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Journal of Virology, April 2005, p. 4599-4609, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4599-4609.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Inhibition of Flavivirus Infections by Antisense Oligomers Specifically Suppressing Viral Translation and RNA Replication

Tia S. Deas,1,{dagger} Iwona Binduga-Gajewska,2,{dagger} Mark Tilgner,2,{dagger} Ping Ren,2 David A. Stein,3 Hong M. Moulton,3 Patrick L. Iversen,3 Elizabeth B. Kauffman,2 Laura D. Kramer,1,2 and Pei-Yong Shi1,2*

Wadsworth Center, New York State Department of Health,1 Department of Biomedical Sciences, University at Albany, State University of New York, Albany, New York,2 AVI BioPharma, Inc., Corvallis, Oregon3

Received 21 October 2004/ Accepted 3 December 2004


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ABSTRACT
 
RNA elements within flavivirus genomes are potential targets for antiviral therapy. A panel of phosphorodiamidate morpholino oligomers (PMOs), whose sequences are complementary to RNA elements located in the 5'- and 3'-termini of the West Nile (WN) virus genome, were designed to anneal to important cis-acting elements and potentially to inhibit WN infection. A novel Arg-rich peptide was conjugated to each PMO for efficient cellular delivery. These PMOs exhibited various degrees of antiviral activity upon incubation with a WN virus luciferase-replicon-containing cell line. Among them, PMOs targeting the 5'-terminal 20 nucleotides (5'End) or targeting the 3'-terminal element involved in a potential genome cyclizing interaction (3'CSI) exhibited the greatest potency. When cells infected with an epidemic strain of WN virus were treated with the 5'End or 3'CSI PMO, virus titers were reduced by approximately 5 to 6 logs at a 5 µM concentration without apparent cytotoxicity. The 3'CSI PMO also inhibited mosquito-borne flaviviruses other than WN virus, and the antiviral potency correlated with the conservation of the targeted 3'CSI sequences of specific viruses. Mode-of-action analyses showed that the 5'End and 3'CSI PMOs suppressed viral infection through two distinct mechanisms. The 5'End PMO inhibited viral translation, whereas the 3'CSI PMO did not significantly affect viral translation but suppressed RNA replication. The results suggest that antisense PMO-mediated blocking of cis-acting elements of flavivirus genomes can potentially be developed into an anti-flavivirus therapy. In addition, we report that although a full-length WN virus containing a luciferase reporter (engineered at the 3' untranslated region of the genome) is not stable, an early passage of this reporting virus can be used to screen for inhibitors against any step of the virus life cycle.


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INTRODUCTION
 
Many members of the Flavivirus genus, a group of arthropod-borne viruses in the family Flaviridae, cause significant human diseases; among these, West Nile (WN), dengue (DEN), Japanese encephalitis (JE), yellow fever (YF), Murray Valley encephalitis, and tick-borne encephalitis (TBE) viruses are emerging and reemerging pathogens (7). Approximately 50 to 100 million human cases of DEN virus infection occur annually (29). The recent epidemics of WN virus have caused significant morbidity and mortality in the United States (9). Vaccines for humans are available only for YF, JE, and TBE viruses (7). No drug therapy is currently available to treat flavivirus infections. It is therefore of great importance to public health to develop an efficacious drug therapy against flaviviruses.

Flavivirus virions are spherical in shape, with a diameter of approximately 50 nm (21). The flavivirus genome is a single-stranded, plus-sense RNA of approximately 11 kb in length. The genomic RNA consists of a 5' untranslated region (UTR), a single long open reading frame (ORF), and a 3' UTR (43). The single ORF encodes a polyprotein that is co- and posttranslationally processed by viral and cellular proteases into three structural proteins (capsid [C], premembrane or membrane, and envelope) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (10). Nonstructural proteins are primarily involved in viral RNA replication as components of a replicase complex, but they may also function in viral assembly (22, 24) and antiimmune responses (38). Upon flavivirus infection, the genomic plus-strand RNA is translated to produce viral proteins and transcribed into a complementary minus-strand RNA, which in turn serves as the template for the synthesis of more plus-strand genomic RNA (10).

The 5' and 3' UTRs of the flavivirus genome are approximately 100 and 400 to 700 nucleotides (nt) in length, respectively. Besides several conserved sequence (CS) elements, both the 5'- and 3'-terminal sequences of the genomic RNA can form highly conserved stem-loop structures (5, 6, 27, 41, 43, 45, 54). Previous studies showed that the conserved RNA sequence and structures are critical for flavivirus replication (3, 8, 12, 19, 20, 25, 28, 31, 52, 53, 56, 57). Host and viral proteins (including NS3 and NS5) were found to interact with the 3' UTR of the flavivirus genome (2, 11, 14, 23, 50, 51). These results suggest that the 5' and 3' UTRs direct the processes of flavivirus translation and RNA replication through interactions with viral RNA elements, viral proteins, and the host protein(s) in a regulated manner. However, the molecular details of how these conserved sequence and structural elements modulate viral translation and RNA replication remain unknown.

Using WN virus, we recently showed that specific sequence and structural elements within the 3'-terminal region of the genomic RNA are required for viral replication. (i) Potential genome cyclization mediated by base-pairing interaction (Fig. 1A) between the 5'CS (within the C coding sequence) and the 3'CSI is essential for WN viral RNA synthesis (25). (ii) The flavivirus-conserved pentanucleotide in the 3' stem-loop of the genome (Fig. 1A) requires a specific sequence and structure for RNA synthesis (52). (iii) The 3'-terminal 5-nt region of the genome also requires specific sequence and structure for WN virus replication (53). These findings, together with earlier results from other flaviviruses, suggest that RNA elements within the terminal regions of the flavivirus genome are critical for viral reproduction and therefore can potentially be targeted for antiviral therapy. In the present study, we explore this possibility by testing the antiviral activities of several phosphorodiamidate morpholino oligomers (PMOs) whose sequences are complementary to various regions of the 5' and 3' termini of the WN virus genome (Fig. 1A to C). PMOs are uncharged, water-soluble, nuclease-resistant antisense agents that are typically synthesized to a length of about 20 subunits and contain purine and pyrimidine bases attached to a backbone composed of morpholine rings joined by phosphorodiamidate intersubunit linkages (Fig. 1C) (49). We show that conjugation of an Arg-rich peptide (Fig. 1C) to the 5' end of the PMOs greatly facilitates the delivery of the molecules into cultured cells. PMOs targeting various regions of the 5' and 3' termini of the viral genome inhibited WN virus infection to various degrees. Among them, a PMO targeting the 5'-terminal 20 nt (5'End) and a PMO targeting the 3'CSI showed the greatest antiviral activities. The 5'End and 3'CSI PMOs inhibit the WN virus life cycle primarily through suppression of viral translation and RNA replication, respectively.



