Control of nonsegmented negative-strand RNA virus replication by siRNA

Virus Research 102 (2004) 27–35

Control of nonsegmented negative-strand RNA virus replication by siRNA Sailen Barik∗ Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, 307 University Blvd., Mobile, AL 36688, USA I dedicate this article to my Ph.D. advisor and mentor, Professor N.C. Mandal, on the eve of his retirement from Bose Institute, Calcutta; his love of science continues to be a guiding light

Abstract Our laboratory provided the first proof-of-concept that double-stranded short interfering RNA (ds-siRNA) can act as potent and specific antiviral agents. Designed against specific mRNAs of nonsegmented negative-stranded RNA (NNR) viruses, siRNAs abrogated expression of the corresponding viral proteins, and generated the predicted viral phenotypes. Knockdown was demonstrated across different genera: respiratory syncytial virus (RSV), a pneumovirus; vesicular stomatitis virus (VSV), a rhabdovirus; and human parainfluenza virus (HPIV), a paramyxovirus. The targeted genes could have a wide range of functions, thus documenting the versatility of the technique. Interestingly, antisense single-stranded siRNA (ss-siRNA) was also effective, albeit at a higher concentration. NNR viral genomic and antigenomic RNA, which are encapsidated by nucleocapsid protein and serve as templates for viral RNA-dependent RNA polymerase, were resistant to siRNA. Together, siRNAs offer complementary advantages over traditional mutational analyses that are difficult to perform in NNR viruses, and are also an important new tool to dissect host–virus interactive pathways. © 2004 Elsevier B.V. All rights reserved. Keywords: Respiratory syncytial virus; Vesicular stomatitis virus; Parainfluenza virus; RNA interference; siRNA; Host–virus interaction

1. Introduction RNA interference (RNAi), mediated by short interfering RNA molecules (siRNA), is now recognized as a major mechanism of post-transcriptional gene silencing (PTGS) in all metazoan eukaryotes (Hannon, 2002; Pickford and Cogoni, 2003). The application of the siRNA technology in mammalian cell culture received a major impetus following the discovery that specific double-stranded siRNA molecules in the 21-nucleotide range effectively degrade the cognate mRNAs, and thus abrogate the expression of the corresponding proteins (Elbashir et al., 2001). These siRNAs are apparently too short to activate the cellular “interferon response” that is known to cause a general inhibition of the cellular translation machinery (Elbashir et al., 2001; Bitko and Barik, 2001b).

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Tel.: +1-251-460-6860; fax: +1-251-460-6127. E-mail address: [email protected] (S. Barik).

0168-1702/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2004.01.012

The specificity and efficacy of siRNA-mediated PTGS against intracellular cytoplasmic parasites was originally documented using respiratory syncytial virus (RSV) as a test model (Bitko and Barik, 2001b). Subsequently, a flurry of exciting studies reproduced similar success in a variety of important animal and plant viruses (See other articles of this volume). The NNR virus family, in particular, encompasses a large roster of members that infect practically every terrestrial species and cause diseases of enormous consequence in human and animal health (Banerjee et al., 1991; Palese et al., 1996). Two major families of the NNR viruses are: Paramyxoviridae and Rhabodoviridae. Clinically important paramyxoviruses include human and bovine respiratory syncytial viruses (RSV) and parainfluenza viruses (PIV), measles virus, mumps virus, canine distemper virus, and the encephalitis-causing Nipah virus. The rhaboviruses include vesicular stomatitis (VSV) and rabies viruses. This review summarizes progress in the use of siRNA in the phenotypic knockdown of representative NNR viral genes, technical considerations, and the extension of the technology to studies of host–virus interaction.

