VP35 knockdown inhibits Ebola virus amplification and protects against lethal infection in mice

Abstract

Phosphorodiamidate morpholino oligomers (PMO) are a class of uncharged single-stranded DNA analogs modified such that each subunit includes a phosphorodiamidate linkage and morpholine ring. PMO antisense agents have been reported to effectively interfere with the replication of several positive-strand RNA viruses in cell culture. The filoviruses, Marburg virus and Ebola virus (EBOV), are negative-strand RNA viruses that cause up to 90% lethality in human outbreaks. There is currently no commercially available vaccine or efficacious therapeutic for any filovirus. In this study, PMO conjugated to arginine-rich cell-penetrating peptide (P-PMO) and nonconjugated PMO were assayed for the ability to inhibit EBOV infection in cell culture and in a mouse model of lethal EBOV infection. A 22-mer P-PMO designed to base pair with the translation start site region of EBOV VP35 positive-sense RNA generated sequence-specific and time- and dose-dependent inhibition of EBOV amplification in cell culture. The same oligomer provided complete protection to mice when administered before or after an otherwise lethal infection of EBOV. A corresponding nonconjugated PMO, as well as nonconjugated truncated versions of 16 and 19 base residues, provided length-dependent protection to mice when administered prophylactically. Together, these data suggest that antisense PMO and P-PMO have the potential to control EBOV infection and are promising therapeutic candidates.

FIG. 1.

Inhibition of viral replication by EBOV-specific P-PMO. (A) Schematic drawing of the EBOV Zaire genome and PMO target sequences. Genes that encode proteins directly involved in viral RNA synthesis are shaded in light gray. PMO compounds were designed to target either genomic (−) RNA () or antigenomic (+) RNA and mRNA (). Compounds binding to plus-stranded RNA were directed against the translation start site regions of either the VP35 gene (3136) or the L gene (11588) and the trailer region (18959). The 3′ end of the genome is magnified to show the target locations of the three negative-strand sequences in detail: leader terminus (leader), transcription start signal of the NP gene (tss-NP), and a region promoting efficient replication (rep pro, 108). Nucleotide numbers refer to GenBank accession number AF086833. (B) Vero cells were pretreated with 5.0 μM of the indicated compound for 3 h and subsequently infected with EBOV at an MOI of 5 TCID₅₀ per cell. After the inoculum was removed, DMEM with 2% FCS containing 5 μM P-PMO was added to the cells. Oligomer sequences and their target sites are listed in Table 1. At 48 hpi, supernatant was used to infect Vero cells in 96-well plates for a TCID₅₀ assay. On day 12 postinfection, the CPE was evaluated and the TCID₅₀/ml calculated. The experiment was performed twice with similar outcomes.

FIG. 2.

Evaluation of cytotoxicity and efficacy of EBOV-specific P-PMO compounds. (A) Vero cells were incubated with 0 to 20 μM of the various P-PMO. After 24 h, the cells were examined for viability using an MTT assay. The data are plotted as percent changes in viability in comparison to untreated control Vero cells. Standard deviations of the means of triplicate samples are indicated by bars. (B) Light microscopy was employed to observe the CPE caused by either EBOV infection or cytotoxicity of the oligomers. Vero cells were preincubated with 5.0 μM of 3136 or scramble P-PMO for 3 h. Cells were then infected with EBOV at an MOI of 5 TCID₅₀ per cell. The inoculum was removed, fresh DMEM was supplemented with 2% FCS, and the appropriate P-PMO at 5.0 μM was added. Cells were checked daily for CPE and photographed under light microscopy. Pictures shown here were taken at 6 dpi.

FIG. 3.