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  		FIG. 1. Sequence selection and cellular delivery of PMOs. (A) WN virus genome. Terminal stem-loop structures and a potential genome cyclization are enlarged to display regions where antisense PMOs target to. Individual PMOs are indicated by their names and targeted regions (bracketed lines). WN virus genome cyclization is mediated through a perfect 12-nt base-pairing interaction (dotted lines) between the 5'CS and 3'CSI sequences (boxed sequence). A pseudoknot (Pskt) interaction within the 3' stem-loop (45) is indicated by dashed lines. The major binding sites of host protein EF1{alpha} (2) is depicted by ovals. The flavivirus-conserved pentanucleotide located at the top of the 3' stem-loop (55) is underlined. The AUG initiation codon of the ORF is in boldface type. (B) PMO sequences. All PMOs contain an Arg-rich peptide at their 5' ends, designated P007. An extra C (italicized) was added to the 3' end of the 5'End PMO to potentially interact with the 7-methylguanylate of the 5' cap and to further stabilize its hybridization with the viral genome. (C) Structure of P007-PMOs. In a PMO, the ribose of RNA is replaced by a morpholine ring, and the phosphodiester internucleotide bond is replaced by a phosphorodiamidate linkage. The CH3CONH-(RAhxR)4-Ahx-ßAla sequence was attached to each PMO through a piperazine linker, where R stands for arginine, Ahx stands for 6-aminohexanoic acid, and ßAla stands for beta-alanine. (D) Enhancement of cellular delivery of PMOs by P007 conjugation. BHK cells were incubated with the fluorescein-labeled scramble PMO with or without a 5' P007 conjugation at a concentration of 10 µM. After 15 min of incubation, cells incubated with the P007-conjugated PMO are fluorescence positive (left), whereas no fluorescence is observed in cells incubated with unconjugated PMOs (right).


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MATERIALS AND METHODS
 
Cells and viruses. Three types of cells were used in this study. (i) BHK-21 (baby hamster kidney) cells were grown in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS). (ii) A reporting BHK cell line containing persistently replicating WN virus replicon with dual reporter genes, Renilla luciferase (Rluc) and neomycin phosphotransferase (RlucNeoRep) (Fig. 2A), was maintained in DMEM with 10% FBS and 1 mg of G418/ml (26). (iii) Vero cells (African green monkey kidney cells; ATCC CCL-81) were cultured in minimal essential medium supplemented with 2% FBS, 2 mM L-glutamine, 0.3% sodium bicarbonate, 100 U of penicillin/ml, and 100 µg of streptomycin/ml. All cells were maintained in 5% CO2 at 37°C. WN virus strain 3356, an isolate from the kidney of an American crow collected in 2000 from New York (15), was used for antiviral assays. Viral RNA derived from the same isolate was used to prepare all constructs used in this study, including various reporting replicons (46) and the full-length luciferase virus.



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  		FIG. 2. Antiviral activities of PMOs in a WN virus replicon-reporting cell line. (A) An Rluc and Neo gene were inserted into a WN virus replicon, resulting in RlucNeoRep. A cell line containing persistently replicating RlucNeoRep was established previously (26). Antiviral activities were analyzed by incubation of the RlucNeoRep cells with PMOs at indicated concentrations for 24 h and assayed for Rluc activities. PMOs targeting the 5' (B) and 3' (C) regions of the genome are presented.

Construction of a full-length WN virus that contains a luciferase reporter. An infectious cDNA clone of the epidemic WN virus isolate 3356 (47) was used to construct a luciferase-reporting full-length virus. An Rluc reporter driven by an encephalomyocarditis virus internal ribosomal entry site (EMCV IRES) was inserted into the WN virus 3' UTR at nucleotide position 10,436 (GenBank accession number AF404756), resulting in RlucWN virus (Fig. 3A). The RlucWN cDNA plasmid was prepared by swapping the SpeI-SacII fragment (nt 8,022 to 10,822) between the wild-type infectious clone (47) and a replicon clone containing the IRES-Rluc insertion (25). The RlucWN RNA was in vitro transcribed and transfected into BHK cells as previously described (25). The resulting RlucWN virus was harvested at day 3 posttransfection (p.t.) when the cytopathic effect (CPE) of the transfected cells was apparent.



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  		FIG. 3. A full-length luciferase-reporting WN virus can be used for antiviral drug screening. (A) A full-length WN virus containing a luciferase reporter. An Rluc gene driven by an EMCV IRES was engineered at the upstream region of the 3 ' UTR of the genome, resulting in RlucWN virus. The numbers indicate nucleotide positions in the WN virus genome (GenBank accession number AF404756). (B) RT-PCR analyses of RlucWN virus. Five sets of RT-PCR covering the entire WN virus genome were performed with template RNAs extracted from a wild-type isolate of WN virus (WT WN), the first passage of RlucWN virus possessing a high level of Rluc activity (RlucWN-P1), and the seventh passage of RlucWN virus lacking Rluc activity (RlucWN-P7). DNA fragments amplified from nt 8706 to 10515 indicate that the IRES-Rluc (1,520 bp) insertion was retained in RlucWN-P1 but was subsequently deleted at RlucWN-P7. The RT-PCR products were analyzed with a 1% agarose gel. (C) Antiviral assay with RlucWN virus. Vero cells were infected with RlucWN-P1 at an MOI of 0.5, immediately treated with known WN inhibitors as indicated, and assayed for Rluc activity at 24 h post infection. Rluc activities were plotted against compound concentrations. An average of two experiments is presented.

