Mutations within the hepatitis B virus (HBV) polymerase gene conferring drug-resistance are selected during prolonged lamivudine (3TC) or adefovir dipivoxil (ADV) treatment. Because there is no other approved drug against HBV, treatments with 3TC or ADV are used either sequentially or in addition, depending on treatment response or failure. Considering the use of de novo or add-on 3TC؉ADV bitherapy, we investigated the possibility of the emergence of an HBV strain harboring polymerase mutations conferring resistance to both 3TC (rtL180M؉M204V) and ADV (rtN236T). We constructed the L180M؉M204V؉N236T mutant and determined its replication capacity and its susceptibility to different nucleos(t)ide analogs in transiently transfected hepatoma cell lines. The triple mutant replicates its genome in vitro, but less efficiently than either the wild-type (wt) HBV or L180M؉M204V and N236T mutants. Phenotypic assays indicated that the L180M؉M204V؉N236T mutant is resistant to pyrimidine analogs (3TC, -FTC, ␤-L-FD4C, L-FMAU). Compared with wt HBV, this mutant displays a 6-fold decreased susceptibility to ADV and entecavir and a 4-fold decreased susceptibility to tenofovir. Interferon alfa inhibited equally the replication of wt and L180M؉M204V؉N236T HBV. In conclusion, the combination of rtL180M؉M204V and rtN236T mutations impairs HBV replication and confers resistance to both 3TC and ADV in vitro. These results suggest that the emergence of the triple mutant may be delayed and associated with viral resistance in patients treated with 3TC؉ADV. T he main goals of the treatment of chronic hepatitis B virus (HBV) infection are the sustained suppression of HBV replication and a remission of liver disease. An immunomodulator, interferon alfa (IFN-␣), and two synthetic nucleos(t)ide analogs, lamivudine (3TC) and adefovir dipivoxil (ADV), are currently approved for the treatment of chronic hepatitis B. With respect to IFN-␣, the overall response rate to this drug is less than 40%, and IFN-␣ therapy is associated with a number of adverse effects. 1 3TC, a nucleoside analog of L-deoxycitydine, and ADV, a phosphonate nucleotide analog of adenosine monophosphate, target the HBV reverse transcriptase (RT) activity, thus inhibiting viral replication. They are both powerful antivirals, and monotherapy with 3TC or ADV results in significant improvement in virological, biochemical, and histological status in most of the patients. [2][3][4][5] However, long-term 3TC or ADV monotherapy leads to the emergence of drug-resistant HBV strains. 3TC-resistance increases at a rate of approximately 20% of patients per year, to reach approximately 70% of treated patients after 4 years. 6,7 Mutations conferring resistance to 3TC have been mapped in the conserved YMDD motif within the C domain of the viral RT (M204I/V). [8][9][10] They are frequently associated with compensatory mutations in the conserved B domain (V173L, L180M) that restore

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 ␣-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) ␣-glucosidases. We demonstrate the lack of correlation between the ability of long-alkyl-chain DNJ derivatives to inhibit ER ␣-glucosidases and their antiviral effect, ruling out ER ␣-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.Iminosugar derivatives containing the glucose analogue deoxynojirimycin (DNJ) exert antiviral effects against viruses of different families, including Human immunodeficiency virus (HIV) (9-12, 16), Hepatitis B virus (HBV) (3,18,19), Woodchuck hepatitis virus (4), Bovine viral diarrhea virus (BVDV) (29), and Dengue virus (8). The antiviral effects were either proven (10, 11) or assumed to be associated with the inhibitory action of DNJ-containing iminosugar derivatives on enzymes in the endoplasmic reticulum (ER), ␣-glucosidases I and II. DNJ, a ring-nitrogen-containing and unmetabolizable glucose analogue, competitively binds to these enzymes and prevents them from performing the stepwise removal of three glucose residues attached to the N-linked glycans carried by newly synthesized polypeptides. This in turn prevents these polypeptides from interacting with the ER chaperones calnexin and calreticulin, which bind to monoglucosylated glycoproteins (14). Interaction with these ER chaperones is crucial for the correct folding of some but not all glycoproteins (13,17,22). Potentially all viruses which encode glycoproteins that depend on calnexin interaction for prope...

