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Journal of Virology, September 2005, p. 11071-11081, Vol. 79, No. 17
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.17.11071-11081.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

RNA Interference-Mediated Virus Clearance from Cells both Acutely and Chronically Infected with the Prototypic Arenavirus Lymphocytic Choriomeningitis Virus

Ana B. Sánchez, Mar Perez, Tatjana Cornu, and Juan Carlos de la Torre*

Scripps Research Institute, La Jolla, California

Received 4 February 2005/ Accepted 25 May 2005


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ABSTRACT
 
Several arenaviruses, including Lassa fever virus, cause severe, often lethal hemorrhagic fever in humans. No licensed vaccines are available in the United States, and currently there is no efficacious therapy to treat this viral infection. Therefore the importance of developing effective antiviral approaches to combat pathogenic arenaviruses is clear. Moreover, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important model for the study of viral persistence and associated diseases, as well as for exploring therapies to treat viral chronic infections. The use of small interfering RNAs (siRNAs) to downregulate gene expression via RNA interference (RNAi) has emerged as a powerful genetic tool for the study of gene function. In addition, the successful use of siRNAs to target a variety of animal viruses has led us to consider RNAi as a potential novel antiviral strategy. We have investigated the use of RNAi therapy against LCMV. Here, we show that siRNAs targeting sequences within the viral L polymerase and Z mRNAs inhibit LCMV multiplication in cultured cells. Unexpectedly, the antiviral efficacy of RNAi-based therapy against LCMV was highly dependent on the method used to deliver effector siRNA molecules. Thus, transfection of chemically synthesized siRNA pools to L and Z was ineffective in preventing virus multiplication. In contrast, targeting of the same viral L and Z gene products with siRNAs produced inside cells using a replication-deficient recombinant adenovirus expression system inhibited LCMV multiplication very efficiently. Notably, transduction with the replication-deficient recombinant adenovirus expression system to Z and L effectively cured persistently LCMV-infected cells, suggesting the feasibility of using RNAi therapy to combat viral chronic infections by riboviruses.


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INTRODUCTION
 
RNA interference (RNAi), also known as RNA silencing, is a mechanism by which double-stranded RNA directs sequence-specific degradation of mRNA in mammalian cells. This phenomenon had been known for many years in plants (dubbed posttranscriptional gene silencing, or PTGS) and fungi (called "quelling"). The role of double-stranded RNA in RNAi was first identified in the nematode Caenorhabditis elegans (18), and has since been documented in a variety of eukaryotes including mammals (35, 40, 41, 52). The RNAi pathway involves the production of small interfering RNAs (siRNAs) (21 to 23 nucleotides) from structured or larger double-stranded RNA by the RNase III enzyme DICER (3). These siRNAs can then guide the specific targeting of mRNAs in a sequence homology-dependent manner by means of an RNA-induced silencing complex (23).

RNAi has become an extremely powerful genetic tool for the analysis of gene function in eukaryotes, including mammals. In addition, recent evidence indicates that RNAi can be also harnessed to target viruses, hence its potential use as a novel antiviral approach. Thus, RNAi has been successfully used to inhibit replication of several animal viruses, including human immunodeficiency virus (30, 51), Rous sarcoma virus (27), influenza virus (20, 53), hepatitis B virus (48), hepatitis C virus (34, 46), herpesviruses (31), polyomaviruses (44), respiratory syncytial virus (4), poliovirus (22), human papillomavirus (32), foot-and-mouth disease virus (33), severe acute respiratory syndrome coronavirus (55) and coxsackievirus (56). These findings led us to explore whether RNAi could be also used as an effective antiviral tool against the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV).

Arenaviruses include clinically important human pathogens that cause severe hemorrhagic fever, such as Lassa fever virus and the South American hemorrhagic fever viruses (19). Increased traveling to and from endemic areas in sub-Saharan Africa has led to the importation of Lassa fever virus into unexpected areas, including the United States, Europe, Japan, and Canada (28). Moreover, because of its severe morbidity and high mortality, lack of immunization and effective treatment, together with ease of introduction into a susceptible population, Lassa fever virus is included in category A of potential bioterrorism weapons (5, 7). Therefore, the importance of developing novel effective antiviral approaches to combat hemorrhagic fever arenaviruses is clear. For such purposes, LCMV provides us with an excellent model system. In addition, LCMV is an important model system to study persistent viral infection and associated disease (26, 42), which make LCMV also an attractive model for the investigation of novel approaches to combat chronic viral infections. Moreover, evidence indicates that LCMV itself is a prevalent, and neglected, human pathogen (9, 10).

