytoplasm of liver cells without integration into the host genome. Because the HCV genome is a single-stranded RNA that functions both as a messenger RNA and as a viral replication template, destruction of HCV RNA could eliminate not only virally directed protein synthesis, but also viral replication. It has been demonstrated that siRNAs interfere with HCV gene expression and replication, and this review will describe the use of RNAi as a tool to inhibit HCV gene expression.     

Specific small interfering RNAs-mediated inhibition of replication of porcine encephalomyocarditis virus in BHK-21 cells

Encephalomyocarditis virus (EMCV) is recognized as a pathogen inducing acute myocarditis and sudden death in preweaned piglets and severe reproductive failure in sows. In this study, eight specific small interfering RNA (siRNA) duplexes targeting different genomic regions of EMCV BJC3 were designed and their ability to inhibit virus replication in BHK-21 cells was investigated. The results showed that BHK-21 cells transfected with siRNA duplexes to 2C gene (JH-4,666, BJC-1,739), 2B gene (BJC-807), 3C gene (BJC-2,363) and 3D gene (BJC-3269) were specifically resistant to EMCV infection when exposed to 500 times the 50% cell culture infective dose (CCID(50)) of EMCV. The levels of the 3D gene in the transfected cells were obviously decreased. IFA and Western blotting analysis confirmed that the expression of VP1 protein in cell culture transfected with the siRNAs was apparently reduced. Of the five siRNAs, JH-4,666, BJC-2,363 and BJC-3,269 were the most effective. Combination of the siRNA duplexes enhanced the inhibition of EMCV replication. Our data indicated that specific siRNAs are able to inhibit the replication of porcine encephalomyocarditis virus in BHK-21 cells, suggesting that RNAi might provide a new approach to prevent EMCV infection.     

Enhanced potency and efficacy of 29-mer shRNAs in inhibition of Enterovirus 71

Enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) in young children. It has been associated with severe neurological complications and has caused significant mortalities in large-scale outbreaks in Asia. In this study, we demonstrated an enhanced silencing of EV71 through the use of chemically synthesized 29-mer shRNAs. The 29-mer shRNAs were designed to target three highly conserved regions of EV71 genome. Transfection of rhabdomyosarcoma (RD) cells with the 29-mer shRNAs significantly inhibited EV71 replication in a dose-dependent manner as demonstrated by reduction of viral RNA, VP1 protein and plaque forming units. The inhibitory effects were more potent and were achieved at 10-fold lower concentrations when compared to 19-mer siRNAs reported previously [Sim, A.C.N., Luhur, A., Tan, T.M.C., Chow, V.T.K., Poh, C.L., 2005. RNA interference against Enterovirus 71 infection. Virology 341, 72-79]. The viral inhibitory effects lasted 72 h post-infection and there was no adverse off-target silencing effect. Gene silencing by 29-mer shRNAs targeted at the 3D(pol) region (sh-3D) was the most effective, achieving 91% viral inhibition. Further evaluation found that no enhanced inhibitory effects were observed when sh-3D was cotransfected with each of the other two candidates. This study showed an improvement in triggering RNAi using the more potent 29-mer shRNAs, indicating its therapeutic potential against EV71.     

siRNA pool targeting different sites of human hepatitis B surface antigen efficiently inhibits HBV infection

The main objective was to determine whether a pool of small interfering RNAs (siRNAs) targeting different regions of hepatitis B virus surface antigen (HBsAg) efficiently inhibits hepatitis B virus (HBV) infection. siRNAs targeting different regions of HBsAg were transfected into HBV-producing HepG2.2.15 cells and at 72 h post-transfection, the culture medium was collected for ELISA to determine HBsAg, while total RNA was isolated from the cells for real-time PCR. Three siRNA sequences that efficiently inhibited HBV infection were converted into small hairpin RNAs (shRNAs) and then cloned into a single plasmid psiSTRIKE driven by a single U6 promoter. These shRNA expressing plasmids were tested for HBsAg gene silencing in HepG2.2.15 cells. A pool of siRNAs targeting HBsAg efficiently inhibited HBV replication and antigen expression when transfected into HepG2.2.15 cells, compared with the use of single siRNA. Similarly, the plasmid encoding three different shRNAs driven by a single U6 promoter was more effective in silencing HBsAg at DNA, mRNA and protein levels compared with the plasmid encoding single shRNA. No apoptotic change was observed in the cells when the plasmid was transfected at a dose of 0.5-2 microg/1 x 10(6) cells after complex formation with Lipofectamine LTX. Furthermore, transfection with siRNA or shRNA did not increase interferon-gamma (IFNs-gamma) release, suggesting no induction of IFN response. In conclusion, a pool of chemically synthesised siRNAs as well as the shRNA expression plasmid encoding multiple shRNAs targeting different regions of HBsAg showed high gene silencing in HepG2.2.15 cells.     

