Potent inhibition of respiratory syncytial virus replication using a 2-5A-antisense chimera targeted to signals within the virus genomic RNA

Abstract

The 2-5A system is a recognized mechanistic component of the antiviral action of interferon. Interferon-induced 2-5A synthetase generates 2-5A, which, in turn, activates the latent constitutive RNase L that degrades viral RNA. Chemical conjugation of 2-5A to an antisense oligonucleotide can target the 2-5A-dependent RNase L to the antisense-specified RNA and effect its selective destruction. Such a 2-5A-antisense chimera (NIH351) has been developed that targets a consensus sequence within the respiratory syncytial virus (RSV) genomic RNA. NIH351 was 50- to 90-fold more potent against RSV strain A2 than was ribavirin, the presently approved drug for clinical management of RSV infection. It was similarly active against a variety of RSV strains of both A and B subgroups and possessed a cell culture selectivity index comparable to ribavirin. In addition, the anti-RSV activity of NIH351 was shown to be virus-specific and a result of a true antisense effect, because a scrambled nucleotide sequence in the antisense domain of NIH351 caused a significant decrease in antiviral activity. The 2-5A system’s RNase L was implicated in the mechanism of action of NIH351 because a congener with a disabled 2-5A moiety was of greatly reduced anti-RSV effectiveness. These findings represent an innovative approach to the control of RSV replication.

The most important cause of viral lower respiratory tract disease in children and infants is respiratory syncytial virus (RSV). This virus is responsible for 300,000 hospitalizations and 4,500 deaths annually in the United States (1) and more than 1 million deaths every year worldwide. Institutionalized elderly (2) and immunodeficient (1) adults also are at increasing risk for serious sequelae from RSV infection. The only anti-RSV chemotherapeutic agent approved in the U.S. is ribavirin, which acts to reduce virus shedding in infected children, but its beneficial effect on mortality or duration of hospitalization sometimes has been questioned (3). Because ribavirin is administered as an aerosol, there also is concern about its teratogenic hazard to pregnant health care workers. In the search for alternative antiviral strategies, we have investigated the potential of novel chimeric antisense molecules, 2-5A-antisense, in the control of RSV infections.§ We already have reported the effectiveness of 2-5A-antisense (4) against an M2 RSV mRNA (5). Herein, we describe a drug design approach that employs 2-5A-antisense directed against consensus sequences in the RSV genomic RNA.

The RNA genome of RSV, a member of the pneumovirus subfamily of the family Paramyxoviridae, codes for 10 virus-specific proteins (3) and is a single strand of negative-sense RNA of 15,222 nt. The RSV strain A2 has been sequenced completely (refs. 6 and 7; GenBank accession no. M74568, RSHSEQ). After infection, the RSV genome becomes a template for transcription of the RSV mRNAs, and within the 15,222-nt RNA there are gene-end, intergenic, and gene-start signals that lead to mRNA initiations and terminations. However, during replication, the complete 15,222-nt sequence of the negative-strand RNA template must be duplicated in an intact form. Those intergenic sequences not expressed as mRNA during transcription (3) must be incorporated into the full-length, positive-strand RNA (antigenomic RNA) because this RNA serves as a template for the synthesis of progeny negative-strand RSV genomic RNA. Therefore, the RNA synthesis must enter an “antitermination mode,” i.e., all of the signals at gene boundaries and at the boundary between the leader RNA template and various viral genes are ignored to generate a full-length 15,222-nt RNA template (3).

The unique 2′,5′-phosphodiester bond-linked oligonucleotide, 2-5A [p_n5′A2′(p5′A2′)_mp5′A], plays a key role in the anti-encephalomyocarditis virus action of interferon (8). After generation by interferon-induced and dsRNA-activated 2-5A synthetase, 2-5A functions as a potent inhibitor of translation through the activation of a constitutive latent endonuclease, the 2-5A-dependent RNase (RNase L), which degrades RNAs. We have paired the RNA cleavage capacity of the 2-5A system with the specificity of the antisense approach by constructing synthetic adducts between 2-5A and antisense oligonucleotides (4). Such composite nucleic acids could, through the antisense domain, target the chimera to a particular mRNA sequence, which then would be targeted for destruction by the 2-5A component through a localized activation of the latent 2-5A-dependent RNase L.

