Antiviral effects of antisense morpholino oligomers in murine coronavirus infection models - PubMed (original) (raw)

Antiviral effects of antisense morpholino oligomers in murine coronavirus infection models

Renaud Burrer et al. J Virol. 2007 Jun.

Abstract

The recent emergence of novel pathogenic human and animal coronaviruses has highlighted the need for antiviral therapies that are effective against a spectrum of these viruses. We have used several strains of murine hepatitis virus (MHV) in cell culture and in vivo in mouse models to investigate the antiviral characteristics of peptide-conjugated antisense phosphorodiamidate morpholino oligomers (P-PMOs). Ten P-PMOs directed against various target sites in the viral genome were tested in cell culture, and one of these (5TERM), which was complementary to the 5' terminus of the genomic RNA, was effective against six strains of MHV. Further studies were carried out with various arginine-rich peptides conjugated to the 5TERM PMO sequence in order to evaluate efficacy and toxicity and thereby select candidates for in vivo testing. In uninfected mice, prolonged P-PMO treatment did not result in weight loss or detectable histopathologic changes. 5TERM P-PMO treatment reduced viral titers in target organs and protected mice against virus-induced tissue damage. Prophylactic 5TERM P-PMO treatment decreased the amount of weight loss associated with infection under most experimental conditions. Treatment also prolonged survival in two lethal challenge models. In some cases of high-dose viral inoculation followed by delayed treatment, 5TERM P-PMO treatment was not protective and increased morbidity in the treated group, suggesting that P-PMO may cause toxic effects in diseased mice that were not apparent in the uninfected animals. However, the strong antiviral effect observed suggests that with further development, P-PMO may provide an effective therapeutic approach against a broad range of coronavirus infections.

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Figures

FIG. 1.

FIG. 1.

P-PMO target sites and efficacy evaluation in a reporter assay. (A) Locations targeted by each PMO compound on the genomic (+) and antigenomic (−) viral RNA (top) and on the subgenomic RNAs (lower right; example given is RNA6). (B) Comparison of translation inhibitory activities by P-PMOs against in vitro-transcribed RNA consisting of the MHV 5′-UTR sequence fused to luciferase in rabbit reticulocyte lysate in vitro translation reactions. Activities of P-PMOs complementary to the genomic MHV 5′-UTR (left) and other regions of viral RNA (right) from the same experiment are depicted. Error bars indicate standard errors of the means.

FIG. 2.

FIG. 2.

Evaluation of PMO sequences and various peptide conjugates in MHV-infected cells. (A) Dose-dependent inhibition of MHV plaque expansion. DBT cell cultures were inoculated with fixed doses of MHV-A59, and an R9F2-PMO was added with each agarose overlay. The plaque diameter was then measured 72 h after the overlay was applied. (B) R9F2-PMO treatment reduces syncytium formation by MHV-1 and MHV-4. Qualitative changes in cell morphology and density were compared against untreated, uninfected (left) and untreated, infected (right [column marked untreated]) controls. Representative images show cells pretreated for 3 h with 10 μM R9F2-PMO and fixed for 24 h after inoculation. (C) Effects of delivery peptide conjugation on PMO activity. DBT cells were treated with 20 μM P-PMO for 3 h before inoculation at a multiplicity of 0.1 PFU/cell with MHV-A59. The virus yield was quantified 24 h after inoculation. 5′-Terminal peptide conjugates follow standard single-letter amino acid naming, except as follows: X, 6-aminohexanoic acid; B, beta-alanine; and Man, mannose. Asterisks indicate significant differences (P < 0.05 by Student's t test) with respect to mock-treated controls. (D) Dose-response experiment comparing the effects of R9F2- and (RXR)4-conjugated 5TERM, TRS1, and RND P-PMOs on viral titers. Cells were treated with various P-PMOs at 10 μM for 3 h, infected with MHV-A59 for 1 h, and then incubated again in the presence of P-PMOs for 24 h. Error bars throughout indicate standard errors of the means.

FIG. 3.

FIG. 3.

Single-dose and long-term inhibition of coronavirus multiplication. (A) Inhibition of replication of various MHV strains (inoculated at multiplicities of 0.01 to 1 PFU/cell) in DBT cells after a 3-h preinoculation treatment with 10 μM P-PMO. The virus yield was quantified 24 h after inoculation. (B) Dose-activity comparison of the effects of SARS-5TERM and SARS-TRS1 P-PMOs on SARS-CoV (1 PFU/cell) in Vero-E6 cells. Cells were treated with P-PMOs for 6 h prior to inoculation, and viral titers were analyzed 48 h later. (C) Effects of long-term P-PMO treatment on MHV replication. DBT cells were treated for 6 h with P-PMOs and then inoculated with plaque-purified MHV-A59 (0.1 PFU/cell). Culture medium was used to inoculate fresh cultures of P-PMO-treated cells every 24 h for a total of 10 viral passages. The virus yield was quantified as described for panel A. Error bars throughout indicate standard errors of the means.