PMOs. Nine PMOs of 20 to 23 bases in length were synthesized. The sequences of the PMOs and their potential base pairing regions are detailed in Fig. 1A and B. A scramble PMO containing a random sequence was synthesized as a nonviral negative control (Fig. 1B). For efficient delivery of PMOs into cells, an Arg-rich peptide [CH3CONH-(RAhxR)4-Ahx-ßAla, designated P007; R stands for arginine, Ahx stands for 6-aminohexanoic acid, and ßAla stands for beta-alanine] was covalently conjugated to the 5' end of the PMOs through a noncleavable piperazine linker (Fig. 1C). The effect of the Arg-rich peptide on cellular delivery of PMOs was examined by incubating BHK cells with a 3'-fluoresceinated scramble PMO with or without the 5' P007 conjugation (Fig. 1D). The methods for synthesis of PMOs, conjugation of P007, and purification and analysis of P007-PMOs were similar to those previously described (37, 49). The PMOs were dissolved in sterile water at 2 mM, aliquoted, stored in the dark at –20°C, and used in antiviral assays without multiple rounds of freeze and thaw. All assays were performed by direct addition of the PMOs to cell culture medium (described above) without changing the FBS concentration.

Antiviral assays. Three types of antiviral assays were performed in this study. (i) A WN virus reporting cell line containing persistently replicating replicons with dual reporter genes (RlucNeoRep) (Fig. 2A) was used to screen PMOs for antiviral activities. Construction and characterization of the WN virus reporting cell line were described previously (26). For assay of the antiviral activities of PMOs, approximately 2 x 105 cells (in a total volume of 0.8 ml) were seeded in each well of 12-well plates in DMEM with 10% FBS without G418. PMOs were added to the medium at 16 h postseeding. After 24 h of PMO treatment, cells were lysed and assayed for Rluc activities with an Rluc assay kit (Promega, Madison, Wis.).

(ii) A full-length WN virus containing a luciferase reporter (RlucWN) (Fig. 3A) was developed to screen potential inhibitors against all steps of the virus life cycle. Five compounds of known potency against WN virus were used to verify RlucWN virus as an authentic reporter for antiviral assays. Mycophenolic acid, 6-azauridine, ribavirin, and glycyrrhizin were purchased from Sigma (St. Louis, Mo.), and 3-deazauridine was purchased from MP Biomedicals, Inc. (Ëschwege, Germany). All compounds were dissolved in dimethyl sulfoxide (DMSO) and added to cells in medium with a final DMSO concentration of 1%. Cells not treated with compounds were also treated with 1% DMSO as a negative control. Briefly, compounds were added to Vero cells at 80% confluency in 12-well plates with 1.5 ml of minimal essential medium plus 2% FBS per well. Immediately after compound addition, cells were infected with RlucWN virus at a multiplicity of infection (MOI) of 0.5. Rluc activities were assayed at 24 h postincubation (p.i.). Based on the Rluc signal and compound concentration curves (Fig. 3C), regression analysis (SAS, version 6.12; SAS Institute, Inc., Cary, N.C.) was performed to calculate the compound concentration required for 50% inhibition of Rluc activity (EC50) for each inhibitor. Once the RlucWN-virus-based assay had been validated with known WN virus inhibitors as described above, a similar procedure was used to evaluate the antiviral activities of selected PMOs (Fig. 4A).



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  		FIG. 4. Antiviral and cytotoxic analyses of 5'End, 3'CSI, and AUG-I PMOs. (A) Antiviral activities of PMOs were analyzed by the RlucWN-infection assay. PMOs were added to Vero cells during inoculation of RlucWN-P1 virus at an MOI of 0.5. At 24 h postinfection, cells were lysed and assayed for Rluc activity. Antiviral activity was indicated by the reduction of Rluc signal upon PMO treatment. A scramble PMO was included as a negative control. The keys to PMO identity shown in panel B are the same in panel A. (B) Cytotoxicity was evaluated by incubation of Vero cells with the indicated concentration of 5'End, 3'CSI, AUG-I, or scramble PMO. At 24 h p.i., viability of treated cells was measured by an MTT assay and presented as a percentage of colorimetric absorbance derived from untreated cells (see details in Materials and Methods).

(iii) WN virus isolate 3356 was used to verify the effectiveness of the two most potent PMOs (5'End and 3'CSI) and a moderately active PMO (AUG-I) in a viral yield reduction assay (Fig. 5). A protocol similar to that used for the RlucWN virus infection assay was performed (described above) except that (i) Vero cells were infected at an MOI of 0.1, and (ii) virus titer reductions were measured at 42 h postinfection, when CPE was clearly observed in untreated cells. The titer of the virus yield in supernatant was determined by plaque assays on Vero cells (47). A similar protocol was used for analyzing the antiviral activities of PMOs in St. Louis encephalitis (SLE; Kern217.3.1.1), DEN-2 (New Guinea), and YF (17D) viruses. All assays in this study were performed in duplicate or triplicate.



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  		FIG. 5. Inhibition of an epidemic strain WN virus infection by PMOs. Epidemic WN virus isolate 3356 was used to verify the potencies of the 5'End, 3'CSI, and AUG-I PMOs in a virus yield reduction assay. Each PMO was incubated with Vero cells infected with WN virus isolate strain 3356 at an MOI of 0.1. Antiviral activities of the PMOs were measured by virus titer reduction at 42 h postinfection. The scramble PMO was used as a negative control. One set of data representative of duplicate experiments is presented.

MTT cytotoxicity assay. Vero cells grown to confluence in 96-well plates were incubated with selected PMOs in medium identical to that used for the viral infection assay. After incubation with PMOs for 24 h, cell proliferation (viability) was assessed with a tetrazolium colorimetric assay kit (American Type Culture Collection, Manassas, Va.). Briefly, 10 µl of MTT reagent [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] was added to each well. Cells were incubated for 2.5 h at 37°C until the purple precipitate was clearly visible by light microscopy. Detergent reagent was then added, and cells were further incubated overnight at room temperature in the dark to ensure that all formazan crystals were dissolved. The plates were read at 570 nm with an automatic plate reader. Cell viability was presented as the percentage of absorbance at 570 nm derived from the PMO-treated cells relative to absorbance derived from the untreated cells.

