Design of P-PMO.
Considerations in P-PMO sequence design for this study included our current understanding of both the function of various FLUAV genomic regions and P-PMO mechanisms of action. Previous reports indicate that the translation start site region of mRNA of viral nonstructural genes (1
), or sequence involved in viral long-range RNA-RNA interaction (8
), may include effectual P-PMO target sites. The P-PMO for this study were therefore designed to base pair with the AUG translation start site regions of the mRNAs involved in FLUAV RNA synthesis (PA, PB1, PB2, or NP) or one of the four terminal regions of the NP vRNA or cRNA (Fig. ; Table ).
Eight P4-PMO were designed against the PR/8 (H1N1) sequence for the initial experiments in this study. The four AUG-targeted P4-PMO cover at least four nucleotides on either side of the AUG translation initiation codon of their respective mRNA target sequences and are also complementary to corresponding cRNAs.
The FLUAV RNA polymerase complex requires that the two termini of an RNA segment be partially duplexed in order to efficiently initiate RNA synthesis (37
). Each segment of the FLUAV genome is believed to undergo a long-range RNA-RNA interactive event between the 5′ and 3′ ends during RNA synthesis, both in the production of cRNA or mRNA from vRNA and likewise from cRNA back to vRNA (49
). NP has been reported to play critical roles in FLUAV genome replication, intracellular trafficking, packaging of the viral genome, and virus-host interactions (36
). Therefore, four P4-PMO compounds were designed to base pair with one of the 5′- or 3′-terminal sequences of the NP vRNA or cRNA. In addition to the antisense P-PMO, a random-sequence P-PMO (designated “Dscr”) was synthesized to serve as a negative control compound.
The degree of sequence conservation between six FLUAV subtypes and between individual strains within those subtypes was also considered during P-PMO design. FLUAV subtypes selected for sequence conservation analysis included the three human-infecting subtypes H1N1, H2N2, and H3N2, the three avian influenza virus subtypes that are considered to be the largest threat to humans, H5N1, H7N7 and H9N2, and the primarily equine subtype H3N8. Table summarizes the degree of sequence conservation at the target sites in FLUAV of the eight P-PMO antisense sequences used in this study. Near-perfect homology across all subtypes and strains was most pronounced for the target of the PB1-AUG P-PMO.
Conservation of P-PMO target sequences in FLUAV subtypesa
Identification of highly active P-PMO.
Eight antisense and the random-sequence Dscr P4-PMO (Table ) were initially compared at a single concentration for their ability to inhibit PR/8 (H1N1) production over a period of 48 h in Vero cells. FLUAV titers were monitored by HA assay at 24, 36, and 48 hpi. In a trial comparing the four AUG region-targeting compounds, all but the PA-AUG-targeted P-PMO produced at least eightfold reductions in virus titer at 48 hpi (Fig. ). The four P-PMO targeting the terminal ends of NP were compared in a separate trial under conditions identical to those of the AUG-targeting (Fig. ). Again, all but one P4-PMO (NP-c5′) was capable of significantly reducing virus titers, some by as much as 16-fold.
FIG. 2. Effect of 20 μM AUG- and terminal region-targeted P4-PMO on A/PR/8/34 (H1N1) production in Vero cells over time as measured by HA assay. Cultures were incubated with 20 μM P4-PMO or mock treatment (NT) for 6 h before infection with PR/8 (more ...) Dose-response studies with PR/8 (H1N1).
Seven of the eight P4-PMO were selected for further dose-response studies by HA and plaque assays. PA-AUG was not tested further because of its relatively low anti-PR/8 activity in the single-concentration survey described above. The procedures and conditions used for the PR/8 dose-response studies were identical to those described for the previous single-dose experiment, except that titers were measured at 48 h only. An overall dose-response effect was observed for all seven antisense P4-PMO tested by HA assays (Fig. ). At a concentration of 10 μM, PB1-AUG, NP-AUG, NP-c3′, NP-v3′, and NP-v5′ all caused a greater than eightfold reduction in titer compared to controls. Plaque assay of the same P4-PMO yielded similar results (Fig. ). At 10 μM, NP-AUG, PB1-AUG, and NP-v3′ inhibited virus titer by over 3 log10 PFU/ml compared to a peak titer of approximately 6.5 log10 in the mock- or Dscr-treated controls (Fig. ).
FIG. 3. Growth charts of A/PR/8/34 (H1N1) titer in Vero cells in the presence of various concentrations of P4-PMO compounds. (A and C) AUG region-targeted P4-PMO; (B and D) NP terminal region-targeted P4-PMO. (A and B) Results of HA assays; see the inset boxes (more ...) Dose-response studies on a panel of virus strains.
Based on their high activity against PR/8 (H1N1) and high conservation of target site sequence across and within FLUAV subtypes, notably H1N1 and H5N1 (see Table ), two of the P-PMO sequences, PB1-AUG and NP-v3′, were selected for evaluation against several other strains of FLUAV. The Dscr negative control and the two antisense sequences were prepared as P7-PMO and evaluated by plaque assays against WSN/33 (H1N1) and Mem/88 (H3N2), by HA assays against Miami/63 (H3N8) and Prague/56 (H7N7), and by ELISA against KAN-1 (H5N1). The recently developed P7 was selected as the conjugation peptide for these subsequent experiments, as it has been reported to transport PMO into cells with equal or greater efficiency than that provided by P4 peptide, yet it is more stable (31
), less affected by serum (8
), and appears to be less cytotoxic than P4 (Hong Moulton, AVI BioPharma, unpublished data; also see below).