Here we present evidence that RNAi-mediated targeting of the LCMV L and Z mRNAs can effectively, and specifically, inhibit LCMV multiplication in cell culture. Intriguingly, the antiviral efficacy of RNAi against LCMV was highly dependent on the method used to deliver effector siRNA molecules. Thus, chemically synthesized anti-LCMV Z and L siRNA pools that efficiently inhibited expression of the Z and L proteins, respectively, in cotransfection assays, were unable to control virus multiplication in cultured cells. In contrast, the use of a recombinant adenovirus system to deliver anti-Z and L siRNAs into cells resulted in both reduced levels of Z and L protein expression and a dramatic inhibition of LCMV multiplication. We also show that RNAi therapy can effectively cure an already established persistent LCMV infection. Our results provide a first and necessary step in support of future studies exploring RNAi-based therapy to combat arenavirus infections and in general chronic infections by noncytolytic riboviruses.


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MATERIALS AND METHODS
 
Cell lines and virus strains. BHK-21 cells were maintained in high-glucose Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated (55°C for 30 min) fetal calf serum, 2 mM L-glutamine, 1x tryptose phosphate broth (Life Technologies), 1 mM sodium pyruvate, and 0.5% glucose. Vero cells were maintained in 199 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, and 1.2 g of sodium bicarbonate/liter. HEK 293T and A549 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, and 10 mM HEPES. Infections with LCMV were done using a plaque isolates of strains Armstrong (ARM) or WE.

siRNA. Smart pools of siRNA were synthesized by Dharmacon Research (Lafayette, CO). www.dharmacon.com. Sequences of siRNA used for the generation of the smart pools are available upon request.

Plasmids. Plasmids pC-L, pC-NP, and pC-Z expressing the polymerase (L), nucleoprotein (NP), and Z of LCMV, respectively, as well as plasmid pMG#7{Delta}2G have been described (37). Plasmid pSUPER (8) was obtained from R. Agami. Plasmids pSUP-L and pSUP-Z, expressing siRNA to L and Z, were constructed by cloning the corresponding target sequences into the BglII and HindIII sites of pSUPER. The sequences targeting L (5'-GGCCCGGATGGTCATTTAA-3') and Z (5'-ACCTTCTGCTGTCAGTATCCG-3') of LCMV-ARM were selected using the software OLIGOENGINE WORKSTATION (www.oligoengine.com). Selected siRNA sequences were subjected to a BLAST search and found to lack significant complementarily to known cellular mRNAs. Details about the primers, target sequences, and experimental procedures are available from the authors.

Generation of recombinant adenovirus vectors. Replication-deficient recombinant adenovirus expressing siRNA to LCMV-ARM L (rAd-riL) and Z (rAd-riZ) were generated using AdEasy technology (Quantum Biotechnologies). The expression cassette containing the RNA polymerase III (H1) promoter and sequences for expression of small hairpin RNA to produce siRNA to Z and L were excised from pSUP-Z and pSUP-L, respectively, with KpnI and SpeI and ligated into KpnI- and XbaI-digested pShuttle. AdEasy1 plasmid (100 ng) was combined with PmeI-linearized recombinant pShuttle (2 µg) and electroporated into Escherichia coli BJ5183 cells (12). Kanamycin-resistant colonies were selected and analyzed by restriction digestion. Plasmid DNA (5 µg) from correct clones was linearized with PacI and transfected into 293 cells for the generation of recombinant adenovirus vectors. Both Ad-riL and Ad-riZ recombinants were plaque purified and amplified.

Transfection of siRNA. Cells (105/per cm2) were seeded into 24-well plates, and transfected with siRNA (50 to 200 nM range) using Lipofectamine 2000.

CAT assays. Cell extracts were prepared by three freeze-thaw cycles in a dry ice-ethanol bath and a 37°C water bath. Cell extracts were clarified by centrifugation at 12,000 x g for 5 min at 4°C. Equal amounts of each sample were incubated for 30 min at 37°C in the presence of 0.25 M Tris (pH 7.8), 0.6 mg of acetyl-coenzyme A (Roche)/ml, and 0.05 µCi of [14C]chloramphenicol (ICN). The reaction was stopped by the addition of 1 ml of ethyl acetate, and chloramphenicol (CAT) was extracted by separating the phases by centrifugation; 900 µl of supernatant was dried, resuspended in 25 µl of ethyl acetate, and analyzed by thin-layer chromatography. Samples were run for 30 min in CHCl3-methanol (95:5). The plate was dried and exposed to an X-ray film.

Generation of LCMV virus-like particles. Virus-like particles were generated as described (37). Briefly, 293T cells were transfected with plasmids encoding NP (0.4 µg), L (0.3 µg), GP (0.2 µg), Z (50 ng), T7RP (0.5 µg), and MG#7{Delta}2G (0.25 µg); 72 hours later, 500 µl of VLP containing supernatant (SP) was passaged onto fresh BHK-21 cell monolayer. After 4 hours adsorption, cells were infected with LCMV ARM helper virus (multiplicity of infection, 2). Forty-eight hours later, cell lysates were prepared and assayed for CAT activity.

