Enhanced inhibition of porcine reproductive and respiratory syndrome virus replication by combination of morpholino oligomers

Abstract

Porcine reproductive and respiratory syndrome (PRRS) has caused heavy economic losses in the swine industry worldwide and current strategies to control PRRS are inadequate. Previous studies have shown that peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) can be an effective antiviral against the PRRS virus (PRRSV). PPMO is structurally similar to DNA with modified backbone and is resistant to nuclease. This study was designed to examine increasing inhibitory effect of PPMO combination. Two pairs of PPMOs were identified to have enhanced suppression of PRRSV replication in cell culture, while individual constituents did not work under the same testing conditions. PPMO 5UP1 that is complementary to 5' terminus of PRRSV genome was paired with 4P1 or 7P1 that are complementary to sequence in the translation initiation regions of ORFs 4 and 7, respectively. The PPMO combination also inhibited replication of heterologous strains in the North American PRRSV genotype. Treatment of the cells with the combinations reduced PRRSV RNA and protein levels. In cell-free or cell-based luciferase reporter assays, the PPMO combination suppressed target mRNA translation more effectively than individual constituents, indicating that the suppression was due to their antisense effect. These results suggest potential application of these PPMO combinations for PRRS control.

Fig. 1

Schematic illustration of PPMO target sequence locations in PRRSV genome. The arrows indicate the 5′–3′ orientation of the PPMOs in relation to the PRRSV RNA genome.

Fig. 2

Enhanced inhibition of PRRSV replication in infected CRL11171 cells by PPMO combination. (A) Titration of virus yields from CRL11171 cells after PRRSV infection and indicated PPMO treatment. Virus titers are shown as TCID₅₀ (log₁₀/ml). PPMO 5UP1 was used at 0.5 μM, and the other PPMOs used at 8 μM. VC indicates cells with virus infection but no PPMO treatment. (B) Virus yields from cells treated with combination of 5UP1 at 0.5 μM and the other PPMOs used at 16 μM. Cells that were treated with PPMO combination 5UP1 + 4P1 and 5UP1 + 7P1 had virus yield below detection level (ND), and a bar is arbitrarily drawn to show the samples in the graph. The significance of difference in viral yields between the treatments is indicated by **P < 0.01. Error bars indicate variations.

Fig. 3

Effect of PPMO combination on PRRSV RNA synthesis detected by real-time RT-PCR. The 5UP1 was used at 0.5 μM and the concentrations of the other PPMOs are indicated in parenthesis. The significant difference in viral RNA copies between the treatments is indicated by *P < 0.05 and **P < 0.01. The PRRSV RNA copy numbers were calculated based on a standard curve after normalization with transcript of β-actin.

Fig. 4

Immunofluorescence assay with N-specific antibody. Cells were infected with PRRSV and treated with PPMOs as indicated. IFA was conducted at 24 h p.i. PPMO combination of 5UP1 0.5 μM with 4P1 or 7P1 at 16 μM resulted in inhibition of PRRSV replication below detection level. The images below the green fluorescence images were taken under phase contrast from the same field to show total number of cells.

Fig. 5

Inhibition of target RNA translation by PPMOs in luciferase reporter assay. (A) Cell-free luciferase reporter assay. Relative percentages of inhibition were calculated in comparison with signal from mock-treatment control. PPMO concentrations of 1, 5, 50, and 100 nM were used in this assay and shown in different bars. PPMO 5UP1 at 1 and 5 nM was combined with different concentrations of 7P1 or CP1. Error bars show variation between three replicates. (B) Luciferase reporter assay in transfected cells. PciNeoluc-5UTR plasmid was transfected into CRL11171 cells and luciferase yield was measured. Relative percentages of luciferase yield were calculated in comparison with signal of none PPMO control. Combination of 5UP1 0.5 μM + 7P1 16 μM shows enhanced inhibitory effect, while 5UP1 + CP1 has no such effect.

Fig. 6

Cross-strain inhibition assay. (A) Virus yield titration shows inhibition of nine North American PRRSV strains by PPMO combination of 5UP1 + 4P1 or 5UP1 + 7P1. Lelystad is a prototype of European PRRSV genotype. All other strains are North American PRRSV genotype. “Mock” is virus infection control without PPMO. Treatment of the cells with the two PPMO combinations led to suppression of PRRSV replication of all North American strains, which had virus yields not detectable in this assay, and bars are arbitrarily drawn to show the samples in the graph. (B) Sequence aligment of complementary sequences of 5UP1, 4P1, and 7P1, respectively, to show nucleotide mismatches between PRRSV strains. PRRSV strain names are listed in the first column. “Lely” stands for Lelystad strain, which has little similarity in the 5′ UTR to other strains, as indicated by “-” in the alignment of 5UP1 target sequence. PPMO names are listed above the sequence. VR2385 sequence is used as the reference sequence, as the PPMOs were designed against it. For all other sequences, only nucleotides differing from the reference sequence are shown, and identical nucleotides are indicated as “.”. Missing nucleotides are indicated as “-”. The initiation codon ATG of ORFs 4 and 7 are underlined. GenBank accession numbers for PRRSV strains in the alignment are listed in parenthesis: 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.