Mode-of-action analysis. A reporting replicon containing an Rluc gene (fused in frame with the ORF in the position where the viral structural region was deleted) was used to differentiate PMO-mediated inhibition of viral translation from inhibition of RNA replication (RlucRep) (Fig. 6A). We previously demonstrated that Rluc signals derived from the RlucRep-transfected cells at 2 to 10 h and after 24 h p.t. represent initial translation of input RNA and translation of replicating viral RNA, respectively (25). To adapt the system to a mode-of-action study, we electroporated 8 x 106 BHK cells with 10 µg of RNA, as reported previously (25). The transfected cells were suspended in 25 ml of DMEM with 10% FBS. Three milliliters of cell suspension was added to six-well plates containing preseeded PMOs to a final concentration of 7.5 µM and was assayed for Rluc activity at 2 h p.t. (representing peak translation) and 72 h p.t. (representing RNA replication). Inhibition of viral translation and inhibition of RNA replication in anti-WN virus PMO-treated cells were each expressed as the percentage of Rluc signal derived from the PMO-treated cells compared with the signal derived from the untreated cells at an equivalent time point.



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  		FIG. 6. Mode-of-action analyses. (A) A WN virus reporting replicon containing an Rluc gene (fused in frame with the ORF; RlucRep) was used to analyze the modes of action of 5'End, 3'CSI, and AUG-I PMOs. BHK cells transfected with RlucRep exhibit two distinctive Rluc peaks at 2 to 10 h and after 24 h p.t., representing viral translation and RNA replication of input RlucRep, respectively (25). (B) Differentiation between PMO-mediated inhibition of viral translation and RNA synthesis. RlucRep-transfected BHK cells were incubated with 7.5 µM PMOs and assayed for Rluc activity at 2 h p.t. (viral translation) and 72 h p.t. (RNA replication). Values for PMO-mediated inhibition of viral translation and RNA replication are each presented as a percentage of the Rluc signal derived from the untreated cells at an equivalent time point.


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RESULTS
 
Sequence selection of PMOs for targeting terminal regions of the WN virus genome. Nine antisense PMOs were synthesized to target various RNA elements within the terminal regions of the WN virus genome (Fig. 1A and B). The 5'End PMO was designed to contain a sequence capable of base-pairing with the first 20 nt of the genome. An extra C (Fig. 1B) was added to the 3' end of the 5'End PMO to potentially interact with the 7-methylguanylate of the 5' cap and to further stabilize its hybridization with the viral genome. Two PMOs, AUG-I and AUG-II, were prepared to block viral translation through base pairing with a region spanning the initiation codon (bolded, Fig. 1A) of the viral ORF. The 5'CS and 3'CSI PMOs were synthesized to interfere with a potential genome cyclization (17) mediated through a perfect 12-nt base-pair interaction (Fig. 1A). The pseudoknot (Pskt) PMO was designed to invade a region that had been previously reported to form a pseudoknot structure (Fig. 1A) (45). The 3'Stem PMO was synthesized to base pair with a region containing the major binding site of the host protein EF1{alpha} (5'-CACA-3') (Fig. 1A) (2). The 3'Loop PMO was prepared to target a flavivirus-conserved pentanucleotide (Fig. 1A) located at the top loop of the 3' stem-loop (55). The 3'End PMO was designed to base pair with the 3'-terminal 20 nt of the genome. In addition, a scramble PMO was synthesized to contain a random sequence as a nonviral negative control.

Enhancement of cellular delivery of PMOs by an Arg-rich peptide. PMOs conjugated with a short positively charged peptide resembling a putative translocation domain of the human immunodeficiency virus type 1 Tat protein were shown to penetrate cultured cells much more efficiently than PMOs lacking such a peptide conjugation (36, 37). A new type of Arg-rich peptide conjugate, designated P007 (Fig. 1C), was synthesized and examined for its effect on cellular delivery of PMOs. BHK cells were incubated with a fluorescein-labeled scramble PMO conjugated with or without a P007 peptide. Remarkably, fluorescent microscopic analyses showed that all cells incubated with the P007-conjugated PMO (at 10 µM) were fluorescence positive at 15 min p.i. (Fig. 1D, left). The fluorescent intensity increased during the first 2 h of incubation and reached saturation afterwards (data not shown). Although the fluorescence was distributed throughout the cells, signals were concentrated at the nucleus. In contrast, no fluorescence was observed in cells incubated with the PMO lacking a P007-conjugation (Fig. 1D, right). Longer incubation of cells with non-P007-conjugated PMO (up to 4 h) did not elicit any fluorescent signal (data not shown). The results clearly demonstrated that P007 enhanced delivery of PMOs into cultured cells. Therefore, all PMOs used in the following experiments were synthesized to contain a P007 conjugation.

PMOs targeting various RNA elements within the termini of WN virus genome inhibit viral replication to various degrees. We initially screened the antiviral activities of the PMOs in a WN virus replicon-reporting cell line (26). The reporting cell line harbors a persistently replicating replicon that contains dual reporter genes, Renilla luciferase (Rluc) and neomycin phosphotransferase (RlucNeoRep) (Fig. 2A). We previously showed that Rluc activity derived from the replication of RlucNeoRep within the cell line could be used to estimate the antiviral activities of WN virus inhibitors (26). To examine the effects of PMOs on WN virus replication, we incubated the PMOs with RlucNeoRep-containing cells and assayed for Rluc activity at 24 h after PMO addition. The PMO-mediated inhibition of viral replication was reflected by an inverse dose response between the PMO concentration and the Rluc signal intensity. All four PMOs targeting 5'-terminal regions of the genome exhibited antiviral activities with potency decreasing in the order of 5'End, AUG-I, AUG-II, and 5'CS PMOs (Fig. 2B). Among the five PMOs targeting the 3'-terminal regions of the genome, 3'CSI showed greatest potency. Three other PMOs (Pskt, 3'Stem, and 3'Loop) exhibited moderate inhibition, whereas 3'End PMO (targeting the 3'-terminal 20 nt of the genome) did not consistently show inhibition (Fig. 2C). As a control, the scramble PMO with nonviral sequence did not exhibit any inhibition of Rluc activity (Fig. 2B and C). No significant cytotoxicity was observed at 10 µM, the highest concentration tested in the above experiments (see below), indicating that the observed antiviral activities of the PMOs were not due to cytotoxicity. Comparison of the Rluc curves derived from the various PMOs suggests that (i) PMOs targeting different regions of the WN genome inhibit viral replication to differing degrees and (ii) 5'End and 3'CSI are the two most potent inhibitors. Therefore, the following experiments were focused on further analysis of the 5'End and 3'CSI PMOs.

