Inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers

Abstract

Delivery of phosphorodiamidate morpholino oligomers (PMO) into fish cells in vitro and tissues in vivo was examined. Uptake was evaluated by fluorescence microscopy and flow cytometry after treating cultured cells or live rainbow trout with 3' fluorescein-tagged PMO. Arginine-rich peptide conjugated to the 5' end of the PMO markedly enhanced cellular uptake in culture by 8- to 20-fold compared with non-peptide-conjugated PMO as determined by flow cytometry. Enhanced uptake of PMO conjugated to peptide was also observed in tissues of fish treated by immersion. The efficacy of PMO as inhibitors of infectious haematopoietic necrosis virus (IHNV) replication was determined in vitro. Peptide-conjugated PMOs targeting sequences within the IHNV genomic RNA (negative polarity) or antigenomic RNA (positive polarity) significantly inhibited replication in a dose-dependent and sequence-specific manner. A PMO complementary to sequence near the 5' end of IHNV genomic RNA was the most effective, diminishing titre by 97%, as measured by plaque assay and Western blot. These data demonstrate that replication of a negative-stranded non-segmented RNA virus can be inhibited by antisense compounds that target positive polarity viral RNA, or by a compound that targets negative polarity viral RNA.

Figure 1

(a) Chemical structure of phosphorodiamidate morpholino oligomers (PMO). The deoxyribose of DNA is replaced by a 6‐membered morpholine ring, and the phosphodiester internucleoside bond is replaced by a phosphorodiamidate linkage in the PMO structure. Arginine‐rich peptide, when present, is conjugated to the 5′ end of a PMO through the illustrated linker. Arginine‐rich peptide sequences used in this work are described in Materials and methods. Fluorescein, when present, is conjugated directly to the nitrogen of the morpholine ring, on the 3′ end of the PMO. (b) Targeting localizations in the IHNV genome of the PMOs used in this study.

Figure 2

Microscopy of CHSE‐214 and EPC cells incubated O/N with 10 μ m fluorescein‐conjugated PMOs. Left‐hand side panel is bright‐field image and right‐hand side is the same field under fluorescent illumination. (a) CHSE‐214 cells incubated with P002‐PMO‐FITC. (b) CHSE‐214 cells incubated with P003‐PMO‐FITC. (c) EPC cells incubated with P002‐PMO‐FITC. (d) EPC cells incubated with P003‐PMO‐FITC. Compound names/sequences are as in Table 1.

Figure 3

Flow cytometric analysis of cells treated with fluorescein‐conjugated PMOs. Cultured cells; (a) CHSE‐214 or (b) EPC were treated with either 10 μ m fluorescein‐conjugated PMO‐FITC (solid histogram), P002‐PMO‐FITC (—) or P003‐PMO‐FITC (‐ ‐ ‐) for 1 h. The cells were analysed by flow cytometry after trypsinization and washing. (c) The mean fluorescent intensity (m.f.i.) for each cell type and treatment are calculated from the histogram shown.

Figure 4

Inhibition of IHNV titre by PMOs. Representative experiments from the several trials carried out to determine the effect of different PMO compounds on viral titre. The data are presented as two separate graphs, as these two series of samples were tested at different times and the mock‐treated controls achieved differing final titre readings. Single wells of CHSE‐214 cells were treated with the designated PMOs at a concentration of 15 μ m overnight and then infected with IHNV. Final viral titres were assessed by plaque assay as described in Materials and methods. (a) Lanes: 0, No PMO; 1, P003‐G PMO; 2, P003‐L PMO; 3, P003‐L 3′ PMO; 4, G PMO; 5, L PMO; 6, L 3′ PMO. (b) IHNV viral titres in CHSE‐214 treated with N‐targeted PMOs. Lanes: 0, No PMO; 7, P003‐N PMO; 8, P003‐N Scramble PMO and 9, P003‐irrelevant sequence PMO. In the lower portion of panels a and b, the results are expressed as the percentage inhibition of titre of treated cells compared with untreated cells. (c) Immunoblots of CHSE‐14 cells receiving PMO treatment and subsequent IHNV infection as above were harvested 10 days post‐inoculation. Cell lysates were prepared and used for immunoblots. Blots were probed with an antibody against the 67‐kDa IHNV glycoprotein (upper blot). The lower blot shows the 43‐kDa band generated with a β‐actin antibody. Sample designations that appear above the lanes apply to both blots and correspond to the sample numbers above in Fig. 4a.

Figure 5

Plaque assay results from PMO dose–response challenge of IHNV‐infected cells. Details of experimental time‐line as for Fig. 4a,b and as described in text. Duplicate wells of CHSE‐214 cells treated with various concentrations of PMOs were infected with log dilutions of IHNV and the final infected titres were evaluated by plaque assay. Each bar graph represents cells treated with a different PMO: (a) P003‐L PMO, (b) P003‐L 3′ PMO, and (c) P003‐N PMO. Dose designations appear under the x‐axis of the graph.

Figure 6

Fluorescence microscopy of gills from rainbow trout. Approximately 0.3 g fish were exposed to 10 μ m of the stated compounds by immersion for 20 min as described in Materials and methods. (a) PMO‐FITC treatment, (b) P002‐PMO‐FITC and (c) P003‐PMO‐FITC treatment. The left‐hand side images are bright field and the right‐hand side images are the identical fields under fluorescent illumination.

Figure 7

Predicted secondary structure of the P003‐L 3′ PMO binding region and surrounding sequence in the 5′ end of the viral RNA‐negative strand. The P003‐L 3′ PMO sequence target is designated as a black line bordering the super‐structure.