The 2-5A-antisense strategy has been demonstrated in several cell-free systems: HIV TAR-A₂₅-vif RNA in CEM cell cytosol (4), PKR RNA (9, 10), BCR RNA (11), and HIV gag RNA (12) with purified recombinant human RNase L. 2-5A-antisense chimeras also have effected ablation of PKR mRNA, protein, and biologic function in intact cells (9) and caused the specific degradation of RSV mRNA and an accompanying blockade of RSV replication (5, 13).

There are some specific features of the genomic RNA of RSV that are of interest for an RSV control strategy that uses 2-5A-antisense to target RNA for catalytic destruction. Examination of the RSV genomic RNA shows that each gene begins with a conserved 9-nt gene-start signal, 3′-CCCCGUUUA. This generality holds for all but the L gene, which has the signal 3′-CCCUGUUUUA (the differences are underlined). Transcription always starts at the first nucleotide of the signal (3). Each RSV gene terminates with a semiconserved 12- to 13-nt gene-end signal, 3′-UCA AU UN AU AU AU UUU. This latter signal directs transcriptional termination and polyadenylation (3). The A2 strain RSV intergenic regions range in size from 1 to 52 nt and do not contain any conserved sequences (3). However, these intergenic sequences are uridylate-rich, making them a potential substrate for the established uridylate-preferring RNase L (8) of the 2-5A-antisense approach (4, 5, 9–12).

MATERIALS AND METHODS

2-5A-Antisense Chimeras and Other Oligonucleotides.

Reagents and procedures for synthesis of all oligonucleotides were as described previously (14–16). Phosphorothioate and mixed phosphorothioate backbone 2-5A-antisense chimeras were prepared by using [³H]1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent) (17). These 2-5A-antisense chimeras have the general formula sp5′A2′[p5′A2′]₃O(CH₂)₄OpO(CH₂)₄Op5′(dN)_m, and are abbreviated 2-5A₄-Bu₂-(dN)_m. The 5′ terminus of the 2-5A moiety bears a 5-monothiophosphoryl group (15), and the antisense domain is of varying nucleotide composition and/or substituted in the backbone with phosphorothioate (PS). Oligonucleotides were purified and characterized as described (16).

Cells and Virus.

Embryonic African green monkey kidney cells (MA-104) were a continuous cell line obtained from BioWhittaker. HEp-2 cells were a continuous cell line derived from a human epidermoid carcinoma of the larynx (American Type Culture Collection, ATCC; Manassas, VA), and EBTr cells (ATCC) were initiated from male bovine fetal tracheas. The cells were grown in minimal essential medium (MEM, GIBCO/BRL) supplemented with 0.1% NaHCO₃ and 10% fetal bovine serum (FBS, HyClone). During antiviral assays, serum was reduced to 2% and gentamicin (Sigma) was added to the medium at a final concentration of 50 μg/ml. With the exception of RSV strain 393, which was generously donated by Richard Weltzin (Orovax., Cambridge, MA), all RSV strains as well as bovine RSV (BRSV) were acquired from ATCC. For antiviral assays, all oligonucleotides were dissolved in sterile water at 1-mM concentrations and then diluted to test concentrations in media.

Cytopathic Effect (CPE) Inhibition Assay.

