Development of peptide-conjugated morpholino oligomers as pan-arenavirus inhibitors - PubMed (original) (raw)

Development of peptide-conjugated morpholino oligomers as pan-arenavirus inhibitors

Benjamin W Neuman et al. Antimicrob Agents Chemother. 2011 Oct.

Abstract

Members of the Arenaviridae family are a threat to public health and can cause meningitis and hemorrhagic fever, and yet treatment options remain limited by a lack of effective antivirals. In this study, we found that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) complementary to viral genomic RNA were effective in reducing arenavirus replication in cell cultures and in vivo. PPMO complementary to the Junín virus genome were designed to interfere with viral RNA synthesis or translation or both. However, only PPMO designed to potentially interfere with translation were effective in reducing virus replication. PPMO complementary to sequences that are highly conserved across the arenaviruses and located at the 5' termini of both genomic segments were effective against Junín virus, Tacaribe virus, Pichinde virus, and lymphocytic choriomeningitis virus (LCMV)-infected cell cultures and suppressed viral titers in the livers of LCMV-infected mice. These results suggest that arenavirus 5' genomic termini represent promising targets for pan-arenavirus antiviral therapeutic development.

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Figures

Fig. 1.

Fig. 1.

Schematic representation of PPMO target sites on both arenavirus genomic RNA segments. (A) PPMO are shown in red, and genes are labeled in the 5′-to-3′ orientation. Additional potential binding sites for TERM-L, TERM-S, TERM-L-REP, 3′TERM-L, and 3′TERM-S PPMO at the viral genomic termini (black and white rectangles) are not shown on this diagram but are listed in Fig. S1 in the supplemental material. (B) Hypothetical mechanisms of action for each PPMO are indicated. Abbreviations: GR, genomic replication; T, subgenomic mRNA transcription.

Fig. 2.

Fig. 2.

Effect of PPMO on Vero E6 cell viability. MTT cytotoxicity assays were performed using uninfected Vero E6 cells incubated for 24 h (A) or 96 h (B) with increasing concentrations of the indicated PPMO before incubation with MTT. After MTT treatment for 40 min, cells were solubilized with DMSO and insoluble compound formation levels were determined photometrically at 560 nm. Viability is charted relative to mock-treated cell results (set at 1.0). Error bars denote standard deviations calculated from the results of three replicate assays.

Fig. 3.

Fig. 3.

Effect of arenaviruses on cellular adhesion and morphology. Cells that remained adherent after three saline solution washes and crystal violet staining are shown to illustrate changes in cellular adherence 48 h after mock infection (left panel) or infection with LCMV (middle panel). An example of cell fusion induced by modified JUNV is shown in the right panel. Arrowheads point to clusters of nuclei in the center of each syncytium.

Fig. 4.

Fig. 4.

Antiviral effects of PPMO treatment in cell culture. Dose-inhibition curves for LCMV-infected cultures treated with the indicated PPMO are shown. Error bars indicate standard deviations for the results from three replicate samples.

Fig. 5.

Fig. 5.

Reduction of LCMV protein and RNA expression following PPMO treatment. Vero-E6 cells were pretreated with TERM-L PPMO (A and B) or TERM-L-REP PPMO (C and D) 3 h before inoculation with LCMV. Cell lysates were harvested for protein analysis 24 h (A and C) and 48 h (B and D) after inoculation. Protein from purified virions appears in the leftmost lane of each panel (V). Quantitative RT-PCR was performed to measure the amount of RNA in which the NP gene was in the positive sense, which would include both NP mRNA and S segment vcRNA. (E) Levels of NP RNA relative to GAPDH mRNA controls are expressed as percentages ± standard deviations of the results from three replicate samples relative to infected untreated control NP RNA levels 24 h after inoculation. (F) NP, GPC, and GP2 protein levels were measured by densitometry analysis of Western blots, including those shown in panel A, and are expressed as percentages ± standard errors relative to infected untreated control protein levels. Panel G compares NP and NP RNA levels 24 h after inoculation.

Fig. 6.

Fig. 6.

Antiviral efficacy of PPMO against LCMV in mice. Mice were administered sterile saline solution or PPMO at ∼6 mg/kg (A) or ∼9 mg/kg (B) by intraperitoneal (i.p.) injection at 3 h before inoculation with LCMV and daily for 3 days thereafter. Tissues were collected 96 h after inoculation. Virus titers were determined by plaque assay and normalized to tissue weight. The dotted lines indicate the threshold of detection in our plaque assays.

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