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2. Nonsegmented negative-strand RNA (NNR) viruses 2.1. An overview of the NNR viral life cycle: transcription versus replication Historically, the early siRNA targets were all cellular mRNAs, transcribed in the nucleus by DNA-dependent RNA polymerase (Cogoni and Macino, 2000). In contrast, the vast majority of viral RNA genomes are transcribed exclusively in the cytoplasm, catalyzed by virally encoded RNA-dependent RNA polymerases (RdRP) (Barik et al., 1990; O’Reilly and Kao, 1998; Ahlquist, 2002). Fig. 1 summarizes the generalized scheme of transcription and replication of the NNR viruses; a more comprehensive treatise can be found in earlier reviews (Banerjee et al., 1991; Banerjee and Barik, 1992). A scholarly minireview by Ahlquist (2002) has recently discussed these steps with special reference to siRNA. The minimal RdRP of NNR viruses consists of the large viral protein L and the accessory phosphoprotein P (Banerjee and Barik, 1992; Barik et al., 1995). Depending on the particular virus, optimal transcription may require additional viral proteins, cytoskeletal proteins (such as tubulin in VSV and actin in RSV) (Moyer et al., 1986; Burke et al., 1998), and possibly additional cellular proteins (such as profilin in RSV) (Burke et al., 2000; Bitko et al., 2003). The viral genome is a single, linear, negative-sense (anti-mRNA sense) RNA molecule, ranging roughly from 10 to 15 kb in

size in different NNR viruses, and is intimately wrapped with the nucleocapsid protein N. The resultant nucleoprotein complex, termed N-RNA template, associates itself with the RdRP subunits, primarily L and P, and is finally packaged inside the structural shell of the virion, mainly made up of viral glycoproteins. The nucleocapsid inside the virion is, therefore, transcription-ready, and thus, primary viral transcription begins shortly after infection. A notable physical property of the N-RNA template is its extreme resistance to RNases, suggesting that the tightly bound N protein offers protection. Only this N-RNA complex serves as the biological template for the viral RdRP, which would not recognize pure deproteinized genomic RNA. The RdRP functions in two operationally distinct modes: transcription and replication (Banerjee and Barik, 1992). In the transcription mode, it copies the negative-sense genome to produce mRNAs corresponding to each individual gene by a stop–restart mechanism (Barr et al., 2002). The mRNAs have the features of standard eukaryotic mRNAs, i.e., they are 5 -capped and 3 -polyadenylated (Bisaillon and Lemay, 1997; Barik, 1993). The transcription process exhibits “polarity,” such that the genes most proximal to the promoter (3 end of the negative-strand genome) are transcribed most abundantly (Barik, 1992). This is also reflected in the relative abundance of the corresponding proteins, and fits the function of the different proteins while allowing transcriptional economy. For example, the N gene, usually

Fig. 1. NNR viral life cycle (see text for details). A hypothetical NNR virus genome is shown for the purposes of illustration only. In specific viruses some of these genes may be absent and others present. For example, VSV does not contain F, and RSV contains additional genes such as SH, HPIV-3 contains HN, etc. Note that in contrast to the mRNA, the full-length genomic and antigenomic RNA (− and + sense, respectively) are neither capped nor polyadenylated, and are wrapped with N protein and hence, are not accessible to siRNA.

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being the closest to the promoter, produces the most mRNA and protein, which befits the stoichiometric need of N protein to wrap the large full-length genomic and antigenomic (see below) RNA. The L gene, coding for the large subunit of the RdRP, is usually the farthest (i.e., at the 5 end of the negative-strand genome), and produces the least amounts of mRNA. As the RdRP is only needed in catalytic quantities, small amounts of the L mRNA suffice the functional need. The NNR viral mRNAs are translated by the host’s translation machinery. Since the virion particles do not package any mRNA (or any positive-sense RNA, for that matter), viral transcription is an obligatory pre-requisite to viral protein synthesis. Transcription and translation eventually generate more of the RdRP, boosting transcription further (Fig. 1). Once sufficient N protein is synthesized, it binds to the newly transcribed nascent RNA, and this somehow provokes the leading RdRP to switch from the transcription to the replication mode, such that the RdRP now ignores the intergenic stop signals, producing full-length complementary positive strand (mRNA-sense) RNA, called antigenome. Like the genome, the antigenome is also encapsidated with the N protein. In contrast to the genome, it is not transcribed, but simply serves as a replication template to produce more of the full-length negative-strand genome. At the end of the intracellular life cycle of the virus, the encapsidated negative-strand genomes, along with other structural proteins are packaged into virion particles. All siRNAs used against NNR viruses in published papers so far have been double-stranded and synthetic, and designed according to the recommendations of Elbashir et al. (2001); this issue and its exceptions are further discussed in detail in Section 4.1. In most experiments, the cells were pre-transfected with the antiviral siRNA 10–24 h before infection. 2.2. Silencing of viral RdRP Based on the foregoing, silencing of the minimal essential RdRP subunits, namely L and P, should lead to a nearly total loss of all RNA synthesis, and this is what we have observed in all three test viruses (RSV, VSV, and HPIV-3). RSV is shown as an example in Fig. 2, in which the ablation of P protein by siRNA led to a drastic inhibition of overall viral gene expression (Bitko and Barik, 2001b). It is logical to assume that primary transcription, which is catalyzed by preformed viral RdRP, would be unaffected by siRNAs targeting the RdRP. However, in routine infections at low m.o.i., primary transcription produces only small amounts of viral mRNA, and it is the secondary transcription that contributes to the bulk of viral mRNA and protein synthesis. Thus, abolition of new RdRP synthesis by siRNA results in a strong and sustained antiviral effect at an early intracellular step in the NNR viral life cycle while still allowing a small amount of viral transcription and translation to occur. In our experience ex vivo, the antiviral effect persisted for at least

