Study of the mechanism of antiviral action of iminosugar derivatives against bovine viral diarrhea virus

Abstract

The glucose-derived iminosugar derivatives N-butyl- and N-nonyl-deoxynojirimycin (DNJ) have an antiviral effect against a broad spectrum of viruses including Bovine viral diarrhea virus (BVDV). For BVDV, this effect has been attributed to the reduction of viral secretion due to an impairment of viral morphogenesis caused by the ability of DNJ-based iminosugar derivatives to inhibit ER alpha-glucosidases (N. Zitzmann, A. S. Mehta, S. Carrouée, T. D. Butters, F. M. Platt, J. McCauley, B. S. Blumberg, R. A. Dwek, and T. M. Block, Proc. Natl. Acad. Sci. USA 96:11878-11882, 1999). Here we present the antiviral features of newly designed DNJ derivatives and report for the first time the antiviral activity of long-alkyl-chain derivatives of deoxygalactonojirimycin (DGJ), a class of iminosugars derived from galactose which does not inhibit endoplasmic reticulum (ER) alpha-glucosidases. We demonstrate the lack of correlation between the ability of long-alkyl-chain DNJ derivatives to inhibit ER alpha-glucosidases and their antiviral effect, ruling out ER alpha-glucosidase inhibition as the sole mechanism responsible. Using short- and long-alkyl-chain DNJ and DGJ derivatives, we investigated the mechanisms of action of these drugs. First, we excluded their potential action at the level of the replication, protein synthesis, and protein processing. Second, we demonstrated that DNJ derivatives cause both a reduction in viral secretion and a reduction in the infectivity of newly released viral particles. Long-alkyl-chain DGJ derivatives exert their antiviral effect solely via the production of viral particles with reduced infectivity. We demonstrate that long-alkyl-chain DNJ and DGJ derivatives induce an increase in the quantity of E2-E2 dimers accumulated within the ER. The subsequent enrichment of these homodimers in secreted virus particles correlates with their reduced infectivity.

Figures

FIG. 1

Chemical structures of the iminosugar derivatives used in this study. (A) Iminosugar derivatives based on the glucose analogue DNJ. (B) Iminosugar derivatives based on the galactose analogue DGJ.

FIG. 2

Antiviral effect of DNJ derivatives in the “in vitro” BVDV system. DNJ derivatives containing alkyl side chains of different lengths ranging from C4 to C18 (x axis) were tested for their ability to inhibit BVDV plaque formation on MDBK cells as described in Materials and Methods. An MOI of 0.01 was used. Hexadecan-DNJ and octadecan-DNJ are unsaturated. The IC50 of each molecule is shown on the y axis.

FIG. 3

ER α-glucosidase inhibition and antiviral effect of iminosugar derivatives. BVDV-infected MDBK cells (MOI of 1) were not treated (No) or were treated with NB-DNJ (NB) and NN-DNJ (NN) at their respective IC50s (left panel) and IC90s (right panel). Cells were lysed at 18 h p.i., and proteins were separated by SDS-PAGE (10% polyacrylamide) under reducing conditions. A Western blot analysis was performed using the anti-E2 MAb WB214 (diluted 1:1,000). The two polypeptides detected (E2 and E2-p7) are indicated by arrows.

FIG. 4

Biosynthesis and processing of BVDV envelope glycoproteins in the presence of NN-DNJ or NN-DGJ. MDBK cells were infected with BVDV at an MOI of 1. At 18 h p.i., the cells were not treated (−) or were treated (+) with 100 μM of NN-DNJ (A) or NN-DGJ (B). At 2 h later, the cells were pulse-labeled with [35S]methionine-[35S]cysteine for 15 min, chased for the times indicated in the continuous presence of the drug, and immunoprecipitated with anti-E2 MAb WB214. The proteins were separated by SDS-PAGE (10% polyacrylamide) under reducing conditions and visualized by autoradiography. The two main polypeptides detected (E2 and E2-p7) are indicated by arrows.

FIG. 5

Effect of iminosugar derivatives on viral secretion. MDBK cells were infected with either a cp (NADL) (A) or ncp (Pe515) (B) strain of BVDV at an MOI of 1 and grown for 24 h (A) or 48 h (B) in the absence or presence of different iminosugar derivatives at the concentrations indicated. Viral RNA was purified from the supernatant and used to perform a single-tube RT-PCR analysis. Then 10 μl (A) or 20 μl (B) of the RT-PCR product was loaded and run on a 2% agarose gel stained with ethidium bromide. (A) Result obtained using the cp NADL strain. The quantity of cDNA was determined by densitometry analysis. The experimental titer was measured by the plaque reduction assay, while the theoretical titer was calculated using the linear regression curve obtained with the standard curve (established from a virus stock with a titer of at 2 × 107 PFU/ml). A summary of these results is presented in the table shown below the gels. (B) Result obtained using the ncp Pe515 strain. The quantity of cDNA was determined by densitometry analysis, and the experimental titer was measured by the plaque reduction assay. The values corresponding to the reduction in cDNA quantity and the reduction in the titer, obtained by comparison of treated and nontreated samples, are presented in the table below the gels.