A full-length WN virus containing a luciferase reporter can be used to screen for potential inhibitors of the WN virus life cycle. The RlucNeoRep reporting cell line described above could only be used to screen for inhibitors against viral replication (including viral translation, polyprotein processing, and minus- and plus-strand RNA synthesis), but not viral entry and assembly (due to a deletion of structural C-prM-E genes in the replicon). To facilitate antiviral screening for potential inhibitors against any step of the virus life cycle, we attempted to construct an infectious reporting WN virus by engineering a luciferase gene into the full-length WN virus genome (47). An Rluc gene driven by an EMCV IRES was inserted at the upstream region of the 3' UTR of the genome, resulting in RlucWN virus (Fig. 3A). Transfection of BHK cells with RlucWN RNA yielded a high-titer reporting virus (up to 109 PFU/ml at day 3 p.t.). Infection of cells with the initial virus stock at an MOI of 0.5 resulted in an Rluc signal over 105-fold higher than background level (Fig. 3C). However, multiple passaging of the reporting virus in BHK cells resulted in sequential decreases in the Rluc signal. By the third round of infection, the Rluc signal was reduced to background level (data not shown). Meanwhile, a high level of infectious virus was maintained during multiple passaging of the virus, as indicated by plaque assays and CPEs from infected cells (data not shown). The results suggest that the RlucWN virus does not stably retain the nonviral reporter.

To determine the exact region(s) of mutation or deletion during the passaging of the reporting virus, we extracted RNAs from wild-type virus (without reporter), first passage of the RlucWN virus (containing a high level of luciferase activity), and seventh passage of the virus (lacking any luciferase activity). Five sets of RT-PCR covering the entire WN virus genome were performed on the extracted RNAs (Fig. 3B). The results showed that the IRES-Rluc fragment (a total length of 1,520 bp) inserted at nucleotide position 10463 was present at the first passage of the RlucWN virus but was subsequently deleted by the seventh passage of the virus. Sequencing of the reverse transcription-PCR (RT-PCR) product derived from the seventh passage of the reporting virus showed that only the 5'- or 3'-terminal residues of the IRES-Rluc cassette were retained at the insertion site. Since we did not sequence the entire genome, it is also possible that mutations exist in other regions of the seventh-passage virus.

Although the RlucWN virus is not stable, we argue that the reporting virus could still be useful as a reagent for antiviral screening assays. Incubation of Vero cells (infected with reporting virus stock derived directly from the RlucWN-RNA-transfected cells) with potential inhibitors should reduce the Rluc signal. Comparison of Rluc signals derived from the compound-treated infection with the untreated infection should reveal the potency of the inhibitor. To explore this possibility, we incubated the RlucWN-infected cells (at an MOI of 0.5) with several known WN virus inhibitors (Fig. 3C). The compounds were added to culture medium at the time of viral infection and were retained in cell culture throughout the assay. As expected, Rluc activities assayed at 24 h p.i. showed an inverse correlation with PMO concentration. Based on the Rluc curves, the EC50s were estimated to be 0.34, 0.74, 12, 19, and >300 µM for mycophenolic acid, 6-azauridine, 3-deazauridine, ribavirin, and glycyrrhizin, respectively. The EC50s for 6-azauridine (0.74 µM), ribavirin (19 µM), and glycyrrhizin (>300 µM) agreed well with the EC50s derived from an authentic virus infection assay (0.82 µM, 3 to 60 µM, and 486 µM, respectively) (13, 18). Thus, at early passage, the reporting virus (RlucWN) can potentially be used to screen for inhibitors against WN virus reproduction.

It should be noted that EC50 values derived from the RlucWN virus assay are lower than those derived from the RlucNeoRep cell line assay (Fig. 2A). As described above, the EC50s derived from the RlucWN virus assay were estimated to be 0.34, 0.74, and 19 µM for mycophenolic acid, 6-azauridine, and ribavirin, respectively (Fig. 3C), whereas the EC50s derived from RlucNeoRep cell line were determined to be 5.4, 11, and 140 µM (26). The discrepancy in EC50s between the two assay systems is most likely due to the difference in viral replication status when the compound was added. Specifically, in the RlucNeoRep cell-based assay, a high level of replicons has already been replicating inside cells before treatment with a compound, whereas in the RlucWN infection-based assay, compound and virus were added simultaneously to naïve cells.

Once the RlucWN-infection assay had been validated, we employed the assay to evaluate the antiviral activities of the two most potent PMOs (5'End and 3'CSI) and a moderately active PMO (AUG-I). A similar procedure for analyzing known WN inhibitors (Fig. 3C) was performed. As shown in Fig. 4A, Rluc signals were significantly suppressed by the 5'End, 3'CSI, and AUG-II PMOs at 1 µM and were further decreased at 5 µM. The relative potencies of these PMOs derived from the RlucWN infection assay correlated well with those derived from the replicon-reporting cell line (Fig. 2). In contrast, the scramble PMO did not exhibit substantial suppression of the Rluc signal. These results again suggest that the RlucWN-reporting virus can be used as an antiviral reagent.

Cytotoxicity of PMOs. An MTT assay was performed to exclude the possibility that the observed inhibition of WN viral infection was due to PMO-mediated cytotoxicity. Vero cells were incubated with various concentrations of 5'End, 3'CSI, AUG-I, or scramble PMO for 24 h. Viability of PMO-treated cells was quantified by cellular metabolism of MTT tetrazolium salt. The results showed that cells incubated in PMO at a concentration up to 10 µM maintained a cell viability of around 85%. However, a minor change in cell morphology began to appear when cells were incubated in 10 µM PMO (but not at a concentration of ≤7.5 µM; data not shown), indicative of a low level of cytotoxicity at 10 µM. The cytotoxicity-mediated morphological change of treated cells became more pronounced at a PMO concentration of 20 µM (data not shown). The latter observation agreed with the MTT result that cell viability was reduced to about 70% when cells were incubated in 20 µM PMO (Fig. 4B). The results suggest that PMOs cause minor cytotoxicity at 10 µM or higher. Therefore, the PMO-mediated inhibition of a WN virus reporting replicon (Fig. 2) or reporting virus (Fig. 4A) was not due to cytotoxicity at concentrations of <10 µM.