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Fig. 2. Inhibition of RSV gene expression by siRNA (see Bitko and Barik, 2001b for details). A549 cells were transfected with indicated concentrations of anti-P siRNA, and infected with RSV 16 h thereafter. Lane ‘U’ indicates control, uninfected cells. Top: Immunoblot (Western) of total cell extracts was performed with either rabbit anti-P or monoclonal anti-actin antibody (Boehringer-Mannheim), as indicated. Bottom: Viral protein synthesis in siRNA-treated cells was measured by standard immunoprecipitation procedures. Cells were metabolically labeled with 35 S-(methionine plus cysteine) at 18 h post-infection, followed by immunoprecipitation with anti-RSV antibody, and analysis of the labeled proteins by SDS–PAGE and autoradiography. ‘La’ represents treatment with 100 nM anti-lamin A/C siRNA. The different viral protein bands are so indicated.

5 days, which was the longest time point in the experiment. In fact, siRNA-treated infected cells were virtually indistinguishable from uninfected ones (Bitko and Barik, 2001b). On a clinical note, one can envisage two classes of antivirals that would inhibit infection at even earlier steps, and both have pros and cons. (i) Inhibitors of virus adsorption: Recently, a novel aromatic compound, dubbed RFI-641, has been shown to inhibit RSV infection (Huntley et al., 2002) by blocking the RSV glycoprotein, F, essential for fusion of the viral envelope with the host cell membrane (see Section 2.3). Complete exclusion of the virus, however, may affect natural immunity, which is still the best form of protection against many NNR viruses, especially RSV and HPIV-3 (Crowe and Williams, 2003). (ii) Direct inhibitors of RdRP activity: The unique sequence features of NNR viral L and P proteins (Barik et al., 1990; Delarue et al., 1990; Banerjee and Barik, 1992) and the absence of similar proteins in mammalian cells make them ideal candidates for structure-based drug design. However, technical difficulties such as problems of