FIG. 6

Effect of iminosugar derivatives on viral secretion: a dose-response analysis. MDBK cells were infected with either a cp (NADL) or ncp (Pe515) strain of BVDV at an MOI of 1 and grown for 24 h (cp) or 48 h (ncp) in the absence or presence of increasing amounts of two different iminosugar derivatives. Viral RNA was purified from the supernatant and used to perform a single-tube RT-PCR analysis, as described in Materials and Methods. Then 10 μl (cp) or 20 μl (ncp) was loaded and run on an ethidium bromide-stained 2% agarose gel. Computerized-inverted images of the gels are presented. The type of virus and drug used are indicated in boxes within the frames of the gels.

FIG. 7

Effect of iminosugar derivatives on the accumulation of E2-E2 homodimers and E1-E2 heterodimers in the ER. (A) MDBK cells were mock infected (lane 1) or infected with the ncp strain (Pe515) at an MOI of 1 and grown in the absence (lane 2) or presence of 100 μM iminosugar derivatives (NN-DNJ, lane 3; N7-oxadecyl-DNJ, lane 4; NN-DGJ, lane 5; N7-oxanonyl-6deoxy-DGJ, lane 6). Ribavirin, which inhibits viral RNA replication, was used as a control (lane 7). At 48 h p.i., the cells were lysed and proteins were separated by SDS-PAGE (10% polyacrylamide) under nonreducing conditions. A Western blot analysis was performed using MAb WB166 (diluted 1:1,000), which recognizes the E2 glycoprotein regardless of its state of folding. In the bottom panel, the same blot was hybridized with an anti-actin antibody (loading control). (B) The same experiment as described for panel A was performed, but this time both cp and a ncp strains of BVDV were used for the infection. N7-oxadecyl-DNJ (abbreviated N7-DNJ) and N7-oxanonyl-6deoxy-DGJ (abbreviated N7-DGJ) were used at the concentrations indicated. The autoradiographs were quantified. The ratio of the amount of E2-E2 to the amount of E1-E2 was established, and the percentages, with the ratio of the amount of E2-E2 to the amount of E1-E2 without inhibitor present set at 100%, are shown at the bottom of each gel. Size standards are indicated on the left, and immunoreactive proteins and complexes are indicated on the right.

FIG. 8

Correlation between the presence of a long alkyl chain branched on DNJ or DGJ head groups and the abnormal accumulation of E2-E2 dimers. MDBK cells were mock infected or infected with the cp (NADL) strain of BVDV at an MOI of 1 and grown for 24 h in the absence or presence of iminosugar derivatives at the concentrations indicated. The cells were lysed, and proteins were separated by SDS-PAGE (10% polyacrylamide) under nonreducing conditions. A Western blot analysis was performed using MAb WB166 (diluted 1:1000). The IC50 and IC90 of each drug are indicated by arrows. No IC50 or IC90 is indicated for NB-DGJ, since this compound is not antiviral.

FIG. 9

Analysis of the envelope glycoprotein dimer composition of the virus particles released under drug treatment. MDBK cells were infected with the cp (NADL) strain of BVDV at an MOI of 1 and grown in the absence (lanes 1 and 1′) or presence of NB-DNJ (2.5 mM; lanes 2 and 2′), NN-DNJ (150 μM; lanes 3 and 3′), or NN-DGJ (150 μM; lanes 4 and 4′). After 24 h, the medium was harvested, clarified at 5,000 × g for 30 min, split in two, and processed by PEG 8000 precipitation (10,000 × g) or by centrifugation (90,000 × g) through a 20% sucrose cushion. Proteins from pellets were separated by SDS-PAGE (10% polyacrylamide) under nonreducing conditions and subjected to a Western blot analysis using the anti-E2 MAb WB166. The bands detected were quantified by densitometry. The ratio of the amount of E1-E2 to the amount of E2-E2 was established for every drug concentration used, and the percentages, with the ratio of the amount of E2-E2 to the amount of E1-E2 without inhibitor present set at 100%, are shown in the table below the gel. a, values are combined quantities (determined by densitometry analysis) from both gels; b, values were taken from the previous column.