Potent inhibition of an epidemic strain of WN virus by PMOs. To validate the authenticity of the observed PMO-mediated viral inhibition, we tested the 5'End, 3'CSI, and AUG-I PMOs in a wild-type WN virus infection assay. The PMOs were incubated with Vero cells infected with an epidemic strain of WN virus at an MOI of 0.1. The effects of the PMOs on viral infection were measured by virus titer reduction at 42 h p.t., when the CPE was clearly observed in untreated cells. As shown in Fig. 5, the 5'End PMO reduced the virus titer by about 6 logs at 5 µM or 10 µM. The 3'CSI PMO inhibited virus titer by 5 logs at 5 µM and 6 logs at 10 µM. The AUG-I PMO was less potent, reducing virus titer by nearly 3 logs at 5 µM and 5 logs at 10 µM. Compared with the 5'End, 3'CSI, and AUG-I PMOs, the scramble PMO did not cause a substantial reduction of virus titer at 1 and 5 µM. However, a 2-log reduction of virus titer was observed at a scramble PMO concentration of 10 µM. This nonspecific virus titer reduction was most likely due to a low level of cytotoxicity of PMOs at this concentration, as described above. Nevertheless, the results clearly suggest that the 5'End and 3'CSI PMO potently inhibit WN virus infection without apparent cytotoxicity at concentrations below 10 µM.

A broad spectrum of antiviral activity of 3'CSI PMO against mosquito-borne flaviviruses. Since the sequence targeted by the 3'CSI PMO is mostly conserved among mosquito-borne flaviviruses (17), we asked whether the 3'CSI PMO could inhibit mosquito-borne flaviviruses other than WN virus. Vero cells were infected with SLE, DEN-2, or YF virus (0.1 MOI), treated with 3'CSI PMO (7.5 µM), and assayed for virus titers at 40 h posttreatment. As expected, virus titers derived from the 3'CSI PMO-treated infections were consistently lower than those derived from the untreated infections (Table 1). The extent of reduction in virus titer correlated with the number of base pairs between the 3'CSI PMO and the targeted sequences of specific viruses. For example, targeted sequences from SLE and DEN-2 viruses have one and two mismatches with the 3'CSI PMO, and the corresponding suppression of virus titer was 500 and 640 fold upon 3'CSI PMO treatment, respectively. In contrast, the targeted sequence from YF virus and the 3'CSI PMO has eight mismatches (out of 22 nt), and the virus titer reduction was only 69 fold upon treatment. As controls, scramble PMO at the same concentration did not inhibit any tested viruses. The results showed that the 3'CSI PMO could broadly inhibit mosquito-borne flaviviruses. Experiments are ongoing to optimize the sequence of the CS1 PMO to improve its potency against various mosquito-borne flaviviruses.


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  		TABLE 1. Antiviral activities of 3'CSI PMO against mosquito-borne flavivirusesa

To further analyze the antiviral specificity of PMOs, we examined the activities of 5'End, 3'CSI, and scramble PMOs against Western equine encephalitis (WEE) virus, an alphavirus from family Togaviridae. Vero cells were infected with 0.1 MOI of WEE virus, treated with 7.5 µM of various PMOs, and assayed for virus titers at 40 h posttreatment. None of the PMOs reduced the titer of the WEE virus. Both treated and untreated infections yielded similar virus titers between 8.3 x 106 to 5 x 107 PFU/ml. The lack of antiviral activity of PMOs on WEE virus and the difference in potency of 3'CSI PMO against various mosquito-borne flaviviruses clearly demonstrated the antiviral specificity of the PMOs.

PMOs inhibit WN virus infection through specific suppression of viral translation or RNA replication. To study the mode of action of the two most potent 5'End and 3'CSI PMOs, we analyzed the inhibitors in a previously developed transient replicon system (25). This replicon contains an Rluc gene fused in frame with the ORF in the position where the viral structural genes were deleted (RlucRep) (Fig. 6A). We previously showed that transfection of BHK cells with RlucRep RNA yielded two distinctive Rluc peaks at 2 to 10 h and after 24 h p.t., respectively, representing viral translation and RNA replication of the transfected RlucRep (25). This unique Rluc pattern could reliably be used to differentiate between RNA elements involved in viral translation and those involved in RNA replication (25, 52, 53). To analyze the modes of action of the 5'End and 3'CSI PMOs, we incubated the RlucRep-transfected BHK cells with PMOs immediately after electroporation. At 2 h (peak translation time of input RNA) and 72 h p.t. (RNA synthesis), cells were assayed for Rluc activities. The medium-active AUG-I and scramble PMOs were also included as controls. Compared with the mock-treated cells, 5'End PMO (at 7.5 µM) suppressed Rluc activity by approximately 90% at 2 h p.t., leading to a background level of Rluc activity at 72 h p.t. As expected, AUG-I PMO (targeting the initiation codon of the ORF) inhibited Rluc activity by about 72% at 2 h p.t. Remarkably, 3'CSI did not substantially inhibit Rluc signal (96% of the mock treated) at 2 h p.t. but reduced Rluc activity to background level at 72 h p.t. As a control, the scramble PMO did not significantly affect Rluc signals at the 2- or 72-h time point. These results suggest that the 5'End and 3'CSI PMOs inhibit WN virus infection through two distinct mechanisms: inhibition of viral translation and RNA replication, respectively.

To further verify the specificity of the 5'End PMO-mediated inhibition on viral translation, we in vitro transcribed an RNA, designated 5' UTR-Rluc-3' UTR (Fig. 7A), which contained a reporting Rluc (in-frame fused with the N-terminal 31 amino acids of the C protein) flanked by 5' and 3' UTRs. Compared with the replicon system shown in Fig. 6, the 5' UTR-Rluc-3' UTR RNA allowed us to directly measure the effects of PMOs on translation. Transfection of BHK cells with such reporting RNA showed that Rluc signal peaked between 5 to 10 h p.t. (Fig. 7B). Treatment of the 5' UTR-Rluc-3' UTR RNA-transfected cells with 5'End and AUG-I PMOs (at 7.5 µM for 5 h) resulted in suppression of Rluc signals by approximately 82 and 52%, respectively (Fig. 7C). In contrast, no significant suppression of Rluc activity was observed by treatment with the scramble PMO. These data agreed well with the replicon results (Fig. 6) and again demonstrated that the 5'End PMO exerts its antiviral activity through potent suppression of viral translation.



