David Durantel

Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.

Clearance of Chronic Virus The family of mRNA-editing enzymes, APOBEC, restricts hepatitis B virus (HBV) replication. Lucifora et al. (p. 1221, published online 20 February; see the Perspective by Shlomai and Rice) provide evidence that specific APOBECs mediate the anti-HBV effects of host cytokines, which in turn apparently induce nuclear deaminase activity without damaging host cells. Thus, there may be potential in these findings for developing a therapeutic route to curing chronic HBV infection. Cytokine induction renders viral DNA vulnerable and eliminates infection. Current antiviral agents can control but not eliminate hepatitis B virus (HBV), because HBV establishes a stable nuclear covalently closed circular DNA (cccDNA). Interferon-α treatment can clear HBV but is limited by systemic side effects. We describe how interferon-α can induce specific degradation of the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-β receptor activation as a therapeutic alternative. Interferon-α and lymphotoxin-β receptor activation up-regulated APOBEC3A and APOBEC3B cytidine deaminases, respectively, in HBV-infected cells, primary hepatocytes, and human liver needle biopsies. HBV core protein mediated the interaction with nuclear cccDNA, resulting in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that prevented HBV reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases—for example, by lymphotoxin-β receptor activation—allows the development of new therapeutics that, in combination with existing antivirals, may cure hepatitis B.

Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases

Nucleoside analogues have been in clinical use for almost 50 years and have become cornerstones of treatment for patients with cancer or viral infections. The approval of several additional drugs over the past decade demonstrates that this family still possesses strong potential. Here, we review new nucleoside analogues and associated compounds that are currently in preclinical or clinical development for the treatment of cancer and viral infections, and that aim to provide increased response rates and reduced side effects. We also highlight the different approaches used in the development of these drugs and the potential of personalized therapy.

Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir

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, β‐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. However, other nucleos(t)ide analogs in development showed an antiviral activity against this multiresistant strain in vitro. This provides a rationale for the clinical evaluation of de novo combination therapies. (HEPATOLOGY 2005;41:1391‐1398.)

Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection

BACKGROUND & AIMS The molecular biology of hepatitis B virus (HBV) has been extensively studied but the exact role of the hepatitis B X protein (HBx) in the context of natural HBV infections remains unknown. METHODS Primary human hepatocytes and differentiated HepaRG cells allowing conditional trans complementation of HBx were infected with wild type (HBV(wt)) or HBx deficient (HBV(x-)) HBV particles and establishment of HBV replication was followed. RESULTS We observed that cells inoculated with HBx-deficient HBV particles (HBV(x-)) did not lead to productive HBV infection contrary to cells inoculated with wild type HBV particles (HBV(wt)). Although equal amounts of nuclear covalently closed circular HBV-DNA (cccDNA) demonstrated comparable uptake and nuclear import, active transcription was only observed from HBV(wt) genomes. Trans-complementation of HBx was able to rescue transcription from the HBV(x-) genome and led to antigen and virion secretion, even weeks after infection. Constant expression of HBx was necessary to maintain HBV antigen expression and replication. Finally, we demonstrated that HBx is not packaged into virions during assembly but is expressed after infection within the new host cell to allow epigenetic control of HBV transcription from cccDNA. CONCLUSIONS Our results demonstrate that HBx is required to initiate and maintain HBV replication and highlight HBx as the key regulator during the natural infection process.

Towards an HBV cure: state-of-the-art and unresolved questions—report of the ANRS workshop on HBV cure

HBV infection is a major cause of liver cirrhosis and hepatocellular carcinoma. Although HBV infection can be efficiently prevented by vaccination, and treatments are available, to date there is no reliable cure for the >240 million individuals that are chronically infected worldwide. Current treatments can only achieve viral suppression, and lifelong therapy is needed in the majority of infected persons. In the framework of the French National Agency for Research on AIDS and Viral Hepatitis ‘HBV Cure’ programme, a scientific workshop was held in Paris in June 2014 to define the state-of-the-art and unanswered questions regarding HBV pathobiology, and to develop a concerted strategy towards an HBV cure. This review summarises our current understanding of HBV host-interactions leading to viral persistence, as well as the roadblocks to be overcome to ultimately address unmet medical needs in the treatment of chronic HBV infection.

