The efficacy of generating three independent anti-HIV-1 siRNAs from a single U6 RNA Pol III-expressed long hairpin RNA

RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs.     

Design of extended short hairpin RNAs for HIV-1 inhibition

RNA interference (RNAi) targeted towards viral mRNAs is widely used to block virus replication in mammalian cells. The specific antiviral RNAi response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular expression of short hairpin RNAs (shRNAs). For HIV-1, both approaches resulted in profound inhibition of virus replication. However, the therapeutic use of a single siRNA/shRNA appears limited due to the rapid emergence of RNAi-resistant escape viruses. These variants contain deletions or point mutations within the target sequence that abolish the antiviral effect. To avoid escape from RNAi, the virus should be simultaneously targeted with multiple shRNAs. Alternatively, long hairpin RNAs can be used from which multiple effective siRNAs may be produced. In this study, we constructed extended shRNAs (e-shRNAs) that encode two effective siRNAs against conserved HIV-1 sequences. Activity assays and RNA processing analyses indicate that the positioning of the two siRNAs within the hairpin stem is critical for the generation of two functional siRNAs. E-shRNAs that are efficiently processed into two effective siRNAs showed better inhibition of virus production than the poorly processed e-shRNAs, without inducing the interferon response. These results provide building principles for the design of multi-siRNA hairpin constructs.     

RNAi as a treatment for HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) was the first primate virus shown to be inhibited by RNA interference (RNAi). Early studies used both synthetic and promoter expressed small interfering RNAs (siRNAs) or expressed short hairpin RNAs (shRNAs) to demonstrate that this virus was susceptible to RNAi. In addition to targeting the virus itself RNAi-mediated down-regulation of cellular targets that encode receptors required for viral entry also proved to be effective. The power of RNAi as an anti-HIV agent has propelled development of RNAi-based gene therapy approaches for the treatment of HIV infection in humans. Nevertheless, extensive in vitro experimentation has revealed potential problems of viral escape mutants and other toxicities caused by the si/shRNAs. This review covers the progress and problems in the development of RNAi for the treatment of HIV infection. Potential modalities for clinical application of RNAi in the treatment of HIV-1 infection are also described.     

Hairpin RNA-mediated strategies for silencing of tomato leaf curl virus AC1 and AC4 genes for effective resistance in plants

RNA interference (RNAi) using short interfering RNAs (siRNAs) has been widely explored for the suppression of intracellular viral target mRNAs. On the basis of our previous work with stable silencing of Tomato leaf curl virus, in vivo by the antisense replicase gene (AC1) of the virus and characterizing AC4, as a small RNA regulator, besides its role in pathogenicity, we used four different plasmid vector-based siRNA generation strategies to silence viral genes (AC1 and AC4) of tomato leaf curl viruses. The RNAi target sequence were chosen from DNA A of the Tomato leaf curl virus (ToLCV) on the basis of conserved regions in AC1 with an overlapping sequences of the AC4 gene. Different hairpin RNA-mediated strategies like antisense, self-complementary inverted repeats, intron-spliced hairpin RNAs, and small hairpin RNAs were deployed for efficient and predictable resistance to the viruses. Here we present that appropriately designed siRNAs not only prevents RNAi suppression but also help in developing trait-stable transgenics. These strategies imply that ToLCV rep-driven RNAi, targeting AC4 and conserved viral sequences, provides a promising approach to suppress a wide spectrum ToLCV infection in the tomato.     

Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs

Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It has recently been shown that HCV RNA replication is susceptible to small interfering RNAs (siRNAs), but the antiviral activity of siRNAs depends very much on their complementarity to the target sequence. Thus, the high degree of sequence diversity between different HCV genotypes and the rapid evolution of new quasispecies is a major problem in the development of siRNA-based gene therapies. For this study, we developed two alternative strategies to overcome these obstacles. In one approach, we used endoribonuclease-prepared siRNAs (esiRNAs) to simultaneously target multiple sites of the viral genome. We show that esiRNAs directed against various regions of the HCV coding sequence as well as the 5' nontranslated region (5' NTR) efficiently block the replication of subgenomic and genomic HCV replicons. In an alternative approach, we generated pseudotyped retroviruses encoding short hairpin RNAs (shRNAs). A total of 12 shRNAs, most of them targeting highly conserved sequence motifs within the 5' NTR or the early core coding region, were analyzed for their antiviral activities. After the transduction of Huh-7 cells containing a subgenomic HCV replicon, we found that all shRNAs targeting sequences in domain IV or nearby coding sequences blocked viral replication. In contrast, only one of seven shRNAs targeting sequences in domain II or III had a similar degree of antiviral activity, indicating that large sections of the NTRs are resistant to RNA interference. Moreover, we show that naive Huh-7 cells that stably expressed certain 5' NTR-specific shRNAs were largely resistant to a challenge with HCV replicons. These results demonstrate that the retroviral transduction of HCV-specific shRNAs provides a new possibility for antiviral intervention.     

靶向HIV-1pol的高效人工miRNA的构建与体外抗病毒能力评价

RNAi技术在抗HIV-1治疗研究中已显示出巨大潜力,获得可高效特异抑制HIV-1的RNAi元件是进行相关研究的重要基础。miRNA在抑制和表达方式上相比siRNA具有更多的优势。本研究即探讨构建可高效特异靶向HIV-1的人工miRNA元件。选择以保守性较好的HIV-1pol基因为靶区筛选高效保守的RNAi序列,设计了16个可靶向pol区高保守区段的RNAi靶点,构建表达载体与HIV-1感染性克隆进行共转染抑制实验,筛选显示pol1026序列兼具高保守性及高抑制效率特点。以天然miR-30a为基础骨架构建了靶向pol1026靶点的人工miRNA元件,通过与HIV-1感染性克隆质粒的共转染抑制实验验证获得了可有效抑制HIV-1表达的人工miRNA元件(miR-1026E)。通过与携带靶序列的报告质粒的共转染实验证明miR-1026E具有良好的靶点特异性。本研究进一步构建了携带miR-1026E表达元件的重组慢病毒,转导MT-4细胞并对转导后细胞进行克隆化筛选,获得稳定整合miR-1026E表达元件的MT-4-miR1026E细胞克隆,该细胞在体外攻毒实验中可高效抑制HIV-1的复制,具有显著的抑制HIV-1的能力。同时应用实时RT-PCR方法检测显示,miR-1026E在细胞中不会影响内源性代表miRNA(miR-181与miR-16)的表达水平和干扰素效应相关基因stat1的表达水平,具有良好的特异性。所获得的可特异高效抑制HIV-1复制的人工miRNA元件可为抗HIV-1研究提供重要参考。     

RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences

Potent antiviral RNAi can be induced by intracellular expression of short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs). Expression of shRNA and miRNA results in target mRNA degradation (perfect base pairing) or translational repression (partial base pairing). Although efficient inhibition can be obtained, error-prone viruses such as human immunodeficiency virus type 1 (HIV-1) can escape from RNAi-mediated inhibition by mutating the target sequence. Recently, artificial miRNAs have been shown to be potent RNAi inducers due to their efficient processing by the RNAi machinery. Furthermore, miRNAs may be more proficient in suppressing imperfect targets than shRNAs. In this study, we tested the knockdown efficiency of miRNAs and shRNAs against wild-type and RNAi-escape HIV-1 variants with one or two mutations in the target sequence. ShRNAs and miRNAs can significantly inhibit the production of HIV-1 variants with mutated target sequences in the open reading frame. More pronounced mutation-tolerance was measured for targets in the 3′ untranslated region (3′ UTR). Partially complementary sequences within the 3′ UTR of the HIV-1 RNA genome efficiently act as target sites for miRNAs and shRNAs. These data suggest that targeting imperfect target sites by antiviral miRNAs or shRNAs provides an alternative RNAi approach for inhibition of pathogenic viruses.     