Western blot assay. Cells were harvested in lysis buffer (50 mM Tris-HCl, pH 8, 62.5 mM EDTA,1% NP-40, 0.4% deoxycholate) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by blotting onto an Immobilon-P polyvinylidene difluoride membrane (Millipore). Expression of Z-hemagglutinin (HA) was detected with a rabbit polyclonal serum to HA (Y11, Santa Cruz Biotechnology) used at 1:100 dilution, followed by incubation with a peroxidase-conjugated goat anti-rabbit antibody. Z protein was visualized by enhanced chemiluminescence (ECL-Roche).

Immunofluorescence assay. Cells were grown onto coverslips placed on the bottom of the wells of an M24 plate. Cells were washed once with phosphate-buffered saline and fixed in acetone/methanol (1:1) for 5 min at room temperature. After several washes with PBS and blocking step with 10% normal goat serum in phosphate-buffered saline for 30 min at room temperature, cells were incubated for 1 hours at room temperature with a guinea pig polyclonal serum to LCMV used at dilution 1:100. After several washes with phosphate-buffered saline-0.1% Triton X-100, samples were incubated for 45 min at room temperature with a fluorescein isothiocyanate-labeled goat anti-guinea pig immunoglobulin G used at 1:50 dilution. After extensive washes with phosphate-buffered saline-0.1% Triton X-100, coverslips were mounted using Mowiol and analyzed by fluorescence microscopy. Slides were digitized by using Adobe Photoshop and Canvas software.

Analyses of RNA by Northern blot hybridization and reverse transcription-PCR. RNA was isolated from cells by using TriReagent (Molecular Research Center, Cincinnati) according to the manufacturer's instructions. Northern blots were prepared with appropriate 32P-labeled DNA probes to LCMV NP or green fluorescent protein (GFP) as described (11). the reverse transcriptase reaction was carried out with SuperScript II and random hexamer primers (both from Invitrogen). PCR was performed by using Taq polymerase (Roche Diagnosis, Indianapolis) and NP or glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primers to amplify a segment of 353 or 200 base pairs, respectively.

Determination of virus titers and numbers of productively infected cells. Virus titers were determined by plaque assay as described (17). Briefly, monolayers of Vero cells (5 x 105 cells/9.4 cm2) were washed twice with serum-free Dulbecco's modified Eagle's medium, followed by the addition of serial dilutions of viral samples. The cells were incubated in a 5% CO2 incubator for 90 min at 37°C with rocking, the inoculum was removed, and cells layered with a semisolid (0.25%) agarose-complete medium overlay. Cell monolayers were incubated for 5 days. Monolayers were fixed with 25% formaldehyde and plaques were counted by staining with crystal violet. The number of cells productively infected with LCMV was quantitated using an infectious center assay previously described (16). Briefly, single cell suspensions were prepared from LCMV-Pi cell populations, viable cell numbers were determined and increasing numbers (10 to 104) of LCMV-Pi cells were mixed with 5 x 105 uninfected Vero cells and plated on M6 wells under semisolid agarose-complete medium. Five days later cells were fixed and the numbers of infectious centers were determined by crystal violet staining.


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RESULTS
 
Effect on virus multiplication of synthetic siRNAs targeting the L polymerase and Z protein of LCMV. We first examined whether siRNA to L (siRNA-L) and Z (siRNA-Z) mRNAs could specifically knock down the expression levels of the corresponding LCMV-ARM L and Z gene products. To validate the siRNA-Z we transfected HEK-293 cells with a plasmid expressing a HA-tagged version of LCMV Z protein (pC-ZHA) together with increasing amounts of siRNA-Z, or an siRNA control. Twenty-four hours after transfection, cell lysates were prepared and analyzed by Western blot using an antibody to HA. The lowest concentration (50 nM) of siRNA-Z tested resulted in undetectable levels of Z expression, whereas the highest concentration (200 nM) of siRNA control used did not affect Z expression (Fig. 1A, lane 6). Physiological levels of L protein in LCMV-infected cells are low, and hence difficult to detect by Western blot. Therefore, to validate the siRNA-L we used the LCMV MG rescue assay. In this assay, expression of the MG is an accurate surrogate marker of the viral polymerase activity (36). Cotransfection of siRNA-L at 50 nM, or higher, resulted in complete inhibition of LCMV MG expression, whereas cotransfection of the siRNA control at the highest dose of 200 nM had only a very modest effect (less than 10% reduction) on MG expression (Fig. 1B).