Combination of soluble coxsackievirus-adenovirus receptor and anti-coxsackievirus siRNAs exerts synergistic antiviral activity against coxsackievirus B3

Coxsackievirus B3 (CVB-3) is a major causative agent of chronic heart muscle infections. The present study describes a cell culture system with an ongoing virus infection to evaluate two novel inhibitory strategies, either individually or combined: (1) RNA interference (RNAi) to degrade cytoplasmatic CVB-3 RNA and (2) a vector-delivered soluble variant of the coxsackievirus-adenovirus receptor fused to a human immunoglobulin (sCAR-Fc), which inhibits cellular uptake of CVB-3. Both approaches were capable of inhibiting CVB-3 in persistently infected human myocardial fibroblasts. The antiviral effect of a single treatment lasted for up to one week and could be extended by repeated applications. Each of the single treatments initially reduced the virus titer by approximately 1-log, whereas the combination of both approaches resulted in 4-log inhibition and retained substantial antiviral activity at later time points, when the effect of sCAR-Fc or siRNAs alone had already disappeared. Further analysis revealed that sCAR-Fc protects cells from virus-induced lysis but does not diminish the virus load. Reduction of the virus titer was only achieved with additional destruction of viral RNA by RNAi. Taken together, combination of RNAi and a protein-based antiviral strategy was found to result in a strong synergistic inhibition of an ongoing virus infection.     

Simultaneously inhibition of HIV and HBV replication through a dual small interfering RNA expression system

Human immunodeficiency virus (HIV) is often acquired in individuals already infected with hepatitis B virus (HBV) as a result of shared routes of transmission. Since current options for the treatment of HIV and HBV infections are limited, there is an essential need for the development of effective therapies against HIV/HBV co-infections. RNA interference (RNAi) has been used as a powerful tool to silence genes in cells and animals. In this study, we developed a small interfering RNA generation system that expressed two different siRNAs to target the HBs gene of HBV and the gp120 gene of HIV in Bel-7402 and HEK293T cells, respectively. Our results demonstrated that the two siRNA molecules could simultaneously inhibit the expression of HBs and gp120 by 81% and 89%, respectively. In addition, dual siRNA molecules significantly decreased the production of HBs, and simultaneously inhibited the replication of HBV and HIV. This dual siRNA generation system not only proved to be a novel approach for studying functions of multiple genes simultaneously, but also provides a potential approach for the treatment and prevention of HIV and HBV co-infection.     

Artificial microRNAs can effectively inhibit replication of Venezuelan equine encephalitis virus

Venezuelan equine encephalitis virus is a member of the alphavirus family and genus togaviridae. VEEV is highly infectious in aerosol form and has been weaponized in the past making it a potential biothreat agent. At present, there are no FDA approved antiviral treatments or vaccines for VEEV. Artificial microRNAs are small molecules which are expressed through endogenous microRNA machinery by RNA polymerase II. These artificial microRNAs effectively inhibit gene expression and are non-toxic to the host cell. VEEV RNA dependent RNA polymerase (RdRp) is central to VEEV replication. Therefore, we hypothesize that targeted inhibition of VEEV RdRp using artificial microRNAs may efficiently inhibit VEEV replication. Five artificial microRNAs were tested in vitro in BHK cells. Three of these artificial miRNAs showed significant inhibition of VEEV replication. Further, these microRNAs were cloned into the expression vector in combination to see the synergistic effect on VEEV replication. Combination of more than one miRNA did not result in significant inhibition of virus replication. In conclusion, we have shown that RNAi through artificial microRNAs effectively inhibits VEEV replication and is significantly less toxic in comparison to siRNAs.     

RNA interference and innate immunity

RNA interference is an evolutionarily conserved gene silencing process triggered by double-stranded RNAs. Common to all cell types, is the production of 21-24 nucleotide small interfering RNA (siRNAs), which guide the RNA-induced silencing complex (RISC) to identify and cleave target mRNA sequences. Presently, this biological breakthrough method has revolutionised gene function studies and holds great promise as validating drug targets and treating human diseases. However, despite the success that has been achieved by this technology, studies carried in human blood cells have revealed that siRNAs could generate bystander effects, including the activation of innate immunity and inhibition of unintended target genes. Interestingly, 2' uridine-modified siRNAs did not trigger TLR signalling, but they totally suppressed immune activation by immunostimulatory siRNAs when both molecules where delivered to the same endosomes. This review describes the recent advances in understanding the innate immune response to both single and double-stranded siRNAs. Also, it highlights the spectrum of molecular strategies allowing the design of therapeutic siRNAs with minimal side effects.