Peptide-conjugated morpholino oligomers inhibit porcine reproductive and respiratory syndrome virus replication

Abstract

Porcine reproductive and respiratory syndrome (PRRS) has been devastating the global swine industry for more than a decade, and current strategies to control PRRS are inadequate. In this study we characterized the inhibition of PRRS virus (PRRSV) replication by antisense phosphorodiamidate morpholino oligomers (PMO). Of 12 peptide-conjugated PMO (PPMO), four were found to be highly effective at inhibiting PRRSV replication in cell culture in a dose-dependant and sequence-specific manner. PPMO 5UP2 and 5HP are complementary to sequence in the 5' end of the PRRSV genome, and 6P1 and 7P1 to sequence in the translation initiation regions of ORF6 and ORF7, respectively. Treatment of cells with 5UP2 or 5HP caused a 4.5log(10) reduction in PRRSV yield, compared to a control PPMO. Combination of 6P1 and 7P1 led to higher level reduction than 6P1 or 7P1 alone. 5UP2, 5HP, and a combination of 6P1 and 7P1 inhibited PRRSV replication in porcine alveolar macrophages and protected the cells from PRRSV-induced cytopathic effect. Northern blot and real-time RT-PCR results demonstrated that the effective PPMO led to a reduction of PRRSV RNA level. 5UP2 and 5HP inhibited virus replication of 10 other strains of PRRSV. Results from this study suggest potential applications of PPMO for PRRS control.

Fig. 1

Structure of PPMO and schematic illustration of PPMO target locations in the PRRSV genome. (A) PPMO structure. A morpholine ring and a phosphorodiamidate linkage in PMO replace the deoxyribose and phosphodiester linkage of DNA, respectively. “B” represents the bases A, G, C, or T. An arginine-rich peptide (RXR)₄XB is covalently conjugated to the 5′ end of each PMO. (B) Positions of PPMO target sites in PRRSV genomic RNA. The arrows indicate the 5′ to 3′ orientation of the PPMO in relation to the PRRSV RNA genome.

Fig. 2

PPMO-mediated inhibition of PRRSV replication in infected CRL11171 cells. (A) Titration of virus yield from CRL11171 cells after PRRSV infection and indicated PPMO treatment. Virus titers are shown as TCID₅₀ (log₁₀/ml). “Mock” samples is from cells receiving virus inoculation but no PPMO treatment. Statistical significance of difference in viral yields between PPMO and mock treatments: ^*P < 0.05; ^**P < 0.01. Cells that were treated with 16 μM 5UP2 or 5HP had virus yields not detectable (ND) in this assay, and a bar is arbitrarily drawn to show the presence of the samples in the graph. The experiment was repeated three times and error bars are shown. (B) Immunofluorescence assay with SDOW17 monoclonal antibody. Specific fluorescence is clearly visible at 24 h post PRRSV (VR2385) infection, while no fluorescence is observed in uninfected (−) control. Treatment with PPMO 5UP2, 5HP, 6P1, and 7P1 resulted in reduction of virus replication, while 3UP2 and CP1 did not appear to have any effect under identical conditions. The images below the green fluorescence images were taken with a DAPI filter to show the total number of living cells.

Fig. 3

Dose-dependent inhibition of PRRSV replication. Virus yield in cell culture is reduced concomitantly with increasing concentrations of 5UP2, 5HP, 6P1, and 7P1. Cells receiving some of the treatments had virus yields not detectable (ND) in this assay, and a bar is arbitrarily drawn to show the sample presence in the graph. The dose-dependent inhibition of PRRSV replication by 5UP2, 5HP, 6P1, and 7P1 is significantly different from CP1 (P < 0.05). The experiment was repeated three times and error bars are shown.

Fig. 4

Combinatory inhibition assay of PPMO. Two PPMO were added at equal molar concentration to cells after PRRSV inoculation. The combination treatment of 6P1 and 7P1 led to significant lower virus yield (P = 0.003) than did either PPMO alone. Cells that were treated with 16 or 32 μM 6P1 + 7P1 had virus yields not detectable (ND) in this assay, and a bar is arbitrarily drawn to show the presence of the samples in the graph.