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recombinant expression and lack of three-dimensional structures have hindered progress in this area. Thus, the siRNA is a potential alternative for clinical intervention of NNR virus infection. As elaborated in the viral life cycle (Section 2.1; Fig. 1), NNR virus growth generates both the negative-strand genome and the positive-strand antigenome in the infected cell. However, it has been shown that they are resistant to siRNA (Bitko and Barik, 2001b). The most likely explanation is that their obligatory encapsidation by N protein protects them from annealing to the siRNA or to each other (Fig. 1), just as it protects them from RNase action in vitro. In any case, it is clear that the NNR viral mRNAs, and not the genomes, are viable targets for RNA interference. 2.3. Silencing of viral accessory genes There are a number of NNR viral proteins that are not involved in viral RNA or protein synthesis, but take part in important pre- or post-transcriptional aspects of the viral life cycle. For example, paramyxoviruses initiate infection by attaching to cell surface receptors and fusing viral and cell membranes. Viral attachment proteins, such as hemagglutinin-neuraminidase (HN) or glycoprotein (G) bind receptors, whereas fusion (F) proteins direct membrane fusion (Dutch et al., 2000). The fusion protein, F, is generally essential for cell fusion (formation of syncytia), although the relative importance of the attachment proteins in fusion is under active study. Recombinant expression has sometimes produced variable results. For instance, in transient transfection experiments co-expression of all three RSV glycoproteins—F, G, and SH—was required for optimal fusion (Heminway et al., 1994a,b), whereas recombinant RSV lacking SH formed fused cells as efficiently as the wild type (Bukreyev et al., 1997). It is possible that the exact outcome is dictated by the relative amounts of the recombinant proteins and/or the expression system used. The siRNA approach, in contrast, obviates recombinant expression, and allows phenotypic analysis in a virus-infected cell by removing one protein while leaving all others intact. This has been illustrated by individually abrogating the F or the SH protein in RSV-infected cells by siRNA. Knockdown of F resulted in complete loss of syncytium (Bitko and Barik, 2001b), without affecting G or SH synthesis (unpublished results). Reciprocally, abrogation of SH by siRNA had no effect on F or G, and also had no appreciable effect on syncytium (unpublished results). Together, these results validate an essential role of F and the dispensability of SH in syncytium formation by RSV. In another example, a number of studies using reverse genetics and variant virus strains have shown that both the fusion (F) protein and hemagglutinin-neuraminidase (HN) are essential for syncytium in HPIV-3 infected cells (Heminway et al., 1994a,b; Dutch et al., 2000; Porotto et al., 2003). Curiously, RSV encodes neither a hemagglutinin nor a neu-

Fig. 3. Inhibition of HPIV-3 syncytium formation by siRNA. A549 cell monolayers were transfected with 50 nM of the following double-stranded siRNA against the F or HN sequence of HPIV-3 (GenBank accession numbers X05303 and Z26523, respectively): F: CCAUGAAAUGGAGAGCUGU; HN: CUCAGACUUGGUACCUGAC. Only the N19 portion (Elbashir et al., 2001) of the sense strand of the siRNA is shown. Transfection and subsequent virus infection was carried out as in Fig. 2, and phase-contrast pictures were taken as described (Bitko and Barik, 2001b; Bitko et al., 2003). Top: Note the large fused mass of cells (syncytium) at the center of the panel labeled “HPIV-3,” and prevention of such fusion by ds-siRNA (100 nM) targeting F and HN. Bottom: Immunoblot shows loss of HN upon siRNA-treatment.

raminidase activity, but is obviously capable of forming syncytia. Thus, we retested the role of the HN protein in syncytium formation in wild type HPIV-3-infected cells using siRNA. As shown in Fig. 3, abrogation of either F or HN by the respective siRNA resulted in unfused cells, confirming the need for both proteins for syncytium formation. Although the vast majority of NNR viral mRNAs produce a single polypeptide, there are a few examples of multiple initiations. In a recent study, VSV matrix gene (M) mRNA was shown to produce three proteins M1, M2, and M3 (Jayakar and Whitt, 2002). M1 was the longest polypeptide initiating at the most 5 -proximal ATG; M2 and M3 were shorter, and produced by initiation at internal ATG’s in the same reading frame. While the exact roles of these different proteins remain unclear, the M protein(s) have been shown to inhibit host gene expression (Jayakar and Whitt, 2002; Kopecky and Lyles, 2003). Recent studies have suggested that the M gene is also important for apoptosis of the host cell, manifested by the characteristic rounding and detachment of VSV-infected cells (Jayakar and Whitt, 2002; Kopecky and Lyles, 2003). In an attempt to determine which product of the M gene mRNA is involved in apoptosis and

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(a) Virus-resistant cells: It is possible to generate, by constitutive or inducible expression of a transgene siRNA, a cell line that would resist virus infection. Specialized vectors have been constructed that drive siRNA expression from RNase III-based tRNA or snRNA (mainly U6) gene promoter (Yu et al., 2002; Kawasaki and Taira, 2003), and are marketed by commercial vendors. This would establish a form of “intracellular immunity” (Carmichael, 2002) that can be extended to produce transgenic virus-resistant animals. (b) Complementation experiments: As a rule, siRNAs lead to the loss of the protein rather than its mutational alteration, and thus, cannot be used for structure–function analysis. However, the phenotypic viral mutant can be complemented by transient transfection of the corresponding cDNA clone. The latter can be subjected to site-directed mutagenesis, and the mutants that fail to complement would define the amino acid residues important for function.