The iminosugar N-butyldeoxynojirimycin (NB-DNJ), an endoplasmic reticulum ␣-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.Bovine viral diarrhea virus (BVDV) is a pestivirus member of the Flaviviridae family, which also comprises the genera Flavivirus and Hepacivirus (24). In the absence of an efficient cell culture system able to support hepatitis C virus (HCV) replication, BVDV has been adopted as a model and surrogate for HCV (1), as both viruses share molecular and virological features. HCV and BVDV are small, enveloped viruses with positive single-stranded RNA genomes of approximately 9,600 and 12,600 nucleotides, respectively. The polypeptide precursor is transcribed from a single large open reading frame and subsequently co-and posttranslationally processed into structural and nonstructural proteins. Most BVDV and HCV proteins are functionally homologous. The envelope glycoproteins E1 and E2 interact either noncovalently (HCV) (7) or through disulfide bonds (BVDV) (26, 28) to form a dimer, which has been proposed as the functional complex present on the surfaces of mature virions.We have previously shown that endoplasmic reticulum (ER) ␣-glucosidase inhibitors containing the glucose analogue deoxynojirimycin (DNJ) as the head group have a strong antiviral effect on BVDV (32). Castanospermine (CST), another ␣-glucosidase inhibitor, was shown to cause misfolding of recombinant HCV E1 and E2 glycoproteins expressed in BHK-21 cells and reduce their association into native dimers (5). Similarly, CST affected the morphogenesis and assembly of Dengue virus (DENV), another member of the Flaviviridae family (6).The ER ␣-glucosidases perform the stepwise removal of the three glucose residues on N-linked glycans attached to nascent polypeptides. This removal enables folding intermediates to associate with the lectin-like ER chaperones calnexin and calreticulin, which interact with monoglucosylated glycoproteins and which retain incompletely folded polypeptides and oligomers in the ER (2, 23). However, not all cellular proteins are...

Complete characterization of the biological properties of hepatitis B virus (HBV) variants requires the generation of full-length genomes. The aim of this study was to develop new tools for the efficient full-length genome amplification of virus from samples with low viral loads. Rolling circle amplification (RCA) was used to amplify full-length HBV genomes from both sera and liver biopsy samples from chronic HBV carriers. Serum-derived relaxed circular HBV DNA could be amplified only after completion and ligation of plus-strand DNA. Covalently closed circular DNA (cccDNA) from liver biopsies could be amplified directly from as few as 13 copies, using RCA, followed by a full-length HBV PCR. Three serial liver biopsy samples were obtained from a lamivudine-resistant patient who cleared detectable serum HBV after adefovir dipivoxil was added to the lamivudine therapy and then seroconverted to anti-HBs. Only the genomes from the last biopsy specimen obtained after the emergence of lamivudine resistance contained the lamivudine resistance-associated mutations rtL180M and rtM204V ("rt" indicates reverse transcriptase domain). Defective genomes were also found in this biopsy sample. Genomes cloned from the liver biopsy specimens were transfected into HuH7 cells to study their replication competence and their susceptibility to lamivudine. RCA is a powerful tool for amplifying full-length HBV genomes and will be especially useful for the study of occult or inactive HBV infections and patients undergoing antiviral treatment. It can also be used to probe HBV cccDNA, the crucial intermediate in viral persistence and the archive of resistance mutations. Chronic hepatitis B virus (HBV) infection is widespread,with an estimated 370 million carriers worldwide (1). Despite constraints imposed by a complex genetic architecture involving overlapping genes, the virus is highly variable, with eight known genotypes designated A to H (20).The complete molecular and biological characterization of HBV variants requires the isolation of full-length genomes. This has been done classically by amplifying overlapping fragments from serum-derived relaxed circular genomes (RC-DNA) (4, 21). However, the discontinuous structure of RC-DNA means that the amplification of at least one of the fragments can be problematical. Increasing the number of PCRs ineluctably augments the probability of incorporating PCR errors, and further steps are necessary to construct a full-length HBV genome. Günther et al. have developed an elegant method for amplifying full-length HBV genomes in a one-step PCR (7). However, this method requires serum with viral loads of Ͼ10 4 copies/ml of HBV DNA. In typical chronic HBV infections, viral loads usually range from 10 6 to 10 10 copies/ml. However, for inactive carriers (individuals who are hepatitis B surface antigen positive [HBsAg ϩ ]) with minimal virus replication and for occult HBV-infected patients (HBsAg Ϫ ), serum HBV DNA levels are usually very much lower. Consequently, few occult HBV infections have been studi...