The CPE inhibition assay used in this study was performed as described by Sidwell and Huffman (18) with slight modifications. Varying concentrations of test compounds were added to each plate containing near-confluent cell monolayers (1 × 10⁵ cells per well) followed immediately by the addition of virus at a multiplicity of infection (moi) of 0.01, and then incubated at 37°C. On the following day, if required by experimental protocol, the medium from each plate was removed and fresh compound was added. This was repeated as required. All compounds were assayed for virus inhibition in quadruplicate and for cytotoxicity in duplicate at serial 3-fold dilution ranging from 0.01 to 10 μM for oligonucleotides. For each compound, two wells were set aside as uninfected, untreated cell controls per test and four wells per test received virus only and represented controls for virus replication. The assay was stopped when the virus CPE in the virus-infected untreated control cells affected all cells (usually 6 days, but ranged from 5 to 8 days, depending on the virus strain). Changes because of viral CPE were graded on a scale of 1–4, grade 4 representing a scenario in which the entire (100%) monolayer in a well showed viral cytopathic effect (giant cells, syncytia, or lysed cells). For all CPE-based assays, the 50% EC₅₀ was calculated by regression analysis by using the means of the CPE ratings at each concentration of compound. The inhibitory activity of that assay was confirmed by neutral red uptake assay done on the same plate (vide infra).

Neutral Red (NR) Assay of CPE Inhibition (and Cytotoxicity).

This assay was employed to confirm results of the visual cytopathic effect assay, and the value is reported in Tables 2, 4, and 5. It was performed in stationary cells by a modified method as described by Cavenaugh et al. (19). Briefly, 0.2 ml of neutral red (0.034% in PBS) was added to each of the CPE assay plate wells and the plate was incubated for 2 h at 37°C in the dark. The NR solution and medium were removed from the wells and the wells were rinsed twice with PBS (pH 7.4). Equal volumes (0.1 ml) of absolute ethanol and Sörenson citrate buffer (0.1 M sodium citrate/0.1 M HCl, pH 4.2) were mixed together and added to each well. Plates were incubated in the dark for 30 min at room temperature to solubilize the dye. The plates were then gently mixed on a 96-well plate-adapted vortexer for 1 min. Absorbances at 540 and 450 nm were read with a microplate reader (Bio-Tek EL 1309; Bio-Tek, Burlington, VT). Absorbance values were expressed as percentages of untreated controls, and EC₅₀ and IC₅₀ values were calculated by regression analysis.

Table 2.

Targeting the RSV genome RNA: Evolution of a 2-5A-antisense chimera

Oligonucleotide	Description	Structure/sequence	EC50	IC50, μM ± SD
NIH273	All PS	AsAsAsAsAsTsGsGsGsGsCsAsAsAsTsAsA	3  ± 0.2	>10
NIH317	All PS, G4, scrambled	AsTsAsAsGsGsGsGsAsAsCsAsTsAsAsAsA	1  ± 0.02	5 ± 0.1
NIH318	All PS, no G4, scrambled	GsAsAsGsAsCsAsGsAsAsTsAsAsGsAsTsA	10  ± 2	8 ± 2
NIH320	Gap-mer	AsAsAsAATGGGGCAAAsTsAsA	10  ± 2	>10
NIH351	2-5A-gap-mer	2-5A-Bu2-AsAsAsAATGGGGCAAAsTsAsA	0.3  ± 0.1	>10
Ribavirin nucleoside	Antiviral standard		30  ± 6	56 ± 7

Oligonucleotides were added to the cells twice immediately before virus exposure, again 12 h postinfection, and then every 12 h thereafter for 4 days by removal of medium and addition of fresh medium containing the appropriate oligonucleotide concentration. The assay was stopped at the end of day 6. This protocol was employed because it had been determined previously (D.B. and P.F.T., unpublished observations) that 2-5A-antisense chimeras of an earlier chemical formulation and directed against the RSV mRNA required multiple additions to cell cultures for optimal activity. EC₅₀, μM concentration (±SD) of oligonucleotide that reduced virus replication by 50% as determined by neutral red assay. IC₅₀, μM concentration (±SD) of oligonucleotide that reduced the cell density of the monolayer by 50% as determined by neutral red assay. Oligonucleotide concentrations >10 μM were not tested. Oligonucleotide sequences are in 5′ → 3′ sequence. 