3. Cellular genes important in NNR virus–host interaction 3.1. Profilin and RSV: a case in point

Fig. 4. Inhibition of VSV cytopathic effect by ds-siRNA. Monolayers of HEp-2 cells were transfected with double-stranded siRNA (80 nM) corresponding to the following sequence of the VSV matrix (M) mRNA (Indiana serotype; GenBank accession number X04452): AGGUAAGAAAUCUAAGAAA. Subsequent infection with VSV, phase-contrast micrography, and immunoblot were done as described in Fig. 3. The antibody was made against a C-terminal peptide common to all three M proteins: KFSDFREKALMFGLIVEEE (Jayakar and Whitt, 2002). Top: Note that siRNA-treated cells look as if uninfected. Bottom: Immunoblot shows specific loss of the three M proteins, but not P, upon treatment with anti-M siRNA.

cell-rounding, we targeted the region of the mRNA that is unique to M1 and upstream of M2 and M3. Interestingly, however, the siRNA abrogated the expression of all three proteins (Fig. 4). Cell detachment was concomitantly inhibited, demonstrating the role of the M gene in the process, although we could not identify the exact protein involved. The reason to explain the loss of all three proteins must await further studies, but the most likely possibilities are: (a) loss of one part of the mRNA made the rest susceptible to degradation by RNases, and (b) loss of the 5 -cap and lack of internal ribosome entry inhibited expression of the downstream M2 and M3 ORFs. 2.4. Future directions A variety of creative experiments can be designed by targeting NNR viral genes with siRNA. A few suggestions are offered here.

Silencing of cellular genes by siRNA can be used to address the role of such genes in host–virus interaction. We have recently applied this approach to determine the role of profilin in the RSV life cycle (Bitko et al., 2003). Previous in vitro studies had shown that profilin stimulates RSV transcription, but is not absolutely necessary for virus replication (Burke et al., 2000). Knockdown of profilin showed multiple effects: (a) it had a minor effect on viral transcription (and hence translation), supporting in vitro results; (b) it strongly inhibited virion morphogenesis such that progeny virions were not produced; (c) it inhibited formation of actin stress fibers in cells where such fibers form in response to RSV infection (e.g., HEp-2 and L4 cells); and (d) it abrogated fusion in all cells tested. These results establish an essential role of profilin in the post-gene expression steps of RSV growth, and in the regulation of the actin cytoskeleton and the cell membrane in the infected cell. 3.2. Future directions The ability to quickly and reversibly knock down interactive cellular and viral genes by siRNA will allow single and combinatorial analysis of various interacting host–virus pathways that would have been otherwise impossible. Two examples are considered here. (a) Genes dispensable for cellular viability but essential for NNR virus: Although the loss of profilin was tolerated in cultured cells (Bitko et al., 2003), profilin gene knockout led to embryonic lethality, suggesting its essential role in early development. In a seminal study

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(Harborth et al., 2001) selected nuclear and membrane proteins were targeted by siRNA to test their essential need in a number of cell lines in culture. Two cytoskeleton/membrane regulatory proteins that were found to be nonessential are Ena/VASP and zyxin. The validity of this approach is borne out by the fact that Ena/VASP and zyxin knockout mice are viable (Aszódi et al., 1999; Hoffman et al., 2003). Thus, it would be exciting to determine if such (relatively) nonessential cellular genes are important in NNR viral life cycles. If they are, it would not only enrich our fundamental knowledge of host–virus interaction, but may lead to antivirals that would specifically block such interactions without gross toxicity to the host. (b) Specific viral triggers of cellular pathways: NNR viruses as a class activate a variety of cellular genes and signaling pathways. These include cellular transcription factors such as NF-␬B (Bitko and Barik, 1998; Helin et al., 2001; Tian et al., 2002), protein kinases (Monick et al., 2001; Pazdrak et al., 2002), and apoptosis (Bitko and Barik, 2001a; Esolen et al., 1995; O’Donnell et al., 1999; Cristina et al., 2001). The specific viral proteins (single or multiple) that trigger these pathways can be identified by siRNA knockdown.