Pretreatment of cells with PB1-AUG P7-PMO before infection with either WSN/33 or Mem/88 virus resulted in pronounced antiviral activity. Cells were incubated for 6 h with P7-PMO, infected, and then incubated for 24 h without P7-PMO. At 24 hpi, WSN/33 titers were reduced 12-fold at 10 μM and 82-fold at 20 μM, while Mem/88 titers were reduced 8-fold at 10 μM and 216-fold at 20 μM (Fig. ). No inhibition of viral titer was observed with either virus at the lowest P7-PMO concentration (5 μM) tested. WSN/33, but not Mem/88, titers at 24 hpi were reduced about 20-fold by preinfection treatment of cells with 20 μM NP-v3′, although lower concentrations of this compound did not significantly affect replication titers. Replication of either virus was not affected by the presence of Dscr control P7-PMO at any concentration used throughout the assays. The titers of both viruses in the presence of any of the P7-PMO were similar to mock-treated infection controls by 45 hpi (data not shown), suggesting that there was insufficient intracellular presence of the oligomers to sustain their antiviral activity.
FIG. 4. Dose-response challenge of PB1-AUG or NP-v3′ P7-PMO against A/WSN/33 (H1N1) or A/Memphis/8/88 (H3N2), measured by plaque assay. MDCK cells were incubated with the indicated P7-PMO or mock treatment (NT) for 6 h and then infected at an MOI of 0.001 (more ...)
The effect of preinfection treatment with PB1-AUG and/or NP-v3′ P7-PMO on H3N8 and H7N7 viruses in MDCK cells was measured by HA assay. Cells were incubated for 4 h with P7-PMO, infected, and then incubated for 48 h without P7-PMO. As shown in Fig. , NP-v3′ produced a dose-dependent response, with a greater than 85% reduction in virus titer at 15 μM, against both H3N8 and H7N7. However, the PB1-AUG P-PMO appears to have little or no effect on virus titer, and additional experiments will need to be conducted to further investigate this result. Although PB1-AUG appeared to have no effect by itself, when combined with NP-v3′ at a concentration of 15 μM each an additive effect was realized, producing a reduction in virus titers of more than 90%, which is an effect greater than NP-v3′ alone at 15 μM.
FIG. 5. Dose-response challenge of PB1-AUG and/or NP-v3′ P7-PMO against A/Eq/Miami/63 (H3N8) or A/Eq/Prague/56 (H7N7), measured by HA assay. MDCK cells were treated with the indicated P7-PMO or received mock treatment (NT) for 4 h and then were infected (more ...)
To examine the effect of PB1-AUG and NP-v3′ P7-PMO on the replication of an H5N1 isolate, MDCK cells were incubated with P7-PMO for 4 h, infected with two different levels of KAN-1 (H5N1) virus, incubated again with P7-PMO, and assayed for activity by ELISA at 24 hpi. Both antisense P7-PMO generated dose-dependent inhibition at either virus infection level, with NP-v3′ consistently generating moderately greater antiviral effect at the lower doses than PB1-AUG. At the lower virus infection level (5 TCID50), NP-v3′ at 10 μM produced 88% and PB1-AUG 57% inhibition of viral NP protein levels (Fig. ). In contrast, the Dscr control P7-PMO had less than 8% activity at this dose. At the higher virus infection level (25 TCID50), both antisense P7-PMO produced a greater than 85% reduction in viral NP protein at 20 μM, a dose at which the Dscr control P7-PMO had no activity (Fig. ).
FIG. 6. Dose-response challenge of PB1-AUG and NP-v3′ P7-PMO against A/Thailand/1(KAN-1)/04 (H5N1), measured by ELISA. MDCK cells were incubated with the indicated concentrations of P7-PMO or received mock treatment for 4 h before viral infection and (more ...) Specificity of active anti-FLUAV P-PMO.
In order to assess the sequence specificity and cytotoxicity of the P4-PMO used in this study, each was tested at 20 μM for inhibition activity against influenza B virus (IBV). As with the PR/8 (H1N1) inhibition assay, IBV was inoculated at 0.05 MOI in Vero cells, and titers were determined at 48 hpi by HA assay. IBV grew to nearly as high a titer at 48 h as PR/8 had. No difference in IBV titer between cells treated with any of the P4-PMO and cells receiving mock treatment was observed (data not shown). This was not surprising, as the degree of sequence conservation between IBV and FLUAV at any of the P4-PMO target sites is below 82% (data not shown). We interpret this result as evidence that the P4-PMO had minimal cytopathic or generic antiviral activity, as IBV titer would be expected to diminish compared to that for mock-treated samples if any of the P4-PMO had caused such nonspecific effects.