Table 4.

Anti-RSV activity of NIH351: Effect of 5′-monothiophosphate removal and antisense domain nucleotide sequence scrambling

Oligonucleotide	Description	Structure/sequence	Neutral red assay
			EC50, μM	IC50, μM
NIH351	2-5A-gap-mer	2-5A4-Bu2-AsAsAsAATGGGGCAAAsTsAsA	0.08  ±  0.02	>3.2
NIH426	Scrambled 2-5A-gap-mer	2-5A4-Bu2-GsAsTsAGAAATAGAAAsGsCsA	3.4  ±  1.5	>3
NIH489	Dephospho-2-5A-gap-mer	A3-Bu2-AsAsAsAATGGGGCAAAsTsAsA	>10	>10
NIH320	Gap-mer	AsAsAsAATGGGGCAAAsTsAsA	>10	>10

Oligonucleotides were added to HEp-2 cells according to the protocol of Table 2 for 4 days by removal of medium and addition of fresh medium containing the appropriate oligonucleotide concentration. The assay was stopped at the end of day 6. Under these assay conditions, ribavirin showed an EC₅₀ of <4 μM and an IC₅₀ of 231 μM. EC₅₀ and IC₃₀ see legend to Table 2. 

Table 5.

Antiviral and anticellular activities of NIH351 compared with ribavirin against a spectrum of human RSV strains and bovine RSV

Virus	NIH351			Ribavirin
	EC50, μM	IC50, μM	SI	EC50, μM	IC50, μM	SI
Long*	0.4  ± 0.1	2.0 ± 0.5	5	12 ± 1	280 ±  50	23
9320	0.4  ± 0.2	>10	>25	80 ± 5	1,130 ±  400	>5
A2	0.2  ± 0.1	>3.2	>16	40 ± 6	410 ±  110	10
CH 18537†	0.1  ± 0.1	2.4 ± 5	24	12 ± 1	233 ±  6	19
393†	1.6  ± 0.03	>3.2	>2	4 ± 2	96 ±  17	24
BRSV	1  ± 0.6	>10	>10	80 ± 6	410 ±  110	5

For experiments with Long, A2, 393, and CH 18537, strains of human RSV, NIH351, or ribavirin were added to HEp-2 cells immediately before virus absorption and again, with a medium change, 24 h later. Virus replication was assayed on day 6. For experiments with BRSV and human RSV strain 9320, the compounds were added only once, immediately before virus absorption, and virus replication assays were ended on day 8. Results are from neutral red dye uptake assay. EC₅₀ and IC₅₀ are defined in the legend to Table 2. SI, IC₅₀/EC₅₀. BRSV, bovine RSV. This virus replication was assayed in EBTr cells. 

^*Human RSV A strain. 

^†Human RSV B strain. 

Cytotoxicity.

Cytotoxicity was evaluated by using both visual changes in cell density and appearance and the neutral red dye uptake assay on the same tissue culture plate used to evaluate antiviral activity (vide supra). IC₅₀ values reported in Tables 2, 4, and 5 refer to neutral red uptake assays. Because of limited amounts of 2-5A-antisense oligonucleotide available for evaluation, the 50% cytotoxic dose could not be determined, and this is indicated in the Tables when the IC₅₀ values are given as “>” concentrations.

Cytotoxicity in actively growing cells was determined by cell yield using the neutral red uptake assay. Ninety-six-well tissue culture plates were seeded with 1 × 10⁴ cells suspended in growth medium and incubated for 4 h at 37°C. The cells were approximately 20% confluent. The medium was then replaced with growth medium containing the same concentrations of test compound as in the antiviral experiments. Test compound also was administered to the cell culture according to the number of additions used in the primary antiviral assays. Each concentration of test compound was analyzed in sextuplicate. Untreated controls were also included. The cells and drug were incubated at 37°C for 72 h, after which neutral red was added and the analysis proceeded as described above. An IC₅₀ was determined by regression analysis.