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  		FIG. 1. (A) Inhibition of Z protein expression by chemically synthesized siRNA. HEK-293T cells were cotransfected with pC-ZHA (0.5 µg) and different concentrations of the siRNA-Z or siRNA control. Twenty-four hours later cell lysates were prepared and analyzed by Western blot using antibodies to HA or actin. (B) Inhibition of L protein expression by chemically synthesized siRNA. HEK-293T cells were cotransfected with pC-T7 (0.25 µg), MG#7{Delta}2G (0.125 µg), pC-NP (0.2 µg), pC-L (0.1 µg) and increasing amounts of siRNA-L or siRNA control. Forty-eight hours later cell lysates were prepared for the CAT assay. (C) Inhibition of Z-mediated budding by siRNA-Z. HEK-293T cells were cotransfected with pC-T7 (0.5 µg), MG#7{Delta}2G (0.25 µg), pC-NP (0.4 µg), pC-L (0.3 µg), pC-GP (0.2 µg), pC-Z (50 ng) and siRNA to L or Z, or as the control siRNA. All siRNAs were used at a concentration of 100 nM. Forty-eight hours later supernatants (SP) (1 ml) were saved and cell lysates were prepared for the CAT assay. Supernatant from transfected cells (600 µl) were used to infect fresh monolayer of BHK-21 cells. After adsorption for 4 hours, LCMV-ARM helper virus was added (multiplicity of infection, of 3 PFU/cell), and adsorption continued for another 2 hours. After this, the inoculum was removed and fresh medium was added. Forty-eight hours later cell lysates were prepared for the CAT assay. NAc, nonacetylated chloramphenicol; Mac, monoacetylated chloramphenicol. (D) Effect of siRNA on LCMV-ARM-infected cells. HEK-293T cells were transfected with the indicated siRNA (range, 50 to 200 nM). Seven hours later the transfection medium was removed and cells were infected with LCMV-ARM (multiplicity of infection, 0.05 PFU/cell). Twenty-four hours later cell culture supernatants were collected, and total cell RNA was isolated and equal amounts of each sample were analyzed by Northern blot hybridization (panel Di), using a [32P]dCTP-labeled NP-DNA probe to detect the S genomic RNA and NP mRNA. Supernatants were examined for levels of infectious virus by plaque assay (panel Dii).

We have documented that Z plays a key role in arenavirus budding (43). Consistent with this finding, RNAi targeting of Z did not affect replication of LCMV MG (Fig. 1C, lane 2), but it caused a very significant reduction on VLP production compared to the siRNA control (compare lanes 7 and 9 in Fig. 1C).

These results strongly suggested that siRNA to L and Z could exert a potent antiviral effect against Arenavirus during the natural course of infection. To test this we investigated the effect of siRNA Z and L, and in combination, on LCMV-ARM multiplication in cultured cells. Unexpectedly, treatment of infected cells with siRNA to Z or L, or both together failed to control LCMV multiplication as determined by levels of viral RNA synthesis (Fig. 1Di) and production of infectious virus progeny (Fig. 1Dii). We obtained similar results when cells were treated with siRNA prior to virus infection, or at different times following virus infection.

It seems highly unlikely that this finding was due to LCMV-mediated disturbances of the RNAi pathway because an siRNA to GFP was similarly effective in reducing levels of GFP expression in LCMV-infected and noninfected control cells (Fig. 2A). In addition, treatment with siRNA-GFP, but not with the siRNA control, caused a similar decrease in GFP mRNA levels in both LCMV-infected and uninfected control cells (Fig. 2B). In some experiments we observed an effect of the siRNA control on levels of GFP mRNA. However, phosphorimager quantification of hybridization signal as in Fig. 2B (not shown) indicated that this effect was extremely modest (5 to 15% reduction) compared to the strong inhibition (>90% reduction) exerted by the specific siRNA-GFP. This finding led us to consider that transfection might be, under our experimental conditions, an inefficient delivery system for siRNA and thereby resulting in a significant fraction of cells that harbored insufficient levels of siRNA to protect against a replicating agent.



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  		FIG. 2. Effect of LCMV infection on the activity of the RNAi pathway. HEK-293T cells were infected with LCMV-ARM (multiplicity of infection, 2 PFU/cell), or mock infected. Twelve hours later LCMV-ARM and mock-infected cells were cotransfected with pC-GFP (50 ng) and siRNA-GFP (100 nM) or siRNA control (100 nM). Twenty-four hours later GFP expression was analyzed by epifluorescence (A), and levels of GFP mRNA wre determined by Northern blot hybridization (B) using a [32P]rUTP-labeled antisense GFP-RNA probe.

Effect of recombinant adenovirus expressing siRNA to L and Z on LCMV multiplication. We sought to use a delivery system for siRNA that would reliably target the majority of the cells within the population. For this, we chose the use of a replication-deficient recombinant adenovirus expression system that allows RNA polymerase III-mediated intracellular synthesis of small hairpin RNAs. These small hairpin RNAs can be then processed within the cell into functionally active siRNA. This approach has been already used successfully for RNAi targeting of gene expression in mammalian cells (47).