Study of the Mechanism of Antiviral Action of Iminosugar Derivatives against Bovine Viral Diarrhea Virus

ABSTRACT The glucose-derived iminosugar derivatives N-butyl- andN-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 α-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) α-glucosidases. We demonstrate the lack of correlation between the ability of long-alkyl-chain DNJ derivatives to inhibit ER α-glucosidases and their antiviral effect, ruling out ER α-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.

Receptor Complementation and Mutagenesis Reveal SR-BI as an Essential HCV Entry Factor and Functionally Imply Its Intra- and Extra-Cellular Domains

HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

Control of hepatitis B virus replication by innate response of HepaRG cells

Hepatitis B virus (HBV) is currently viewed as a stealth virus that does not elicit innate immunity in vivo. This assumption has not yet been challenged in vitro because of the lack of a relevant cell culture system. The HepaRG cell line, which is physiologically closer to differentiated hepatocytes and permissive to HBV infection, has opened new perspectives in this respect.HBV baculoviruses were used to initiate an HBV replication in both HepG2 and HepaRG cells. To monitor HBV replication, the synthesis of encapsidated DNA, and secretion of hepatitis B surface antigen (HBsAg), was respectively analyzed by southern blot and enzyme‐linked immunosorbent assay. The induction of a type I interferon (IFN) response was monitored by targeted quantitative reverse transcription polymerase chain reaction (qRT‐PCR), low‐density arrays, and functional assays. The invalidation of type I IFN response was obtained by either antibody neutralization or RNA interference. We demonstrate that HBV elicits a strong and specific innate antiviral response that results in a noncytopathic clearance of HBV DNA in HepaRG cells. Challenge experiment showed that transduction with Bac‐HBV‐WT, but not with control baculoviruses, leads to this antiviral response in HepaRG cells, whereas no antiviral response is observed in HepG2 cells. Cellular gene expression analyses showed that IFN‐β and other IFN‐stimulated genes were up‐regulated in HepG2 and HepaRG cells, but not in cells transduced by control baculoviruses. Interestingly, a rescue of viral replication was observed when IFN‐β action was neutralized by antibodies or RNA interference of type I IFN receptor. Conclusion: Our data suggest that a strong HBV replication is able to elicit a type I IFN response in HepaRG‐transduced cells. (HEPATOLOGY 2009.)

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

ABSTRACT The iminosugar N-butyldeoxynojirimycin (NB-DNJ), an endoplasmic reticulum α-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 ofNB-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.

Hepatitis B Virus Requires Intact Caveolin-1 Function for Productive Infection in HepaRG Cells

ABSTRACT Investigation of the entry pathways of hepatitis B virus (HBV), a member of the family Hepadnaviridae, has been hampered by the lack of versatile in vitro infectivity models. Most concepts of hepadnaviral infection come from the more robust duck HBV system; however, whether the two viruses use the same mechanisms to invade target cells is still a matter of controversy. In this study, we investigate the role of an important plasma membrane component, caveolin-1 (Cav-1), in HBV infection. Caveolins are the main structural components of caveolae, plasma membrane microdomains enriched in cholesterol and sphingolipids, which are involved in the endocytosis of numerous ligands and complex signaling pathways within the cell. We used the HepaRG cell line permissive for HBV infection to stably express dominant-negative Cav-1 and dynamin-2, a GTPase involved in vesicle formation at the plasma membrane and other organelles. The endocytic properties of the newly established cell lines, designated HepaRGCav-1, HepaRGCav-1Δ1-81, HepaRGDyn-2, and HepaRGDyn-2K44A, were validated using specific markers for different entry routes. The cells maintained their properties during cell culture, supported differentiation, and were permissive for HBV infection. The levels of both HBV transcripts and antigens were significantly decreased in cells expressing the mutant proteins, while viral replication was not directly affected. Chemical inhibitors that specifically inhibit clathrin-mediated endocytosis had no effect on HBV infection. We concluded that HBV requires a Cav-1-mediated entry pathway to initiate productive infection in HepaRG cells.