Antisense morpholino-oligomers directed against the 5' end of the genome inhibit coronavirus proliferation and growth - PubMed (original) (raw)

Antisense morpholino-oligomers directed against the 5' end of the genome inhibit coronavirus proliferation and growth

Benjamin W Neuman et al. J Virol. 2004 Jun.

Abstract

Conjugation of a peptide related to the human immunodeficiency virus type 1 Tat represents a novel method for delivery of antisense morpholino-oligomers. Conjugated and unconjugated oligomers were tested to determine sequence-specific antiviral efficacy against a member of the Coronaviridae, Mouse hepatitis virus (MHV). Specific antisense activity designed to block translation of the viral replicase polyprotein was first confirmed by reduction of luciferase expression from a target sequence-containing reporter construct in both cell-free and transfected cell culture assays. Peptide-conjugated morpholino-oligomers exhibited low toxicity in DBT astrocytoma cells used for culturing MHV. Oligomer administered at micromolar concentrations was delivered to >80% of cells and inhibited virus titers 10- to 100-fold in a sequence-specific and dose-responsive manner. In addition, targeted viral protein synthesis, plaque diameter, and cytopathic effect were significantly reduced. Inhibition of virus infectivity by peptide-conjugated morpholino was comparable to the antiviral activity of the aminoglycoside hygromycin B used at a concentration fivefold higher than the oligomer. These results suggest that this composition of antisense compound has therapeutic potential for control of coronavirus infection.

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Figures

FIG. 1.

FIG. 1.

PMO and R9F2-PMO, targeting the MHV polyprotein gene translation initiation codon region, and scrambled control R9F2-PMO were assayed for inhibition of luciferase expression from a chimeric plasmid containing the luciferase gene fused in frame with a 43-base region spanning the MHV polyprotein gene translation initiation codon. The relative percent inhibition by each PMO or R9F2-PMO in one experiment representative of three is shown (A). DBT cells treated with R9F2-PMO-Fl or PMO-Fl were rinsed thoroughly and photographed under mercury vapor illumination to visualize punctate cell-associated fluorescein (B). Fluorescent cells are indicated with black and white arrowheads, respectively, in the phase-contrast and fluorescent image pairs. Fluorescein-positive cells were counted by FACScan. One representative scan from three replicates showing untreated and R9F2-PP PMO-Fl-treated cells is shown (C).

FIG. 2.

FIG. 2.

Cytotoxicity of the R9F2-PP PMO (A) and R9F2-SC PMO (B) was tested by MTT assay at 10 μM (left bar of each pair) or 20 μM (right bar of pair) concentration. The means of three experiments with standard errors are shown. Cells transfected with a plasmid containing a MHV target gene-luciferase fusion were assayed for inhibition of luciferase expression after treatment with R9F2-PMO (C). DBT cells transfected with a plasmid containing a missplicing luciferase reporter gene were assayed in a splice correction assay after treatment in cell culture medium containing R9F2-PMO (D). Relative light units produced from translated luciferase in each monolayer are shown. The dotted line represents the mean of nine controls treated with medium alone. Error bars (C and D) represent the standard errors of the means of three replicates.

FIG. 3.

FIG. 3.

MHV-infected or mock-infected DBT cells treated with R9F2-PMO or water were fixed and stained with crystal violet 24 h after inoculation (A). Multinucleate syncytia were observed and quantified within three randomly placed 4-mm2 windows per flask of three replicates. The number of multinucleate cells per square millimiter is shown for each treatment type and concentration, with error bars representing the standard errors of the means (B). None, cells were inoculated with MHV in the absence of R9F2-PMO treatment; Uninfected, cells were mock inoculated and not treated with R9F2-PMO. (C) Western blot probed with anti-β-actin or anti-MHV N antibody on samples from treated and untreated MHV-infected or mock-infected cells with loading normalized with respect to actin content. Standard molecular weight markers show that each of the proteins detected corresponds to the expected size of murine β-actin (43 kDa) or MHV N (50 kDa).

FIG. 4.

FIG. 4.

Three MHV growth experiments in which serum-free culture medium was substituted for serum-containing culture medium at different points during incubation of R9F2-PMO with DBT cells or virus growth period were performed. Cells were treated in 1 ml of VP-SFM, and then 10 ml of serum-containing medium was added after 3 h (left panel) or 6 h (middle panel). One treatment group received 10 ml of VP-SFM after inoculation (right panel). Results are presented for a minimum of three replicate flasks per treatment, and cells were inoculated with MHV after 6 h of R9F2-PMO treatment. Infectious MHV present in culture supernatant recovered 18 h after inoculation was titrated by plaque assay. Mean titers in PFU per milliliter are shown with error bars corresponding to the standard errors of the means. An MHV growth experiment was performed with incubation in the absence of serum. Results shown are mean values of three replicate flasks in one of four independent assays, with standard errors indicated by error bars.

FIG. 5.

FIG. 5.

Comparison of HYG and PP-P003. MHV was titrated by plaque assay on DBT cells with R9F2-PMO present in the indicated concentrations in the agarose-VP-SFM overlay. The average plaque diameter, measured to the nearest 0.5 mm ± standard error, is shown for each treatment group (top). Results and a selection of plaques (bottom) from 10 μM concentration treatment groups of one experiment representative of two are shown. MHV plaques are visible as large or small holes in the DBT monolayer that has been degraded to some extent by 72-h incubation with R9F2-PMO or HYG.

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