Prior to generating recombinant adenoviruses expressing siRNA for L and Z we verified that the corresponding intracellularly produced small hairpin RNAi, via RNA polymerase III, could be processed into siRNAs that effectively target expression of LCMV L and Z proteins. For this, we used plasmid pSUPER (8) to generate constructs pSUP-Z and pSUP-L, which allowed RNA polymerase III H1-mediated synthesis of siRNA to Z and L, respectively. In cotransfection assays pSUP-Z efficiently inhibited expression of Z (Fig. 3A, lane 1), whereas using the LCMV MG rescue assay we verified that pSUP-L, but not the control pSUP-GFP, could efficiently target the expression of the virus L polymeraseymerase as determined by its effect on MG associated CAT activity (compare lanes 1, 3, and 4 in Fig. 3B).



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  		FIG. 3. Effect of plasmid and recombinant adenovirus-supplied siRNA on the expression of LCMV Z and L gene products. (A) Effect of pSUP-Z and rAd-riZ on Z protein expression. Vero cells transduced with rAd-riZ (lane 2) or nontransduced (lanes 1, 3, and 4) were cotransfected with pC-ZHA (lanes 1 to 3) and pSUP-Z (lane 1) or empty pSUPER (lane 3). Twenty-four hours after transfection cell lysates were prepared and analyzed by Western blot using antibodies to HA or actin. (B) Effect of pSUP-L and rAd-riL on L-mediated MG expression. Vero cells nontransduced (lanes 1 to 4), or transduced with rAd-riL (lane 5) or rAd-Luc (lane 6), were cotransfected with the indicated plasmids. Forty-eight hours later cell lysates were prepared for the CAT assay. NAc, nonacetylated chloramphenicol; Mac, monoacetylated chloramphenicol.

We then used the expression cassettes of pSUP-L and pSUP-Z to engineer the corresponding recombinant adenovirus expressing L and Z siRNA. Consistent with the results obtained using pSUPER-based expression of siRNA, we observed reduced levels of Z expression in cells transduced with recombinant adenovirus-riZ (Fig. 3A, lane 2), and dramatically reduced levels of LCMV MG expression in cells transduced with rAd-riL, but not with the control rAd-riLuc expressing a specific siRNA to the reporter gene luciferase (Fig. 3B, compare lanes 1, 5, and 6).

We next examined whether delivery of siRNA via recombinant adenovirus could effectively interfere with LCMV multiplication in cultured cells. For this, we transduced Vero cells with rAd-riZ, rAd-riL, or a control, rAd-riLuc. Transduced cells were then infected with LCMV-ARM (multiplicity of infection, 0.05) and at 24 hours postinfection. the percentage of viral antigen-positive cells was determined by immunofluorescence.

Compared to nontransduced cells, the number of LCMV antigen-positive cells was severely diminished in cells transduced with rAd-riZ or rAd-riL, but not with rAd-riLuc (Fig. 4A). Likewise, Northern blot analysis showed that cells transduced with rAd-riZ or rAd-riL, but not with rAd-riLuc, had dramatic reduced levels of LCMV replication (S RNA) and transcription (NP mRNA) compared to nontransduced cells (Fig. 4B).



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  		FIG. 4. Inhibition of LCMV-ARM multiplication by recombinant adenovirus-supplied siRNA. Vero cells were transduced with rAd-riZ, rAd-riL or both, or as a control with rAd-riLuc. Twelve hours later transduced cells were infected with LCMV-ARM (multiplicity of infection, 0.05 PFU/cell). Twenty-four hours postinfection, cells were examined for the expression of viral antigens, RNA, and virus production. (A) Detection of LCMV antigen-positive cells. Cells were fixed 24 hours after infection with LCMV ARM and examined by immunofluorescence using a guinea pig serum (dilution, 1:100) to LCMV. The percentage of viral antigen-positive cells was determined by examining several fields (n = 3 to 5, 100 to 300 cells/field) of each sample. (B) Detection of viral RNA by Northern blot hybridization using a [32P]dCTP labeled NP-DNA probe to detect the S genome RNA and the NP mRNA. (C) Cell culture supernatants were harvested at 0, 12, and 24 hours postinfection. and viral titers were determined by plaque assay.

The simultaneous use of more than one siRNA to target different viral sequences simultaneously has been shown to increase the effectiveness of RNAi therapy. In this regard we observed that the combined use of both siRNA to L and Z had a stronger effect on LCMV multiplication than each siRNA alone (compare lanes 1 to 3, Fig. 4B). Consistent with the antigen and RNA data, production of infectious LCMV-ARM was inhibited in cells transduced with recombinant adenovirus expressing siRNA to L or Z, but not to luciferase (Fig. 4C).

One of the features of RNAi is the high sequence specificity required for target recognition. To gain further evidence that the inhibitory effect of rAd-riZ and rAd-riL on LCMV-ARM multiplication was indeed mediated by RNAi, we examined their antiviral activities against the WE strain of LCMV. Sequence analysis revealed that ARM and WE strains have nucleotides differences within the sequences targeted by the siRNA expressed by rAd-riZ and rAd-riL (Fig. 5A). As predicted by the sequence data, WE replicated to similar levels in nontransduced and transduced cells (Fig. 5B). In addition, production of infectious WE was not impaired in cells transduced with either rAd-riL or rAd-riZ (Fig. 5C).