Fig. 5

PPMO inhibition of PRRSV RNA synthesis. (A) Northern blot analysis with probe from PRRSV ORF7. CRL11171 cells were inoculated with VR2385 at 0.5 MOI and treated with PPMO at 16 μM. The cells were harvested at 24 h p.i. No detectable RNA is seen after treatment with 5UP2 or 5HP. RNA molecular weight markers are on the left. PRRSV RNA transcript designations are indicated on the right. As a loading control, the transcript of β-actin was detected and is shown in the lower panel. (B) Quantitation of PRRSV RNA by real-time RT-PCR with primers designed to amplify a region of ORF1a. Treatment of the cells with PPMO 5UP2, 5HP, 6P1 + 7P1, 3UP2, NSP2 and NSP3 at 16 μM led to significant reduction of PRRSV genomic RNA level (P < 0.05). The cells were harvested at 24 h p.i. and total RNA was isolated for real-time RT-PCR. Cells treated with 5UP2 had no PRRSV RNA detectable (ND) in this assay and a bar is arbitrarily drawn to show the sample in the graph. The PRRSV RNA copy numbers in each sample was calculated in comparison with a standard curve, after normalization with β-actin transcript levels (see Section 2.8).

Fig. 6

Inhibition of target RNA translation by PPMO in a cell-free luciferase reporter assay. Relative percentages of luciferase level were calculated in comparison with signal of control PPMO sample at 10 nM. The level of luciferase production decreased in response to increasing concentrations of PPMO 5UP2, 5HP, 6P1, and 7P1, while CP1 has little effect on luciferase RNA translation.

Fig. 7

Cross strain inhibition assay. (A) Virus yield titration shows inhibition of ten North American PRRSV strains by PPMO 5UP2 and 5HP. Lelystad is a prototype of European PRRSV genotype. All other strains are within the North American PRRSV genotype. “Mock” denotes virus infected cells receiving no PPMO. Treatment of cells with PPMO 5UP2 or 5HP led to suppression of PRRSV replication of all North American strains, producing virus yields not detectable (ND) in this assay. Bars are arbitrarily drawn for those samples with no detectable virus (“ND”) to show the presence of samples in the graph. (B) Sequence analysis identifies nucleotide mismatches between PPMO and their complementary target sites in PRRSV RNA. PRRSV strain names are listed in the first column. The sequence of strain VR2385 is used as the reference sequence, as the PPMO were designed against it. PPMO names are listed above the blocks of PRRSV sequences. “Lely” stands for Lelystad strain, which has little similarity in the 5′UTR to other strains, as indicated by symbol “–” in the alignment of 5UP2 and 5HP target sequences. For all other sequences, only nucleotides differing from reference sequence are shown, and identical nucleotides are indicated with “.”. Missing nucleotides are indicated with “-”. The initiation codons ATG of ORFs 6 and 7 are underlined. GenBank accession numbers for PRRSV strains in the alignment are listed in parenthesis: NVSL 97-7895 (AY545985), Lelystad (M96262), FL-12 (derived from infectious clone of AY545985), 16244B (AF046869), 11604 (EF523345), Ingelvac MLV (EF484033), and 16138 (EF523346). Nucleotide sequences for the other strains in the figure are unpublished.

Fig. 8

PPMO-mediated suppression of PRRSV replication in porcine alveolar macrophages. (A) Protection of PAM from PRRSV CPE development. Infection of PAM with VR2385 led to severe CPE development (see +VR2385, −PPMO) in comparison with uninfected control cells (−VR2385, −PPMO). Application of PPMO 5UP2, 5HP, or a combination of 6P1 + 7P1 at 16 μM resulted in inhibition of CPE development, while CP1 did not. (B) Reduced PRRSV infection in PAM detected by IFA with SDOW17 antibody. Specific fluorescence is clearly visible at 12 h after VR2385 inoculation. Treatment with PPMO 5UP2, 5HP, 6P1 + 7P1 resulted in reduction of virus replication, while CP1 did not. The images below the green fluorescence images were taken with a DAPI filter to show the total number of living cells. (C) Titration of virus yield from PAM cells after PRRSV infection and PPMO treatment. Virus titers are shown as TCID₅₀ (log₁₀/ml). “Mock” sample is virus inoculation with no PPMO treatment. Statistical significance of the difference in viral yield compared to mock-treatment ^*P < 0.05. The experiment was repeated three times and error bars are shown.