4. The technique In this section, we first describe the basic procedure that we routinely use. To design siRNA, we generally follow the original recommendations (Elbashir et al., 2001). Interested readers can find a detailed description and alternate siRNA designs in the Tuschl lab web page (http:// www.rockefeller.edu/labheads/tuschl/sirna.html). Briefly, we look for a AA(N)19 TT sequence in the target mRNA that is not too close to the ATG. This RNA and its complementary strand is chemically synthesized and purchased from commercial vendors (such as Dharmacon; http://www.dharmacon.com). For transfection, various amounts of the deprotected, desalted, double-stranded siRNA (ds-siRNA) are transfected using OligofectAMINE Reagent (Life Technologies) in OPTIMEM I (Life Technologies) (Bitko and Barik, 2001b). Care is taken to make a homogeneous mixture of the transfection reagent and RNA by heavy vortexing before addition to the culture. We have recently switched to a newer reagent, TransIT-TKO® , marketed by Mirus (http://www.genetransfer.com), which does not require the use of serum-free media. In our experience, optimization of the DNA to TKO ratio is advised as excessive TKO may cause injury to some cell lines. The virus is generally added 6–8 h after siRNA transfection if a viral mRNA is targeted and 14–18 h after transfection if a cellular mRNA is targeted. In what follows, we describe some recent variations and potential considerations in planning a siRNA experiment, particularly in NNR viruses.

Fig. 5. Relative efficacy of single-stranded and double-stranded siRNAs. The sequence of the siRNA, designed against RSV P, has been published earlier (Bitko and Barik, 2001b). The symbols represent the following: (䊊), ds-siRNA; (䊐) ss-antisense siRNA; () same ss-siRNA but 5 -phosphorylated (Martinez et al., 2002). Transfection of siRNA and infection with RSV were carried out as in Fig. 2. Residual P protein was estimated by immunoblot, and plotted as percent of the untransfected control against the negative logarithm of siRNA concentration in molarity (M). Thus, 9 on the x-axis means 1 nM siRNA, 8 means 10 nM, and so forth. The dotted line marks 50% loss.

4.1. Double-stranded versus single-stranded siRNA Although the double-stranded siRNA is used with success in cell culture, it is the antisense RNA strand that binds to the target mRNA and forms the silencing complex, RISC. A few recent studies have indeed documented the effectiveness of single-stranded antisense RNA (ss-siRNA) (Tijsterman et al., 2002; Martinez et al., 2002). In our hands, ss-siRNA showed decent activity against NNR viruses, although it was required in larger quantities to achieve a quantitatively similar effect (Fig. 5). Using RSV P mRNA as the experimental target, the IC50 (amount of RNA causing 50% reduction of P protein) of ds-siRNA and ss-siRNA were about 10 and 75 nM, respectively. When the 5 end of the antisense ss-siRNA was phosphorylated, the IC50 slightly improved to 50 nM (Fig. 5). As suggested earlier (Martinez et al., 2002), we presume that this reflects the relative stability of the RNA species in the order: dsRNA > 5 -phospho-ssRNA > ss-RNA. 4.2. Mismatched siRNA and position effect: pros and cons As a rule, the siRNA is extremely stringent in its specificity such that a single nucleotide mismatch may abrogate its function (Elbashir et al., 2001; Bitko and Barik, 2001b; Chi et al., 2003; Semizarov et al., 2003). A few recent reports have challenged this stringency (Hamada et al., 2002; Jackson et al., 2003). In an extreme example, direct silencing of non-targeted genes containing as few as 11 contiguous nucleotides of identity to the siRNA was observed (Jackson et al., 2003). In our experience with NNR viruses, the tolerance to mismatch varies from one position to another on the same NNR viral mRNA. In other words, a single nucleotide mismatch at one site of the mRNA may prevent siRNA ac-