FIG. 4.

FIG. 4.

5TERM PMO and P-PMO reduce viral titers in the livers of MHV-infected mice. Mice were inoculated i.p. with MHV-A59 (A and B) or various other MHV strains (C). Starting 5 h before infection and on each of the next 2 days, each animal received one dose of saline (black bars), RND PMO or (RXR)4-PMO (hatched bars), or 5TERM PMO or (RXR)4-PMO (white bars), as indicated in Materials and Methods. (RXR)4-PMO was utilized for panels B and C. Unc, unconjugated. Livers were collected on day 4 (A and C) or at the indicated time points (B), and viral titers were determined by plaque assay. Error bars indicate standard errors of the means. Asterisks indicate significant differences from mock-treated controls (P < 0.05 by Student's t test).

FIG. 5.

FIG. 5.

5TERM-(RXR)4-PMO attenuates viral hepatitis. (A) Livers were obtained from mice infected with the indicated MHV strains at 4 (MHV-2, MHV-3, and MHV-Alb139) or 6 (MHV-A59) days p.i. The numbers of necrotic lesions per unit area were compared between P-PMO-treated and mock-treated groups, as described in Materials and Methods. Data points represent individual mice, and bars indicate the mean severities for the combined mock- and RND P-PMO-treated groups and the 5TERM P-PMO-treated group, respectively. The number of lesions was significantly lower in A59-infected mice treated with 5TERM P-PMO than in mock-treated (P < 0.05) or RND P-PMO-treated (P < 0.005) controls. (B to G) Representative areas of H&E-stained livers from uninfected (B and C) and MHV-A59 (D and E [6 days p.i.])- and MHV-3 (F and G [4 days p.i.])-infected animals. Mice received (RXR)4-5TERM P-PMO treatment (C, E, and G) or mock treatment (B, D, and F). Necrotic lesions and inflammation characteristic of lymphocytes are indicated with arrowheads. Note the regions of bright pink staining that mark necrotic areas in panel E. (H) At a higher magnification, the liver sections show ballooning necrosis (large black arrowheads), lymphocytic infiltration with more moderate necrosis (large white arrowheads), widespread coagulation necrosis with karyorrhectic debris (small black arrowheads), and eosinophilic staining reminiscent of Councilman bodies (small white arrowheads). Bars, 0.1 mm.

FIG. 6.

FIG. 6.

Dual effects of (RXR)4-5TERM on weight loss and survival. Mice were infected with 10 PFU MHV-3 (A) or 100 (B), 10,000 (C), or 50,000 (D) PFU MHV-Alb139. Depending on the experiment, some animals received (RXR)4-5TERM, following either a prophylactic (empty circles) or therapeutic (empty squares and empty triangles) regimen. Controls received either saline (black circles) or (RXR)4-RND (black triangles) daily, with the first injection starting 5 h before inoculation. The relative weight is shown for each treatment group (n = 3 to 7). Error bars indicate standard errors of the means. Each death is represented with the group's corresponding symbol positioned on the time axis at the time of the event. Asterisks indicate significant differences relative to the mock-treated control group. *, P < 0.05; **, P < 0.01. Student's t test (weight) or the log rank survival test (mortality) was used to determine statistical significance.

FIG. 7.

FIG. 7.

P-PMOs in the lungs of MHV-1-infected mice. (A) Comparison of viral titers from lung homogenates collected 4 days after inoculation. As indicated, the treated groups received three doses of 5TERM (RXR)4-PMO i.n. or i.p. starting 5 h before or 24 h after i.n. inoculation with 100 PFU MHV-1. The designation “a” marks a nonsignificant trend of difference from mock-treated controls (P = 0.057 by Student's t test). (B) Clinical disease in A/J mice infected with 50 PFU MHV-1 and treated i.n. as indicated. Daily weight values are expressed as a percentage of the initial weight of each animal. Error bars indicate the standard error of the mean for each group (n = 4/group, except for the RND P-PMO group [n = 1]). Asterisks indicate significant differences relative to the mock-treated control group. *, P < 0.05; **, P < 0.01 (Student's t test).

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