All P4- and P7-PMO were tested for cytotoxicity with standard cell viability assays in the absence of virus, under the same culture conditions, and with the same, or wider, range of P-PMO concentrations as those in the various antiviral experiments. The P4-PMO exhibited a somewhat higher impact on cell viability than P7-PMO did, with concentrations of some P4-PMO in the 10 to 20 μM range, resulting in an approximately 20% loss in cell viability after 24 h of incubation. In a variety of trials with MDCK cells, P7-PMO generated less than 10% reduction in cell viability at all concentrations up to and including 20 μM for all treatment durations (data not shown). However, at 40 μM, with a 24-h treatment under the conditions of the H5N1 antiviral assay, each P7-PMO reduced MDCK cell viability from 15 to 30%. In an attempt to derive a “Selectivity Index” (SI) (the ratio of the concentration of drug causing 50% cytotoxicity [CC50] divided by the concentration of drug causing a 50% inhibition of viral production) relevant to the various conditions of this study, we sought to determine the CC50 for the PB1-AUG and NP-v3′ P7-PMO. MDCK cells were treated for 6 h with concentrations of each P7-PMO from 10 to 400 μM, using culture conditions identical to those under which the H3N8 and H7N7 antiviral experiments were conducted but in the absence of trypsin or virus. Cell viability was determined at 24 h after the treatment period. Surprisingly, we were unable to cause a 50% loss in cell viability under these conditions. The experiment was repeated three times, with similar results. A concentration of 400 μM of either oligomer resulted in a 15 to 30% loss in cell viability (data not shown). An unrelated natural plant extract, 6-prenyl naringenin, generated a dose-responsive pattern of cytotoxicity, with a CC50 of approximately 20 μg/ml. Based on a 50% inhibition of viral production of approximately 10 μM for both PB1-AUG (Fig. ) and NPv3′ (Fig. ), we conclude that the SI for either antisense P7-PMO is over 40 under these conditions.
Together, these results indicate that under the various experimental conditions used in this study P-PMO did not have a significant impact on cell viability, and the observed antiviral activity was sequence specific.
Effect of postinfection P7-PMO treatment on FLUAV.
Having established the potent anti-FLUAV activity of P-PMO in settings in which cells were pretreated with compound for 4 or 6 h before viral infection, we investigated the effect of treating cells only after viral infection. At 1, 2, or 3 h after the completion of a 1-h infection period with H3N8, 15 μM of Dscr, NP-v3′, and/or PB1-AUG P7-PMO was added and allowed to remain in the culture medium (which contained trypsin). The results from these experiments showed that, at 48 hpi, NP-v3′ provided a 70% reduction in virus titer if treatment was begun 1 h after the completion of the infection period (Fig. ). Reduction in titer decreased to 40% or 20% if treatment was begun 2 or 3 h, respectively, after the infection period. As with the preinfection protocol, PB1-AUG and Dscr had no observable effect on H3N8.
FIG. 7. Effect of timing of postinfection addition of P7-PMO on H3N8 virus growth in MDCK cells, as measured by HA assay. Cells were infected at an MOI of 0.0001 for 1 h and then allowed to grow for 1, 2, or 3 h before the addition of P7-PMO or mock treatment (more ...) Tolerance of P4-PMO to mismatches with target RNA.
In an attempt to characterize the loss of P-PMO activity that may result from a variable number of mispairs between the base sequence of a P-PMO and its RNA target sequence, the NP-AUG P4-PMO was tested in cell-free in vitro translation inhibition assays against a panel of in vitro-transcribed RNAs. Five RNAs were produced from a series of plasmids that were constructed such that the target sequence for NP-AUG P4-PMO was fused in frame directly upstream of luciferase and downstream from a T7 promoter. The plasmids differ incrementally in the number of mismatches, from 0 to 4, present in the NP-AUG P4-PMO target “leader-sequence” in relation to the NP-AUG P4-PMO sequence. Table lists the NP AUG region leader sequences of the five plasmids. The mismatched RNA target sequences were designed to reflect the most likely natural sequence variations in the NP-AUG region, as derived from the influenza sequence databases. In vitro translations with 1 nM RNA in rabbit reticulocyte lysate were carried out with the five different RNAs against a 6-point dose-response of NP-AUG and Dscr P4-PMO. The results showed that a single mismatch between P4-PMO and target RNA caused little reduction of P4-PMO activity compared to no mismatches (Fig. ). Two or more mismatches, however, generated a considerable loss of inhibitory activity. The 50% effective concentrations of the NP AUG P4-PMO against the RNAs with which it had zero, one, two, three, or four mismatches were approximately 30, 50, 200, 400 and 550 nM, respectively. All concentrations of Dscr generated little or no inhibition of translation of any of the RNAs used in the in vitro translation assays.
NP-AUG P-PMO sequence (3′-5′) and FLUAV in vitro transcript target sequences (5′-3′)
FIG. 8. Effect of sequence mismatch between a P4-PMO and target RNA on inhibition of translation in cell-free assays. Several in vitro-transcribed reporter RNAs, each having a different number of base mismatches in the target region of the NP-AUG P4-PMO in relation (more ...)