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tion (Bitko and Barik, 2001b), whereas at another site on the same mRNA, one mismatch may be largely tolerated (unpublished results). The reason for the sequence effect is currently unknown, but mismatch tolerance has raised issues of specificity. However, this is probably not a major concern with NNR viruses, since the viral genes have little or no homology with one another or with cellular genes. On the contrary, expansion of the target repertoire of the siRNA may enable it to silence multiple mutational variants of a given NNR virus that commonly occur due to the high mutation rate of the RNA genome (Domingo and Holland, 1997). In any case, when designing a siRNA, a BLAST search against the non-redundant nucleotide databases of the GenBank should be routinely done to verify specificity. Recent studies have also shown that the siRNA effect can be strikingly position-dependent, i.e., siRNAs directed against different regions of the same mRNA may exhibit a wide range of silencing efficiency (Holen et al., 2002). Again, the rules governing the position effect remain unknown, but we have also observed this in silencing the NNR virus. Based on these considerations, it may be wise to design multiple siRNAs against a target mRNA using the prescribed design guidelines, test all of them over a range of concentrations, and then select the most effective one. 4.3. Abundant mRNAs and saturation of the silencing machinery In principle, multiple siRNAs can be transfected or expressed in the same cell to abrogate the respective NNR viral proteins simultaneously. In our experience, this works, but seems to have limits (unpublished result). Silencing of an abundant NNR viral mRNA (for example, that of a promoter proximal gene such as N) often leads to reduced silencing of a promoter distal gene (such as L). The mechanism of this is being investigated. Nonetheless, quantitative analysis has shown that the siRNA effect may reach a plateau with increasing siRNA concentration (Kamath et al., 2001; Bitko and Barik, 2001b), suggesting that the RNAi-induced silencing complexes (RISC) of the cell are saturable. Thus, an abundant viral mRNA may sequester most of the cellular RISC, leaving little for the less abundant ones. Conceivably, such competition can also occur between cellular and viral mRNAs, thus potentially limiting host–virus interaction studies.

5. Conclusions Traditionally, structure–function analyses of RNA genomes, including those of NNR viruses, have made use of fortuitous mutants, natural variants, or chemically mutagenized stocks (e.g., Crowe et al., 1994). Such mutations are unpredictable and must be mapped by difficult complementation analyses or sequencing. Because site-directed

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mutagenesis requires a DNA template, direct mutational analysis of selected NNR viral genes is also not possible. These obstacles have been partially circumvented by the use of cloned viral cDNA (Whelan et al., 1995; Lawson et al., 1995; Pekosz et al., 1999; Marriott and Easton, 1999). Despite its revolutionary effect on NNR viral reverse genetics, however, the cDNA-based strategy is not without limitations. First, expression of recombinant viral genomic RNA and all the viral proteins in the correct ratio that reflects polarity remains a grueling task. As mentioned before, the 10–15 kb long RNA genome of the NNR viruses must have specific sequences at the 5 and 3 termini for transcription and replication and must be properly encapsidated by the nucleocapsid protein (N) in order to be recognized by the viral RdRP. Second, many NNR viral genomes and proteins are currently expressed from vaccinia-based cDNA clones, generally requiring super-infection by vaccinia virus, and vaccinia itself modulates multiple cellular entities, including MAP kinases and the actin cytoskeleton (de Magalhaes et al., 2001). It is, therefore, virtually impossible to study the interaction between cellular signaling pathways and NNR viruses in cells that are super-infected by vaccinia. Third, mutations in the recombinant DNA are permanent, i.e., the mutational phenotype cannot be regulated at different times in infection. For example, if a viral gene product has dual essential functions—one early, and one late in infection—the late function will remain undiscovered, since the mutant virus will never proceed beyond the early stage. In conclusion, properly designed siRNA can be a quicker and simpler alternative to cDNA-based reverse genetics of NNR viruses. Its ability to simultaneously knock down viral and cellular gene products offers particular advantages in the dissection of host–virus signaling pathways. We predict that the siRNA strategy will lead to major advances in these areas in the immediate future.

Acknowledgements The siRNA-related research in the author’s laboratory was supported in part by NIH grants AI049682 from the National Institute of Allergy and Infectious Diseases and EY013826 from the National Eye Institute. A computer program to find the AA(N)19 TT sequences was kindly written and provided by Titus Barik (http://www.barikautomation.com). Apology is offered to many whose original research had advanced the field, but could not be cited due to space constraints. Nevertheless, the majority of the research articles are cross-referenced in the recent reviews cited here.

References Ahlquist, P., 2002. RNA-dependent RNA polymerases, viruses, and RNA silencing. Science 296, 1270–1273.

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