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  		FIG. 5. Resistance of LCMV-WE to siRNA specific for the Z and L mRNAs of LCMV-ARM. (A) Strains ARM and WE contain nucleotide differences within the L and Z sequences targeted by siRNAs to Z and L of ARM. Mismatches are indicated by lowercase letters. (B and C) LCMV-WE RNA replication and virus production were not affected by siRNAs to Z and L of ARM. Vero cells were transduced with rAd-riZ, rAd-riL, or rAd-riLuc, and 12 hours later infected with LCMV-WE (multiplicity of infection, 0.05 PFU/cell). Twenty-four hours postinfection samples were analyzed for levels of viral RNA and virus production. Viral RNA was detected by Northern blot hybridization using a probe to detect the S genome RNA (B). Cell culture supernatants (SP) were harvested at 0, 12, and 24 hours postinfection. and WE titers were determined by plaque assay (C).

RNAi targeting of LCMV persistent infection. As with most arenaviruses, LCMV has the ability to persist in cells and tissues without causing noticeable cytopathic effects. Hence, LCMV provided us with an excellent viral system where to explore the feasibility of using a RNAi-based therapy to clear an established nonlytic chronic viral infection. For this we assessed the effect of rAd-riL and rAd-riZ on two cell lines, Vero and A549 cells, persistently infected with two strains, ARM and WE, of LCMV, designated LCMV-Pi (ARM) and LCMV-Pi (WE), respectively. To generate these LCMV-Pi lines we infected Vero and A549 cells with ARM and WE (multiplicity of infection, 0.1). Seventy-two hours later, we subcultured the cells to generate LCMV-Pi (ARM)p1 and LCMV-Pi (WE)p1 for both Vero and A549 cell lines. After two additional passages LCMV-Pi (ARM) and LCMV-Pi (WE) cell populations of both Vero and A549 cell lines contained a majority of cells that expressed viral antigens (>95%) and contained infectious virus (>90%), as determined by immunofluorescence and infectious center assays, respectively (not shown).

We first determined whether a single transduction protocol could achieve viral clearance from LCMV-Pi cells. For this, we transduced LCMV-Pi (ARM) Vero cells with rAd-riL or rAd-riZ, as well as the control rAd-riLuc. Seventy-two hours after transduction cells were examined for levels of viral antigen and RNA by immunofluorescence and Northern blot analysis, respectively. Both viral antigen (Fig. 6A) and RNA load (Fig. 6B) were dramatically reduced, but not entirely eliminated, in cells transduced with recombinant adenovirus expressing siRNA to LCMV L or Z, or both, but not to the Luc reporter gene. We reasoned that the high viral load present in persistently LCMV infected Vero cells, together with a targeting strategy based on one single siRNA, might explain the failure to achieve viral clearance during the first 72 hours of RNAi therapy.



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  		FIG. 6. Effect of single and short-term RNAi-based therapy on virus load in LCMV-Pi cells. LCMV-Pi (ARM) Vero cells were transduced with the indicated recombinant adenovirus and 72 hours later examined for expression of viral antigen and levels of viral RNA. (A) Viral antigens were detected by immunofluorescence as in Fig. 4A. (B) Levels of viral RNA were determined by Northern blot analysis as in Fig. 4B.

We therefore explored the effectiveness of longer siRNA treatments combined with targeting two different viral sequences (Fig. 7). For this, we subjected both Vero and A549 LCMV-Pi cells to consecutive cotransductions using both rAd-riL and rAd-riZ (T2 [L+Z]). As a control, LCMV-Pi cells were also subjected to consecutive transductions with rAd-riLuc (T2 [Luc]). To facilitate the reappearance of virus that had not been completely cleared by the RNAi treatment, we subcultured cells seventy two hours after the second transduction (T2) and maintained them in the absence of recombinant adenovirus treatment for two additional passages (T2 (L+Z) > P2 (-L/-Z)). Both viral antigen and RNA were below detectable levels of immunofluorescence (Fig. 7A) and Northern blot (Fig.7B 7Bi), respectively, in T2 (L+Z) and T2 (L+Z) >P2 (-L/-Z)-treated both Vero and A549 LCMV-Pi (ARM) cells. In contrast, both viral antigen and RNA were readily detected in T2 (rAd-luc)-treated LCMV-Pi (ARM) cells (Fig. 7A and 7Bi).