Inhibition of EBOV replication by the VP35-specific P-PMO is dose, time, and sequence dependent. (A) Vero cells were pretreated with 5 μM of the 3136 or scramble P-PMO for 3 h prior to infection with EBOV (MOI of 5 and 0.05 TCID₅₀ per cell, respectively). After 1 h, the inoculum was removed and replaced with DMEM containing 2% FCS and P-PMO at 5 μM. At 48 hpi, supernatants from the P-PMO-treated and EBOV-infected cells were used to inoculate Vero cells in 96-well plates for a TCID₅₀ assay. The CPE remained constant after 11 dpi, and the TCID₅₀/ml was calculated (left panel). The cells were lysed, and total RNA was isolated and used as a template for RT-PCR, with primers designed to amplify 1,119 nucleotides of EBOV genomic RNA comprising sequence from the 5′ region of VP35 gene and 3′ region of VP40 gene, or GAPDH mRNA as a control. Products were separated by agarose gel electrophoresis and quantified after staining. The amount of EBOV-specific RNA was normalized with the GAPDH control, and reduction was calculated by setting EBOV-specific signals from infected cells without P-PMO treatment at 100% (right panel). Error bars represent the standard errors of the means of three replicates. (B) Compounds were added to Vero cells seeded in 24-well plates at the indicated time points: 3 h prior to infection, immediately after removal of inoculum (0 hpi), or at 4, 8, or 24 h after infection with EBOV (MOI = 5 TCID₅₀ per cell). Cells were harvested at 48 hpi, total RNA was isolated, RT-PCR was performed, and the products were resolved on a 2% agarose gel stained with ethidium bromide. (C) Vero cells were preincubated with 5 or 10 μM of 3136 or scramble P-PMO. After 3 h, cells were infected with MARV at an MOI of 5 TCID₅₀ per cell. The inoculum was removed at 1 hpi and replaced with fresh DMEM containing 2% FCS and the respective P-PMO. At 24 hpi, cells were harvested and total RNA was isolated. RT-PCRs (of a segment of GP) from genomic viral RNA template and cellular GAPDH mRNA, as a control, were performed. The experiments shown in this figure are representative of three experiments of similar design and outcome.

FIG. 4.

Sequence-specific inhibition with VP35 PMO compounds in cell-free translation assay. RNA representing the 5′ 137 nucleotides of VP35 mRNA sequence, which includes the 3136 target site followed by the coding sequence for firefly luciferase, was in vitro transcribed from a reporter plasmid. Various concentrations of oligomers were added to rabbit reticulocyte lysate in vitro translation reactions in the presence of 1 nM RNA (see figure inset for oligomers tested). The graphed line represents the mean light units of three treatment wells per data point. Data are represented as the mean percent inhibitions of reporter signals of triplicate treatment wells in comparison to the means of water-treated control reactions.

FIG. 5.

Prophylactic treatment with antisense PMO compounds targeting VP35 increases survival of mice infected with EBOV. (A) Kaplan-Meier survival curve showing mice pretreated via intraperitoneal injection at 24 and 4 h before EBOV infection with 500-μg doses of 3136 22-mer (♦), leader (▴), or scramble (×) PMO. The mice were challenged with ∼1,000 PFU of mouse-adapted EBOV, and the results are plotted as percent survival for each group (n = 20 to 30 per group). (B) Survival of mice treated intraperitoneally with 500-μg doses of 3136 22-mer (♦), 19-mer (▪), 16-mer (▴), or scramble (×) PMO at 24 and 4 h before infection with ∼1,000 PFU of mouse-adapted EBOV. The data are plotted as percent survival for each group (n = 10 to 20 per group). (C) Survival curves of mice treated 24 and 4 h via intraperitoneal injection before EBOV infection with 500-μg doses of 3136 22-mer PMO (♦), 3136 22-mer P-PMO (▴), scramble PMO (×), or scramble P-PMO (○). The data are presented as Kaplan-Meier survival curves (n = 10 to 20 per group). All mice were observed for at least 28 dpi, and no changes in survival were noted from 14 to 28 dpi.

FIG. 6.

Prevention of lethal outcome in EBOV-infected mice treated therapeutically with VP35-specific P-PMO. (A) Kaplan-Meier survival curve showing mice treated intraperitoneally with a single 500-μg dose of VP35-specific 3136 22-mer P-PMO (♦) or the scramble P-PMO (×) at 24 h after receiving ∼1,000 PFU of mouse-adapted EBOV infection. The results are plotted as percent survival for each group (n = 12 per group). All mice were observed for at least 28 dpi, and no changes in survival were noted from 21 to 28 dpi. (B) The 3136 (♦) or scramble (×) P-PMO mice were weighed daily following lethal EBOV infection. Data are representative of the mean weight of the surviving mice in each group, which were weighed daily en masse (n = 12 per group). (C and D) Blood samples from the 3136 (white bars) or scramble (black bars) P-PMO mice were obtained on day 3 and day 6 under anesthesia by cardiac puncture. Viral titers in the serum were determined by plaque assay (C), and IFN-α levels were determined using a commercially available enzyme-linked immunosorbent assay (D). Data are shown as means (n = 6 mice per group) and standard deviations (viral titers) or standard errors of the means (IFN). Significant differences (P < 0.05) between the VP35-specific 3136 and scramble P-PMO-treated group are indicated by an asterisk.