Rnai, a New Therapeutic Strategy Against Viral Infection
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REVIEW Cell Research (2004); 14(6):460-466 http://www.cell-research.com RNAi, a new therapeutic strategy against viral infection Fischer L. TAN, James Q. YIN* Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China. ABSTRACT RNA interference (RNAi) is an adaptive defense mechanism triggered by double-stranded RNA (dsRNA). It is a powerful reverse genetic tool that has been widely employed to silence gene expression in mammalian and human cells. RNAi-based gene therapies, especially in viral diseases have become more and more interesting and promising. Recently, small interfering RNA (siRNA) can be used to protect host from viral infection, inhibit the expression of viral antigen and accessory genes, control the transcription and replication of viral genome, hinder the assembly of viral particles, and display influences in virus-host interactions. In this review, we attempt to present recent progresses of this break- through technology in the above fields and summarize the possibilities of siRNA-based drugs. Keywords: RNAi, siRNA, viral infection, gene therapy. INTRODUCTION MACHINERY OF RNAi RNA interference (RNAi), an ancient defense pathway, Biochemical and genetic studies have revealed the is a common denominator for the posttranscriptional gene molecular mechanismes, by which dsRNA causes the silencing (PTGS) phenomenon observed in a variety of degradation of target messenger RNA [11]. RNAi includes species such as plants, fungi and animals [1-3]. The intro- two steps: initiation step and effector step [12]. In the duction of long dsRNA into cells can effectively and initiation step, Dicer, a member of the RNase III family of specifically lead to the degradation of cognate mRNAs in ATP-dependent ribonucleases, binds with high affinity to a gene-dependent manner. This powerful technology has dsRNA containing 2 nucleiotide (nt) 3’ overhangs and chops been widely employed to manipulate gene expression, long dsRNA (introduced directly or via a transgene or virus) elucidate signal pathways and to identify gene functions into small interfering RNAs (siRNAs) duplexes. Generally, in a whole-genome scale. Researchers worldwide have Dicer enzymes contain an N-terminal DEXH-box RNA used RNAi for basic research, and are now developing helicase domain, a domain of unknown function (DUF283), RNAi-based drugs for the prevention and treatment of a PAZ domain, two RIII domains and a dsRNA-bingding human diseases such as viral infection, tumors and meta- domain (dsRBD) [13]. Dicer can cleave dsRNA into siRNAs bolic disorders [4-8]. or microRNAs (miRNAs) from endogenous stem loop pre- Despite significant advances have been made in the cursors [14]. Biochemical studies show that siRNAs are therapy of viral diseases, current drugs and vaccines are 21-23 nt dsRNA duplexes with 2-nt 3’ overhangs, a 5’- restricted with many factors such as toxicity, complexity, monophosphate and a 3’-hydroxyl group [15]. In the ef- cost and resistance [4]. RNAi is a self-defense mecha- fector step, the siRNA duplexes are incorporated into nism of eukaryotic cells, which specially prevent infec- RNA induced silencing complex (RISC). The phospho- tion evoked by viruses [5]. It can inhibit the expression rylation of siRNA 5’-terminal is required to entry into of crucial viral proteins by targeting viral mRNA for RISC [16]. A helicase domain of RISC binds to one end degradation through cellular enzymes [9]. In fact, RNAi of the duplex and unwinds the double-strand in an ATP- does work effectively as an antiviral agent in plants. This dependent manner. The thermodynamic stability of the first breakthrough technology emerges as a powerful tool to few base pairs of siRNA can affect the ratio of RISC con- protect human cells from viral infection [10]. taining the antisense or sense strands of siRNAs [17]. Dicer with R2D2 (Dcr-2-associated protein) binds siRNA and facilitates its loading onto RISC [18]. The active RISC *Correspondence: James Q. YIN Tel: +86-10-64888572, Fax: +86-10-64888572 then targets the homologous transcript by base pairing th E-mail: [email protected] interactions and cleaves the mRNA between the 10 and 460 Cell Research, 14(6), Dec 2004 Tan FL and Yin JQ 11th nucleotide from the 5' terminus of the siRNAs [16, cells of different species. Today, researchers are develop- 19]. ing efficient siRNA delivery systems, which can help MicroRNAs (miRNAs) are endogenous ~22 nt RNAs siRNAs enter efficiently into cells in nearly all organs. that can play important regulatory roles in animals and Fourth, siRNAs do not seem to adversely affect cell control plants by targeting mRNAs for cleavage or translational mechanisms. The given length and high homology of repression. miRNAs