RESULTS

Targeting the RSV Genomic RNA with 2-5A-Antisense.

The conserved sequences that occur in gene-start, intergenic, and gene-end signals of the genomic strand of RSV were chosen as targets for 2-5A-antisense. For instance, the 17-mer, 5′-AAA AAT GGG GCA AAT AA-3′, targets several sequences that occur within the critical gene-end-intergenic-gene-start signals of the RSV genomic RNA. Such critical sequences from the RSV A2 genome (6, 7) are illustrated in Table 1. The above 17-mer antisense cassette is a perfect hybridization match for three vital RSV genomic RNA signal sequences (Table 1, sequences 1, 2, and 3). This consensus oligonucleotide antisense sequence additionally may target other critical regions with lowered but significant stringency. For instance, the nucleotide sequence signal at the F gene end/intergenic/M2 gene start signal (Table 1, sequence 4) has only two mismatches to the consensus antisense sequence. Moreover, one of these is a terminal mismatch, which would have a lesser effect on hybrid duplex stability than a similar internal mismatch. Likewise, the signal at the NS2 gene-end-intergenic-N gene start has three mismatches, but only one is of the more critical, internal type. Following this logic, the expected order of hybridization efficiency of the consensus antisense cassette 17-mer with the different listed targets would be: 1 = 2 = 3 > 4 > 5 > 6, 7 > 8, 9 ≫ 10. The result of this design is that a single 2-5A-antisense chimera would be targeted, with varying degrees of specificity, to a number of nucleotide sequence signals that are critical for transcription of the RSV genome to yield RSV mRNAs.

Table 1.

2-5A-antisense targeting the RSV genomic RNA

Antisense domain		5′-AAA AAT GGG GCA AAT AA-3′	Mismatches†
Sequence number	Potential sense target*	Targeted sequence	Terminal	Internal
1	3′ leader/NS1 start	3′-UUU UUA CCC CGU UUA UU-5′	0	0
2	NS1/NS2 gene start	3′-UUU UUA CCC CGU UUA UU-5′	0	0
3	N/P gene start	3′-UUU UUA CCC CGU UUA UU-5′	0	0
4	F/M2 gene start	3′-UUU UGA CCC CGU UUA UA-5′	1	1
5	NS2/N gene start	3′-CUU CUA CCC CGU UUA UG-5′	2	1
6	M/SH gene start	3′-UGU GUA CCC CGU UUA UU-5′	0	2
7	G/F gene start	3′-U UG UGA CCC CGU UUA UU-5′	0	2
8	P/M gene start	3′-U UC CCA CCC CGU UUA UA-5′	1	3
9	SH/G gene start	3′-U UG UAA CCC CGU UUA CG-5′	1	3
10	L gene start	3′-U CA ACA CCC UGU UUU AC-5′	1	7

^*Gene-end–intergenic–gene-start sequences are described by naming the gene end, with a “/” representing the intergenic sequence, followed by naming the gene start. 

^†Underlined nucleotides are mismatches to the 2-5A-chimera antisense domain. Oligonucleotide target sequences are listed in theoretical order of hybridization efficiency to the antisense domain sequence. Mismatches defined as “internal” or “terminal” refer to the annealing antisense oligonucleotide. 

Oligonucleotide Design and Evolution.

The original formulation of 2-5A-antisense chimeras was an all-phosphodiester-backbone oligonucleotide (4, 14). Later formulations (11, 20) involved modifications to provide additional stability to 3′-exonucleases. For the current application, a phosphorothioate (17, 21) modification to the antisense cassette of the 2-5A-antisense chimera was employed. However, phosphorothioates can have nonspecific effects (22–24); moreover, the G quartet in the consensus antisense domain (see above) might give rise other non-antisense effects (22, 23, 25). Thus, we have developed a 2-5A-antisense structure based on the following experimental structural evolution that attempts to minimize nonspecific effects.