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  		FIG.7. Combinatorial long-term RNAi therapy can cure LCMV-Pi cells. LCMV-Pi (ARM) and LCMV-Pi (WE) Vero and A549 cells were subjected to consecutive (T2) transductions with rAd-riL+Z [T2 (L+Z)] or the control rAd-riLuc. T2 (L+Z) treated cells were subsequently passed (every 3 days) two times in the absence of RNAi therapy [T2(L+Z)>P2 (-L/-Z)]. (A) Expression of viral antigens was determined by immunofluorescence as in Fig. 4A. (B) Levels of RNA replication and transcription were first determined by Northern blot (Bi) using an NP double-stranded DNA probe that allowed detection of both S genomic RNA (replication) and NP mRNA (transcription). reverse transcription-PCR (Bii) using specific primers to amplify a segment of 353 bp within the NP open reading frame was done to uncover putative low levels of viral RNA in T2 (L+Z)-treated LCMV-Pi (ARM) Vero and A549 cells. As a control we amplified a fragment of 200 bp within the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The amount of reverse transcription-PCR NP product loaded for sample 2 [T2 (L+Z)>P2(-L/-Z)] corresponded to that obtained from 10 cells, whereas samples 1 and 3 to 5 corresponded to 104 cells. The same amount of reverse transcription-PCR glyceraldehyde-3-phosphate dehydrogenase product (104 cell equivalents) was loaded for all samples. (C) Infectivity associated with LCMV-Pi cells. The percentage of cells productively infected (IC) was determined by the infectious center assay. Values shown correspond to the average ± standard deviation of two independent experiments. (D) Susceptibility to LCMV infection of T2 (L+Z)>P2(-L/-Z)-treated cells. Naïve (–) and T2 (L+Z)>P2(-L/-Z)-treated (+) A549 and Vero cells were infected with LCMV ARM (multiplicity of infection, 0.1 PFU/cell) and 24 hours later levels of viral RNA and viral antigen expression were determined by Northern blot (Di) and immunofluorescence (Dii), respectively.

As predicted based on sequence differences between WE and ARM, both antigen and RNA of WE was not cleared by T2 (L+Z) in either Vero or A549 LCMV-Pi cells (Fig. 7A and 7Bi). Using the more sensitive reverse transcription-PCR approach we were still unable to detect viral RNA in both T2 (L+Z) and T2 (L+Z) >P2 (-L/-Z) treated LCMV-Pi cells (Fig. 7Bii). Likewise, infectious center assays failed to detect virus infectivity associated with either T2 (L+Z) or T2 (L+Z) >P2 (-L/-Z) treated LCMV-Pi (ARM) cells (Fig. 7C). Moreover, nontransduced naïve cells and T2 (L+Z) >P2 (-L/-Z) treated cells exhibited similar susceptibility to infection with LCMV ARM (Fig. 7D). This finding strongly argues against a persistent expression of siRNA in T2 (L+Z) >P2 (-L/-Z) treated cells as the explanation for the lack of detection of viral RNA and antigen. Together, these results provided strong evidence supporting the feasibility of using a RNAi-based therapy to clear a LCMV chronic infection in cell culture.


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DISCUSSION
 
Results with a variety of viral systems have provided support for the use of RNAi as a promising novel antiviral approach. Here we have shown the feasibility of using RNAi to inhibit multiplication of the prototypic arenavirus LCMV in cultured cells. Moreover, we have documented that a RNAi-based therapy can also cure an already established persistent LCMV infection.

During the course of our studies we unexpectedly observed that chemically synthesized siRNA that effectively inhibited the expression of LCMV L and Z gene products in transfection assays (Fig. 1 A to C) were unable to control LCMV multiplication in newly infected cells (Fig. 1D). This observation raised the question of whether a bona fide LCMV infection could disrupt the RNAi pathway. In this regard, recent evidence showed that both influenza virus NS1 and vaccinia virus E3L proteins are suppressors of the RNA silencing-based antiviral response in Drosophila cells (38). We have not examined directly whether LCMV gene products can exert a similar inhibitory effect on the Drosophila RNA silencing-based antiviral response. However, we found that a chemically synthesized GFP siRNA inhibited expression of GFP to similar levels in LCMV-infected and uninfected control cells (Fig. 2). We therefore favor the explanation that a limited transfection efficiency of siRNA may result in a significant percentage of cells containing levels of siRNA that are insufficient to efficiently target the large levels of LCMV mRNA species produced during the infection cycle, and thereby the failure to control virus multiplication.