针对leader序列的siRNA能够有效抑制SARS冠状病毒多个mRNA的表达

小干扰RNAs(siRNAs)能够有效降解具有互补序列的RNA.在SARS-CoV的基因组RNA和所有亚基因组RNA的5′端均有一段共同的leader序列,而且该leader序列在不同的病毒分离物中高度保守,因此leader序列可作为一个用于抑制SARS-CoV复制的有效靶点.研究表明,针对leader序列化学合成的siRNA和DNA载体表达的shRNA都可以有效抑制SARS-CoV mRNA的表达.Leader序列特异的siRNA或shRNA不仅可以有效抑制leader与报告基因EGFP融合基因的表达,而且还可以有效抑制leader与刺突蛋白(spikeprotein)、膜蛋白(membrane protein)和核衣壳蛋白(nucleocapsid protein)基因的融合转录产物的表达.结果表明,针对leader序列的RNA干扰可以发展成为一种抗SARS-CoV治疗的有效策略.     

Human immunodeficiency virus type 1 escape from RNA interference

Sequence-specific degradation of mRNA by short interfering RNA (siRNA) allows the selective inhibition of viral proteins that are critical for human immunodeficiency virus type 1 (HIV-1) replication. The aim of this study was to characterize the potency and durability of virus-specific RNA interference (RNAi) in cell lines that stably express short hairpin RNA (shRNA) targeting the HIV-1 transactivator protein gene tat. We found that the antiviral activity of tat shRNA was abolished due to the emergence of viral quasispecies harboring a point mutation in the shRNA target region. Our results suggest that, in order for RNAi to durably suppress HIV-1 replication, it may be necessary to target highly conserved regions of the viral genome. Alternatively, similar to present antiviral drug therapy paradigms, DNA constructs expressing multiple siRNAs need to be developed that target different regions of the viral genome, thereby reducing the probability of generating escape mutants.     

Silencing of hepatitis A virus infection by small interfering RNAs

Infection by hepatitis A virus (HAV) can cause acute hepatitis and, rarely, fulminant liver failure, in particular in patients chronically infected with hepatitis C virus. Based on our previous observation that small interfering RNAs (siRNAs) can silence translation and replication of the firefly luciferase-encoding HAV replicon, we now exploited this technology to demonstrate the effect of siRNAs on viral infection in Huh-7 cells. Freshly and persistently infected cells were transfected with siRNAs targeting various sites in the HAV nonstructural genes. Compared to a single application, consecutive siRNA transfections targeting multiple sequences in the viral genome resulted in a more efficient and sustained silencing effect than a single transfection. In most instances, multiple applications of a single siRNA led to the emergence of viral escape mutants with mutated target sites that rendered these genomes resistant to RNA interference (RNAi). Efficient and sustained suppression of the viral infectivity was achieved after consecutive applications of an siRNA targeting a computer-predicted hairpin structure. This siRNA holds promise as a therapeutic tool for severe courses of HAV infection. In addition, the results provide new insight into the structural bases for sequence-specific RNAi.     

Functional and intracellular localization properties of U6 promoter-expressed siRNAs, shRNAs, and chimeric VA1 shRNAs in mammalian cells

RNA polymerase III (Pol III) expression systems for short hairpin RNAs (U6 shRNAs or chimeric VA1 shRNAs) or individually expressed sense/antisense small interfering RNA (siRNA) strands have been used to trigger RNA interference (RNAi) in mammalian cells. Here we show that individually expressed siRNA expression constructs produce 21-nucleotide siRNAs that strongly accumulate as duplex siRNAs in the nucleus of human cells, exerting sequence-specific silencing activity similar to cytoplasmic siRNAs derived from U6 or VA1-expressed hairpin precursors. In contrast, 29-mer siRNAs separately expressed as sense/antisense strands fail to elicit RNAi activity, despite accumulation of these RNAs in the nucleus. Our findings delineate different intracellular accumulation patterns for the three expression strategies and suggest the possibility of a nuclear RNAi pathway that requires 21-mer duplexes.     