The first oligonucleotide synthesized (Table 2) was an all-phosphorothioate 17-mer (NIH273) with the consensus sequence described above. This material was able to inhibit RSV replication in HEp-2 cells (EC₅₀ = 3 μM) with low toxicity (IC₅₀ > 10 μM). However, NIH273 contained all-PS internucleotide linkages and a G quartet, both structural motifs associated with nonspecific effects. This 17-mer (NIH273) was related in sequence to a 20-mer all-PS oligonucleotide reported by Jairath et al. (26) to possess significant anti-RSV activity. A second all-PS oligonucleotide (NIH317) was prepared that retained the same overall nucleotide composition but was scrambled in nucleotide sequence save for conservation of the G quartet. This material (NIH317) also possessed significant antiviral activity (EC₅₀ = 1 μM) and detectable toxicity (IC₅₀ = 5 μM). Because this sequence did not match any within the RSV genome or RSV mRNA, the observed antiviral activity was not a result of a true antisense effect, but probably was related to the G quartet-containing phosphorothioate motif. In support of this, an all-phosphorothioate oligonucleotide (NIH318) was prepared in which the entire nucleotide sequence was scrambled. This alteration caused a 10-fold decrease in anti-RSV activity (EC₅₀ = 10 μM), which was not specific (IC₅₀ = 8 μM). Thus, a significant non-antisense effect was inherent in any completely phosphorothioate-substituted oligonucleotide containing the 17-mer consensus sequence.

To reduce the non-antisense (and nonspecific antiviral) effects associated with the all-PS 17-mer, the extent of sulfur substitution was reduced substantially. Using a “gap-mer” approach, only three internucleotide linkages at the 5′ and 3 termini of the antisense oligonucleotide were thiophosphorylated. The resultant antisense oligonucleotide (NIH320) possessed a significantly reduced anti-RSV activity (EC₅₀ = 10 μM) compared with the original all-PS 17-mer and was of low toxicity (IC₅₀ >10 μM). This antisense cassette then was used to construct a 2-5A-antisense chimera (NIH351). To protect against the inactivating effects of phosphatase, the 2-5A domain of the chimeric oligonucleotide was 5′ functionalized with a monothiophosphate moiety (15). This addition of 2-5A to the parent antisense molecule increased its antiviral activity 30-fold against the A2 strain of RSV in HEp-2 cells (Table 2).

When NIH351 was examined (Table 3) in a virus yield reduction assay under the same conditions as used in the experiments of Table 2 in comparison with the approved anti-RSV drug, ribavirin, it showed an EC₅₀ of 0.3 μM compared with an EC₅₀ of 30 μM for ribavirin in HEp-2 cells. In the same assay, the EC₉₀ for NIH351 was 1 μM and 90 μM for ribavirin.

Table 3.

RSV strain A2 virus yield reduction assay by NIH351 in HEp-2 and MA-104 cells

Compound	HEp-2 cells		MA-104 cells
	EC50	EC90	EC50	EC90
NIH351	0.3  ± 0.1 μM	1  ± 0.25 μM	0.3  ± 0.01 μM	1  ± 0.04 μM
Ribavirin	30  ± 6 μM	90  ± 6 μM	25  ± 3 μM	53  ± 33 μM

Both compounds were added only once, immediately before virus absorption. Infection was with A2 RSV strain. EC₅₀ and EC₉₀, concentration required to reduce virus replication by 50 and 90%, respectively. The EC₅₀ was determined by CPE directly on the primary assay wells that also received the antiviral agent to be tested. The EC₉₀ was determined on the secondary HEp-2 culture wells, where the virus titers from the primary wells were determined. 

In a separate experiment (data not shown), the anti-RSV activity of NIH351 was examined in MA-104 cells, a line of embryonic African Green monkey cells. NIH351 inhibited RSV strain A2 replication in MA104 cells with an EC₅₀ of 1 ± 0.3 μM by CPE assay and 0.2 ± 0.1 μM by NR dye uptake assay. NIH351 was added only once during the entire assay course (6 days) to achieve this effect. In a yield reduction assay in MA-104 cells (Table 3), a single addition of NIH351 immediately before virus absorption gave an EC₅₀ of 0.3 μM and an EC₉₀ of 1 μM versus an EC₅₀ and EC₉₀ of 25 and 53, respectively, for ribavirin. Control experiments (data not shown) established that NIH351 was not virucidal.