It is quite plausible that siRNA delivered via transfection conferred to a fraction of cells the ability to inhibit LCMV multiplication. This effect would be very difficult, if possible at all, to appreciate using cell population-based assays, including Northern blot (Fig. 1Di) and virus titration by plaque assay (Fig. 1Dii). With this hypothesis, one would predict that the use of an alternative method capable of reliably delivering high and sustained levels of siRNA into the vast majority of the cells within the population should result in an effective inhibition of virus multiplication. Consistent with this view we observed that transduction of cells with recombinant adenovirus expressing siRNA to L or Z effectively inhibited LCMV multiplication in newly infected cells (Fig. 4A to C). The use of recombinant adenovirus vectors to deliver siRNA could have also triggered a variety of nonspecific effects, including innate immune cell responses (39), which might have influenced our results. However, cells transduced with rAd-riLuc expressing a nonrelevant siRNA to luciferase were fully susceptible to LCMV ARM compared to nontransduced naïve cells (Fig. 4). Moreover, siRNAs to L and Z inhibited replication of LCMV-ARM in Vero cells, which are unable to produce interferon due to a chromosomal deletion (14). These findings together strongly support the conclusion that our findings reflect a specific RNAi-mediated antiviral effect.

Both chemically synthesized and recombinant adenovirus-produced siRNAs mediated similar levels of knockdown expression of their L and Z targets in transfected cells. Therefore, it seems highly unlikely that the striking differences in antiviral activity between chemically synthesized and recombinant adenovirus-derived siRNAs could be explained based on differences on the specific RNA sequences that they targeted within the L and Z mRNAs.

Recent evidence indicates that RNAi-specific off-target effects are frequently observed (29, 45). Thus, a given siRNA or small hairpin RNA designed to target a virus product might alter the expression of unrelated genes that could be responsible for part or all of the potential antiviral activity associated with the siRNA. In addition, most mammalian cells respond to double-stranded RNA by activating their interferon-mediated innate defense responses (2, 50). However, the use of synthetic siRNA or its generation via processing of small hairpin RNAs produced from plasmids appears to partly overcome this problem. Nevertheless, liposome-based transfection protocols used to deliver siRNA or plasmids into cells frequently result in the induction of the interferon system (6, 49), which can introduce additional confounding factors for the assessment siRNA-mediated antiviral activity. Nevertheless, our finding (Fig. 4C) that strain WE of LCMV was resistant to the same siRNAs to L and Z that effectively inhibited strain ARM would argue against off-target effects as responsible for the antiviral activity of the siRNA produced by rAd-riL and rAd-riZ.

Riboviruses, including arenaviruses, have the potential for rapid evolution (15, 25). The molecular basis for this is extremely high mutation frequencies per average site in RNA virus genomes, which is facilitated by the error-prone nature of the RNA-dependent RNA polymerases. Such high mutation frequencies, combined with small genome sizes, short replication cycles, and high fecundity, dictate that RNA viruses replicate and evolve as dynamic complex mutant distributions termed quasispecies (15, 25). This property confers on riboviruses a pertinacious adaptability reflected by the high-frequency isolation of escape mutants whenever an effective selective constraint is in operation. This, in turn, has a significant impact in antiviral therapies, including RNAi.

Single mismatches between an RNAi and its target sequence can dramatically affect the efficacy of RNAi-mediated silencing (54). Thus, for several RNA viruses the use of RNAi as an antiviral tool was limited by the ease with which viral variants resistant to the RNAi emerged within the population (13, 21). Notably, we did not observe the emergence of escape mutants in persistently LCMV infected cells subjected to RNAi therapy, as determine by the inability to detect viral macromolecules, RNA and antigen, or infectivity in cells that were first subjected to T2 (rAd-riL+Z) and subsequently allowed to grow in the absence of RNAi therapy (Fig. 7). The reasons for this remain to be determined, but evidence indicates that the arenavirus polymerase has low fidelity similar to that of other NS RNA viruses.

It is plausible that a high degree of adaptation of the laboratory strain of LCMV to cultured cells contributed to the selection of a quasispecies where the vast majority of the RNA species corresponded to the sequence being targeted by RNAi. This together with reduced levels of viral RNA synthesis during LCMV persistence could limit the plasticity of the virus population in response to changes in selective pressures due to the introduction of siRNAs to L and Z. Moreover, there is evidence for some degree of mismatch tolerance for siRNA-mediated degradation of target mRNA species (1, 24).

Our results do not rule out that siRNA-resistant viruses might easily emerge in the context of RNAi-based Arenavirus therapy in vivo. This, however, would not necessarily detract from the potential value of RNAi antiviral therapy, but rather stress the importance and need of using combined drug targeting strategies to counteract the genome plasticity of riboviruses, which confer on them an exquisite ability to escape from most forms of single-hit-based therapies. Recent improvements in the design of siRNAs and ways to deliver them into the appropriate tissues support to consider the incorporation of RNAi-based therapies into the current limited arsenal of antiviral weapons.


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ACKNOWLEDGMENTS
 
This work was supported by a fellowship of the Spanish Ministerio de Ciencia y Tecnologia to Ana B. Sanchez and NIH grant AI47140 to J. C. de la Torre.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Neuropharmacology IMM-6, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037. Phone: (858) 784-9462. Fax: (858) 784-9981. E-mail: juanct{at}scripps.edu. Back


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Journal of Virology, September 2005, p. 11071-11081, Vol. 79, No. 17
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.17.11071-11081.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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