and endogenous siRNAs have a shared siRNA to the target region of cognate transcription ensure central biogenesis and can perform interchangeable the selective destruction of only interested transcript. biochemical functions. Therefore, the two classes of siRNAs without suitable targets seem to remain inert within silencing RNAs cannot be distinguished by either their cells. This exclusive specificity without adverse side effects chemical composition or mechanism of action [20]. These is the most attractive feature of RNAi as an antiviral short RNA species are produced by Dicer cleavage of long approach. Fifth, siRNAs can silence gene stably. With the (~70 nt) endogenous precursors with imperfect hairpin application of plasmid vectors and viral vectors, siRNAs RNA structure in animals. Mature miRNAs act as a trans- can display their long-term biological effects [25]. Taken lational repressor by partial base-pairing with 5’ or 3’ ends together, siRNAs synthesis in vivo or in vitro transfected of mRNAs while miRNA completely complementary to its into cultured cells or animals could result in the sequence- target mRNA (endogenous siRNA) can result in the degra- specific silencing of mRNA molecules. With the proof-of- dation of cognate mRNA. concept studies, siRNAs have been widely used as an In addition, many other proteins such as eukaryotic alternate therapeutic strategy. translation initiation factor 2C2 (eIF2C2) and Argonaute proteins are likely to function in both pathways. Argonaute RNAi APPLICATION TO VIRAL INFECTION proteins are the key components of RISC. They are evolu- Although there is still a long way to go before the RNAi tionarily conserved with two distinguishing domains, PAZ technology will be employed to clinical application, and PIWI. The PIWI domain is restricted to Argonautes researchers believe that RNAi will finally be used as a while the PAZ domain is shared with Dicer family proteins viable therapeutic alternative for various diseases in the [21]. near future [6-8, 26]. SILENCING MECHANISMS OF RNAi RNAi prevents viral infection Long dsRNA enables the effectively silencing of gene The ability of synthetic siRNAs to inhibit viral produc- expression by presenting various siRNA sequences to tion was determined in cultured cells by introducing target mRNA. Cells infected by viruses invariably produce siRNAs into the cells before viral infection and then dsRNA, but viruses can escape a profound cellular assaying virus titers in the culture supernatants at differ- response. The dsRNA binds to dsRNA-bing proteins ent times after infection [27]. In the first application of (dsRBPs), which have been shown to counteract the effects RNAi technology to prevent disease, researchers revealed of Interferon (IFN) and the resultant dsRNA-acticated that siRNA-directed inhibition of Fas gene expression could protein kinase (PKR) activation, and to suppress RNAi. protect mice from antibody or concanavalin A-induced Recently, it has become clear that 21-nucleotide siRNAs hepatitis [28]. Nowadays, potential siRNA-based prophy- are so short that they cannot induce an interferon response laxis has been widely used in research of viral diseases. in mammalian cells [22]. So, the antiviral effects of siRNAs Influenza virus causes one of the most prevalent in- have attracted more and more scientists to throw their fections in humans. It is a RNA virus without any DNA interests into this field [23]. intermediates during its entire life cycle and normally in- It has been shown that the inhibition of viral infection fects epithelial cells in the upper airway and the lungs. can be mediated by siRNA molecules that target viral Introducing siRNAs into cultured cells and chicken em- mRNA for degradation. In comparison with other con- bryos before virus infection was able to inhibit influenza ventional drugs, siRNA have many advantages. First, it is virus production, especially those encoding NP, PA, and much easier and more flexible to select target sites because PB1. In addition, siRNA inhibitory effects also occurred target mRNA and siRNA are sequences-specific and in cells that had been infected with virus prior to siRNA complementary. For a given mRNA molecule, the inhibitory introduction [27]. siRNAs can be administered by effects of siRNAs can be achieved by targeting different inhalation. It is convenient and able to achieve so high regions of target mRNA. Second, for gene silencing, only local siRNA concentration that epithelial cells can uptake substoichiometric amount of siRNA is enough to drasti- sufficient siRNA. In order to achieve prophylactic effect, cally decrease homologous mRNA within 24 h. Third, siRNAs may be administered prior to infection or during siRNAs can result in the degradation of cognate mRNA in the early phase of infection. Thus,