Small interfering RNAs against the TAR RNA binding protein, TRBP, a Dicer cofactor, inhibit human immunodeficiency virus type 1 long terminal repeat expression and viral production

RNA interference (RNAi) is now widely used for gene silencing in mammalian cells. The mechanism uses the RNA-induced silencing complex, in which Dicer, Ago2, and the human immunodeficiency virus type 1 (HIV-1) TAR RNA binding protein (TRBP) are the main components. TRBP is a protein that increases HIV-1 expression and replication by inhibition of the interferon-induced protein kinase PKR and by increasing translation of viral mRNA. After HIV infection, TRBP could restrict the viral RNA through its activity in RNAi or could contribute more to the enhancement of viral replication. To determine which function will be predominant in the virological context, we analyzed whether the inhibition of its expression could enhance or decrease HIV replication. We have generated small interfering RNAs (siRNAs) against TRBP and found that they decrease HIV-1 long terminal repeat (LTR) basal expression 2-fold, and the LTR Tat transactivated level up to 10-fold. In the context of HIV replication, siRNAs against TRBP decrease the expression of viral genes and inhibit viral production up to fivefold. The moderate increase in PKR expression and activation indicates that it contributes partially to viral gene inhibition. The moderate decrease in micro-RNA (miRNA) biogenesis by TRBP siRNAs suggests that in the context of HIV replication, TRBP functions other than RNAi are predominant. In addition, siRNAs against Dicer decrease viral production twofold and impede miRNA biogenesis. These results suggest that, in the context of HIV replication, TRBP contributes mainly to the enhancement of virus production and that Dicer does not mediate HIV restriction by RNAi.     

Inhibition of hepatitis B virus replication in cultured cells and in vivo using 2′-O-guanidinopropyl modified siRNAs

Silencing hepatitis B virus (HBV) gene expression with exogenous activators of the RNA interference (RNAi) pathway has shown promise as a new mode of treating infection with the virus. However, optimizing efficacy, specificity, pharmacokinetics and stability of RNAi activators remains a priority before clinical application of this promising therapeutic approach is realised. Chemical modification of synthetic short interfering RNAs (siRNAs) provides the means to address these goals. This study aimed to assess the benefits of incorporating nucleotides with 2′-O-guanidinopropyl (GP) modifications into siRNAs that target HBV. Single GP residues were incorporated at nucleotide positions from 2 to 21 of the antisense strand of a previously characterised effective antiHBV siRNA. When tested in cultured cells, siRNAs with GP moieties at selected positions improved silencing efficacy. Stability of chemically modified siRNAs in 80% serum was moderately improved and better silencing effects were observed without evidence for toxicity or induction of an interferon response. Moreover, partially complementary target sequences were less susceptible to silencing by siRNAs with GP residues located in the seed region. Hydrodynamic co-injection of siRNAs with a replication-competent HBV plasmid resulted in highly effective knock down of markers of viral replication in mice. Evidence for improved efficacy, reduced off target effects and good silencing in vivo indicate that GP-modifications of siRNAs may be used to enhance their therapeutic utility.     

Inhibition of Moloney murine leukemia virus integration using polyamides targeting the long-terminal repeat sequences

The retroviral integrase (IN) carries out the integration of the viral DNA into the host genome. Both IN and the DNA sequences at the viral long-terminal repeat (LTR) are required for the integration function. In this report, a series of minor groove binding hairpin polyamides targeting sequences within terminal inverted repeats of the Moloney murine leukemia virus (M-MuLV) LTR were synthesized, and their effects on integration were analyzed. Using cell-free in vitro integration assays, polyamides targeting the conserved CA dinucleotide with cognate sites closest to the terminal base pairs were effective at blocking 3' processing but not strand transfer. Polyamides which efficiently inhibited 3' processing and strand transfer targeted the LTR sequences through position 9. Polyamides that inhibited integration were effective at nanomolar concentrations and showed subnanomolar affinity for their cognate LTR sites. These studies highlight the role of minor groove interactions within the LTR termini for retroviral integration.