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  		FIG. 7. Inhibition of viral translation by 5'End and AUG-I PMOs. (A) A reporting RNA (designated 5' UTR-Rluc-3' UTR) contains a Rluc gene (shaded box) fused in frame with the coding sequence of the N-terminal 31 amino acids of the C protein (open box). The ORF is flanked by WN virus 5' and 3' UTRs. (B) A time course of Rluc activity from BHK cells transfected with 5' UTR-Rluc-3' UTR RNA. (C) BHK cells were incubated with indicated PMOs (7.5 µM) immediately after transfection of 5' UTR-Rluc-3' UTR RNA, and assayed for Rluc activity at 5 h p.t. Inhibition of PMOs on translation is presented as percentage of the Rluc activity derived from the untreated cells.


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DISCUSSION
 
Prevention and treatment of infections by WN virus and other flaviviruses are public health priorities. A number of strategies can be used in the development of flavivirus therapies (44). The goal of this study was to explore the feasibility of targeting RNA elements within the terminal regions of flavivirus genomes for antiviral drug discovery. Using WN virus as a model, we showed that antisense PMOs targeting various terminal regions of the WN virus genome had various antiviral activities. The two most potent PMOs were 5'End (targeting the 5'-terminal 20 nt) and 3'CSI (targeting a 3' element involved in genome cyclization). Treatment of WN virus-infected cells with 5'End or 3'CSI PMOs reduced virus titers by approximately 5 to 6 logs at a concentration of 5 µM without apparent cytotoxicity. The 5'End and 3'CSI PMOs specifically inhibit viral translation and RNA synthesis, respectively.

Mechanisms of antisense PMOs. The efficacies of the antisense PMOs are dependent on many factors, such as (i) the efficiency of cellular delivery, (ii) the roles of the PMO-targeted sequences in the virus life cycle, and (iii) the accessibility of the targets for base pairing with the PMOs during viral infection. It is remarkable that the Arg-rich peptide P007 greatly facilitates the delivery of the PMOs into cultured cells (Fig. 1D). The mechanism of the P007 peptide-mediated cellular uptake is currently unknown and merits further investigation. Other Arg-rich peptides were recently shown to enhance cellular delivery of PMOs (36). For example, an NH2-(R)9(F)2-CONH2 peptide was shown to enhance PMO delivery to inhibit mouse hepatitis virus (39). However, the efficacy of the NH2-(R)9(F)2-CONH2-conjugated PMOs was reported to depend on the absence of serum in the culture medium. It was speculated that the highly charged peptide mediated attachment of PMOs to serum proteins, thus reducing the number of free PMO molecules competent for cell entry (39). In contrast, the efficacy of the P007-conjugated PMOs designed for the present study did not show significant serum dependency, since all assays were performed in medium containing normal serum concentrations (from 2 to 10%). The improvement of this property in our study is likely due to the different composition of P007 peptide.

The PMOs evaluated in this study were designed to target specific terminal elements of the WN virus genome. Previous mutagenesis studies had shown that the targets for 5'End (19), AUG-I and AUG-II (8), 5'CS (12, 20), 3'CSI (3, 25, 56), 3'Loop (52), and 3'End (19, 53, 57) PMOs were essential for flavivirus replication. The biological relevance of the targets for Pskt (45) and 3'Stem PMOs (2) has not been experimentally demonstrated. Screening of the PMOs in a replicon-reporting cell line (RlucNeoRep) (Fig. 2) showed that all molecules, except the 3'End PMO, have antiviral activities. These results indicate that regions involved in pseudoknot interaction (targeted by Pskt PMO) and EF1{alpha} binding interaction (targeted by 3'Stem PMO) may be important for WN virus replication. However, it should be pointed out that, since the 20- to 23-nt PMOs interact with regions larger than the specific target site (i.e., the nucleotides involved in pseudoknot interaction and the major binding site for EF1{alpha}), we cannot exclude the possibility that PMO base pairing with regions other than the targeted site is contributing to the antiviral activities.

The 5'End PMO exhibited the greatest antiviral activity through its suppression of viral translation (Fig. 6 and 7). Base-pairing interaction between the 5'End PMO and the 5'-terminal 20 nt of the genome may suppress the formation of the 43S preinitiation complex and ribosome scanning during viral translation (16), leading to a suppression of initial viral translation. Interestingly, the translation suppression mediated by the 5'End PMO is greater than that mediated by the AUG-I PMO (decreases of 90 and 72%, respectively). The AUG-I and AUG-II PMOs were expected to block the interaction of the initiator AUG with the Met-tRNAi in the 43S preinitiation complex, thereby preventing the 60S ribosomal subunit from joining the preinitiation complex. However, a study using the Kunjin replicon showed that the conserved AG at the 5' terminus of the flavivirus genome is essential for RNA replication (most likely functioning at the step of plus-strand RNA synthesis) (19). These results imply that, besides suppressing viral translation, the 5'End PMO also interferes with WN virus RNA synthesis. It is difficult to quantify the latter effect on viral inhibition, because the 5'End PMO-mediated reduction in translation could lead to suppression of RNA replication.

The 3'CSI PMO exhibited a potent antiviral activity through its suppression of RNA replication, but not translation (Fig. 6). These data agree well with our previous results that mutations within the 3'CSI region (blocking the potential genome cyclization) do not significantly affect translation but completely abolish RNA replication (25). Unexpectedly, the 5'CS PMO was less potent than the 3'CSI PMO (Fig. 2). The 5'CS and 3'CSI PMOs are of similar length, G-C content, and design rationale to target either one of the two genome cyclization sequences. The lower efficacy of 5'CS PMO is likely due to its location at 32 nt downstream of the AUG initiation codon. A previous study has shown that PMOs targeting regions more than 20 nt downstream from the translation initiation site are much less effective in inhibiting viral translation. The processive translocation of the ribosome complex (which exhibits RNA helicase activity) could dislodge the bound PMOs from the target RNA (48). In contrast, the binding site for the 3'CSI PMO is located 520 nt downstream of the stop codon of the ORF. Therefore, the 3'CSI PMO is less likely to be dislocated through ribosome collision.