A parallel study (data not shown) of the effects of frequency and duration of chimera administration revealed that NIH351 had a significant antiviral effect (EC₅₀ = 1 μM) even when administered to HEp-2 cells just once, immediately after virus infection. However, NIH351 showed the most potent effects when given once a day for 3 days or twice a day for 2 days. In such conditions, the EC₅₀ was 0.03 μM as determined by the NR dye uptake assay, and the selectivity index (SI) was >100. Under the same assay conditions, ribavirin displayed an EC₅₀ of 4 μM and an SI of 130.

To ask whether or not this observed antiviral activity was based on a specific antisense effect, the antisense domain nucleotide sequence of the active NIH351 2-5A-antisense chimera was scrambled while the nucleotide composition was maintained. This 2-5A-antisense control, NIH426, was sp(5′A2′)₄Bu₂d(GsAsTs AGA AAT AGA AAsGs CsA). As shown in Table 4, this antisense domain sequence scrambling resulted in a large decrease in anti-RSV activity.

To address the question of whether or not NIH351 owed its enhancement of activity to the involvement of the 2-5A system’s RNase L, another control 2-5A-antisense chimera was synthesized. This modification (NIH489) possessed a 2′,5′-linked oligoadenylate trimer, but without a 5′ monothiophosphate moiety. Other structural features, including the antisense sequence, were identical to NIH351. The deletion of the 5′ monothiophosphate (or phosphate moiety) of a 2-5A-antisense chimera or of an unmodified 2′,5′-oligoadenylate has been shown to reduce dramatically the congener’s ability to activate the key RNase L enzyme (4, 9, 10, 27). Tetrameric 2′,5′-oligoadenylate was not used in the 2-5A-antisense chimera control because evidence exists that non-5′ phosphorylated tetramer may possess some residual ability to activate RNase L (8, 27). In keeping with the above considerations, when analyzed for its ability to activate human RNase L, NIH489 showed no trace of activity even at a concentration of 10⁻⁶ M, under conditions in which NIH351 effected complete RNA cleavage at 10⁻⁷ M (M.R.P. and P.F.T., unpublished observations). This 5′ unthiophosphorylated chimeric oligonucleotide (NIH489) did not show anti-RSV activity (EC₅₀ >10 μM, Table 4).

Table 5 shows the anti-RSV activity of NIH351 against other strains of RSV as assayed in human HEp-2 cells or EBTr cells (BRSV). NIH351 showed varying inhibitory activities against other RSV strains; nonetheless, NIH351 was a potent inhibitor of representative members of both A and B strains of RSV. It also displayed significant, albeit somewhat less potent, activity when assayed against bovine RSV.

NIH351 was not toxic to cells. Neither ribavirin nor NIH351 caused morphological changes. Both did cause decreases in cell monolayer density, although NIH351 did so only rarely. With rapidly dividing HEp-2 cells, NIH351 displayed an IC₅₀ of >10 μM and ribavirin had an IC₅₀ of 160 μM. Higher concentrations of NIH351 were not evaluated for cytotoxicity due to limited amounts of available material.

DISCUSSION

Herein, the 2-5A-antisense strategy (4, 5, 8–16, 20) has been directed against specifically repeated critical consensus regions in the RSV genomic RNA (Table 1). The result is a highly active 2-5A-antisense chimera (NIH351) that is more potent (i.e., lower EC₅₀ concentration) than the presently employed therapeutic, ribavirin (Tables 2 and 3).

