In vitro characterization of the anti-hepatitis B virus activity and cross-resistance profile of 2',3'-dideoxy-3'-fluoroguanosine.

The fluorinated guanosine analog 2',3'-dideoxy-3'-fluoroguanosine (FLG) was shown to inhibit wild-type (wt) hepatitis B virus (HBV) replication in a human hepatoma cell line permanently expressing HBV. Experiments performed in the duck model of HBV infection also showed its in vivo antiviral activity. In this study, we investigated the mechanism of inhibition of FLG on HBV replication and its profile of antiviral activity against different HBV or duck hepatitis B virus (DHBV) drug-resistant mutants. We found that FLG-triphosphate inhibits weakly the priming of the reverse transcription compared to adefovir-diphosphate in a cell-free system assay allowing the expression of an enzymatically active DHBV reverse transcriptase. It inhibits more potently wt DHBV minus-strand DNA synthesis compared to lamivudine-triphosphate and shows a similar activity compared to adefovir-diphosphate. FLG-triphosphate was most likely a competitive inhibitor of dGTP incorporation and a DNA chain terminator. In Huh7 cells transiently transfected with different HBV constructs, FLG inhibited similarly the replication of wt, lamivudine-resistant, adefovir-resistant, and lamivudine-plus-adefovir-resistant HBV mutants. These results were consistent with those obtained in the DHBV polymerase assay using the same drug-resistant polymerase mutants. In conclusion, our data provide new insights in the mechanism of action of FLG-triphosphate on HBV replication and demonstrate its inhibitory activity on drug-resistant mutant reverse transcriptases in vitro. Furthermore, our results provide the rationale for further clinical evaluation of FLG in the treatment of drug-resistant virus infection and in the setting of combination therapy to prevent or delay drug resistance.

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

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.

Antiviral effect of N-butyldeoxynojirimycin against bovine viral diarrhea virus correlates with misfolding of E2 envelope proteins and impairment of their association into E1-E2 heterodimers.

The iminosugar N-butyldeoxynojirimycin (NB-DNJ), an endoplasmic reticulum alpha-glucosidase inhibitor, has an antiviral effect against bovine viral diarrhea virus (BVDV). In this report, we investigate the molecular mechanism of this inhibition by studying the folding pathway of BVDV envelope glycoproteins in the presence and absence of NB-DNJ. Our results show that, while the disulfide-dependent folding of E2 glycoprotein occurs rapidly (2.5 min), the folding of E1 occurs slowly (30 min). Both BVDV envelope glycoproteins associate rapidly with calnexin and dissociate with different kinetics. The release of E1 from the interaction with calnexin coincides with the beginning of E1 and E2 association into disulfide-linked heterodimers. In the presence of NB-DNJ, the interaction of E1 and E2 with calnexin is prevented, leading to misfolding of the envelope glycoproteins and inefficient formation of E1-E2 heterodimers. The degree of misfolding and the lack of association of E1 and E2 into disulfide-linked complexes in the presence of NB-DNJ correlate with the dose-dependent antiviral effect observed for this iminosugar.