It is puzzling that the 3'End PMO targeting the 3'-terminal 20 nt of the genome is not active against WN virus infection (Fig. 2). Our previous results showed that the specific sequence and structure of the 3'-terminal 5-nt region of the genome are essential for WN virus replication (53). We suspect that the 3' terminal nucleotides are constrained in a conformation that is not accessible for base pairing with the 3'End PMO.

Antiviral assays. Development of reliable and authentic assays is essential for antiviral drug discovery. Besides the traditional virus titer reduction assay, we have employed three reporting antiviral assays in the present study. The type 1 assay uses a previously developed cell line containing a dual-reporter replicon (RlucNeoRep) (Fig. 2A). Incubation of the RlucNeoRep-reporting cells with a compound that blocks WN virus replication suppresses the Rluc expression level (26). The utility of this reporting cell line as an antiviral assay was further confirmed in the present study, which screened the relative potencies of PMOs against WN virus replication (Fig. 2B and C). The type 2 assay is based on a full-length reporting virus (RlucWN) (Fig. 3A). We showed that, although the RlucWN virus is not stable after multiple passages (Fig. 3B), an early passage of this reporting virus can be used to screen for inhibitors of WN virus infection (Fig. 3C and 4A). Compared with the traditional virus titer reduction assay (13, 18, 35), both type 1 and type 2 assays are more sensitive and quantitative and can potentially be used for high-throughput screening of compound libraries.

When the two types of assays described above are compared, the RlucWN virus assay covers more steps of the virus life cycle (including virus entry and virion assembly) than does the RlucNeoRep cell line assay (which primarily involves viral translation and RNA replication). Therefore, the RlucWN virus assay can potentially be used to screen for inhibitors of viral entry and virion assembly. This extra feature of the RlucWN virus assay is particularly important, as recent studies suggest that besides viral replication, viral entry and particle assembly are also attractive targets for antiviral therapy (4, 21, 32, 33, 58, 59). For example, crystallographic studies showed that envelope proteins of DEN (32, 33) and TBE (4) viruses undergo a sequential structural change during the fusion-activating transition, a stage which could be targeted in the search for small-molecule inhibitors of flavivirus entry.

Efforts have been made to stabilize the RlucWN-reporting virus. One approach is to employ multiple rounds of plaque purification of replicating isolates that retain a high level of luciferase activity. The rationale of this approach is that viral evolution during multiple rounds of purification could confer adaptations that may accommodate the Rluc reporter, resulting in stable RlucWN isolates. We have performed such experiments by purifying Rluc-positive isolates (typically exhibiting smaller plaques than those derived from the wild-type virus). Disappointingly, after six rounds of plaque purification, the selected RlucWN isolates were still not stable (data not shown). An alternative approach is to insert the Rluc reporter within the ORF of the virus. One major advantage of this approach is the elimination of the IRES (required to drive the translation of the Rluc) that is present in our current construct, thus minimizing the potential packaging problem due to the oversized recombinant genome. The recent success of using transposons to select specific sites tolerable for insertion of a green fluorescent protein reporter in hepatitis C virus replicon (34) suggests that a similar approach could be useful for development of a stable reporting flavivirus.

The type 3 assay is based on a transient reporting replicon that can differentiate between viral translation and RNA replication (RlucRep) (Fig. 6A) (25). Due to its transient nature, this assay is more sensitive to compound inhibition than the two assays described above. For example, the 5'CS, 3'CSI, and AUG-I PMOs completely knocked out the Rluc activity by 72 h p.t., indicating no replication of RlucRep (Fig. 6B). In contrast, similar concentrations (5 to 10 µM) of the same PMOs in the RlucNeoRep cell line (Fig. 2B and C) or RlucWN virus assay (Fig. 4A) did not suppress viral replication to background levels. Nevertheless, the results using the 5'CS, 3'CSI, AUG-I, and scramble PMOs clearly showed that the transient RlucRep system could be used to differentiate between the effects of PMOs on viral translation and RNA replication (Fig. 6B).

In summary, a combination of the above three assays will greatly facilitate identification of potential inhibitors of any step of the viral life cycle and will be useful for determining the specific step at which an inhibitor functions. The potent anti-WN virus activities of the 5' End and 3'CSI PMOs clearly suggest for the first time that flavivirus infection could be inhibited by specific suppression of viral translation and RNA replication. Besides the PMO-mediated antiviral approach, other nucleic acid-based approaches have been previously reported against flaviviruses. Microinjection of modified phosphorothioate antisense agents into DEN-2 virus-infected cells reduced virus titers by 50 to 75% at 24 h postinfection (42). Mosquitoes transduced with a recombinant Sindbis virus capable of expressing a 567-base antisense RNA to target DEN-2 virus genomic RNA were unable to support replication of DEN-2 virus in their salivary glands and therefore were not able to transmit the virus (40). RNA silencing (small interfering RNA) was recently shown to suppress DEN-2 (1) and WN (30) viruses in tissue cultures. The potent inhibition of WN virus by the 5'End and 3'CSI PMOs described in this study has opened another avenue for antiviral therapy of flavivirus and potentially other RNA virus infections. Further development of these promising antiviral agents requires evaluation in animal models to examine in vivo potency, toxicity, metabolism, and pharmacokinetics. Finally, it is worth noting that, besides the antiviral utility, the PMOs can serve as a useful research tool to probe functions of viral RNA elements controlling translation and replication.


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ACKNOWLEDGMENTS
 
We are grateful to the Chemistry Group at AVI BioPharma for synthesis, purification, and analysis of the PMOs used in this study. We thank the Molecular Genetics Core and the Cell Culture Facility at the Wadsworth Center for DNA sequencing and for maintenance of BHK and Vero cells, respectively.

I.B.-G. was supported by Emerging Infection and HIV Training and Research Program grants TW00915 and TW00233 from the Fogarty International Center. The work was supported by grant 1U01AI061193-01 and contract N01-AI-25490 from the National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address: Wadsworth Center, New York State Department of Health, 120 New Scotland Ave., Albany, NY 12208. Phone: (518) 473-7487. Fax: (518) 473-1326. E-mail: ship{at}wadsworth.org. Back

{dagger} These authors made equal contributions. Back


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Journal of Virology, April 2005, p. 4599-4609, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4599-4609.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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