This antiviral activity was tied to a specific antisense effect, because a chimeric 2-5A-antisense with a scrambled nucleotide sequence antisense domain was at least 10- to 30-fold less active against RSV (Table 4). The inhibitory effect was virus-specific. Two closely related viruses of the Paramyxoviridae family, measles and parainfluenza, were not inhibited by NIH351 (D.B. and P.F.T., unpublished observations). In addition, NIH351 was not virucidal. Furthermore, when the 2-5A moiety was modified to a 5′-nonthiophosphorylated chimera (NIH489) that could not activate RNase L, the resultant oligonucleotide showed no anti-RSV activity (Table 4). This latter finding implies that RNase L has a major role in the antiviral activity of NIH351.

All of the congeners examined in this study, including NIH351, NIH426, and NIH320, would be capable of supporting RNase H (22, 28, 29) degradation of the targeted RNA sequences. However, an additional independent observation strongly implies that 2-5A and RNase L are the primary effectors of the antiviral response: conjugation of the partially phosphorothioate-substituted DNA oligonucleotide to 2-5A tetramer to yield NIH351 effected a 30-fold enhancement of the antiviral activity of the parent NIH320 (Tables 2 and 4). The basis for the relative roles of RNase L and RNase H in the activity of such 2-5A-antisense constructs remains to be defined, but may be related to their differential subcellular distributions and the known cytoplasmic site of replication of RSV (3). RNase L has been found in both nucleus and cytoplasm (8), whereas RNase H may be predominantly nuclear-localized in keeping with its postulated roles in DNA replication (30, 31).

The significant antiviral potency of NIH351 against various A and B strains of human RSV and bovine RSV argues for the validity of the working hypothesis: that is, conserved consensus sequences within the RSV genome provide a target vulnerable to 2-5A-antisense. Among the strains tested in Table 5, only RSV A2 has been sequenced completely. However, a number of the critical gene-end–intergenic–gene-start regions of BRSV are known (32). Within these sequences there are no perfect complements for the antisense domain of NIH351; however, several sequences provide partial matches. For instance, in the N/P, P/M, G/F, and F/M2 gene-end–intergene–gene-start sequences, partial complements exist with one terminal mismatch and one internal mismatch. The more distant sequence relatedness and the lack of a perfect match in the BRSV genome may account for the somewhat reduced potency of NIH351 against BRSV as compared with the human virus strains. An example of the RSV B subgroup sequence also is known. The wild-type RSV strain B1 (GenBank accession no. AF013254) possesses a perfect complement in the genomic RNA 3′ leader and several partial complements with one internal mismatch and either one or two terminal mismatches. A mutagenized strain, a cold-passaged attenuated strain, and a cold-passaged, temperature-sensitive, live-attenuated strain of RSV A2 all contain three perfect complements to NIH351 in the 3′ leader/NS1, intergenic/NS2, and intergenic/P genomic RNA sequences (33, 34). On a practical note, the observed activity of NIH351 against strains of both A and B subgroups of RSV would be important from the perspective of the potential use of NIH351 or some congener thereof as a clinical antiviral agent. Epidemics of RSV are accompanied by the occurrence of numerous strains of both A and B subgroups (35, 36), although subgroup A usually predominates.

In summary, we have described an innovative approach to the control of RSV infection. The 2-5A-antisense strategy has been used to target RNase L activity critical consensus regions of RSV genomic RNA. A 2-5A-antisense chimera has been developed that has 50–90 times the anti-RSV potency of the presently employed anti-RSV therapeutic, ribavirin. 2-5A conjugation potentiates classical antisense activity 30-fold. The 2-5A-antisense chimera shows a sequence-specific antisense effect, and its activity is reduced significantly when the 2-5A domain is functionally disabled. This new anti-RSV 2-5A-antisense chimera (NIH351) is highly active with only a single administration in tissue culture and shows potent activity in both human and monkey cells and against both A and B subgroup strains of RSV.

ABBREVIATIONS

RSV

respiratory syncytial virus

PS

phosphorothioate

CPE

cytopathic effect

NR

neutral red

SI

selectivity index

BRSV

bovine RSV