Norica Branza-Nichita

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.

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.

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (−) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (−) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.

Regulation of Hepatitis B Virus Infection by Rab5, Rab7, and the Endolysosomal Compartment

ABSTRACT Despite important progress toward deciphering human hepatitis B virus (HBV) entry into host cells, many aspects of the early steps of the life cycle remained completely obscure. Following endocytosis, HBV must travel through the complex network of the endocytic pathway to reach the cell nucleus and initiate replication. In addition to guiding the viral particles to the replication site, the endosomal vesicles may play a crucial role in infection, providing the appropriate environment for virus uncoating and nucleocapsid release. In this work, we investigated the trafficking of HBV particles internalized in permissive cells. Expression of key Rab proteins, involved in specific pathways leading to different intracellular locations, was modulated in HepaRG cells, using a stable and inducible short hairpin RNA (shRNA) expression system. The trafficking properties of the newly developed cells were demonstrated by confocal microscopy and flow cytometry using specific markers. The results showed that HBV infection strongly depends on Rab5 and Rab7 expression, indicating that HBV transport from early to mature endosomes is required for a step in the viral life cycle. This may involve reduction of disulfide bond-linked envelope proteins, as alteration of the redox potential of the endocytic pathway resulted in inhibition of infection. Subcellular fractionation of HBV-infected cells showed that viral particles are further transported to lysosomes. Intriguingly, infection was not dependent on the lysosomal activity, suggesting that trafficking to this compartment is a “dead-end” route. Together, these data add to our understanding of the HBV-host cell interactions controlling the early stages of infection.

Effects of interferon, ribavirin, and iminosugar derivatives on cells persistently infected with noncytopathic bovine viral diarrhea virus.

ABSTRACT Persistent infection with hepatitis C virus (HCV) is a major cause of chronic hepatitis in humans. In chronic carriers, the viral infection induces liver damage that predisposes the patient for cirrhosis and can lead to hepatocellular carcinoma. Current chemotherapies are limited to alpha interferon (IFN-α) used either alone or in combination with ribavirin (RBV). In addition to its limited efficacy, this treatment is frequently poorly tolerated because of its side effects. The urgently needed development of new drugs is made difficult by the lack of an in vitro or in vivo infectivity model, and no cell line has been found so far to reliably and reproducibly support HCV infection. For this reason, the closely related pestivirus bovine viral diarrhea virus (BVDV) has sometimes been used as a surrogate in vitro infectivity model. In this study we used an MDBK cell line persistently infected with noncytopathic BVDV to assess the antiviral effect of IFN-α and RBV, the two drugs currently in clinical use against HCV. The same system was then used to evaluate the potential of two classes of iminosugar derivates to clear noncytopathic BVDV infection from MDBK cells. We show that treatment with long-alkyl-chain deoxynojirimycin derivatives, which are inhibitors of the endoplasmic reticulum (ER)-resident α-glucosidases, can greatly reduce the amount of secreted enveloped viral RNA. Long-alkyl-chain deoxygalactonojirimycin derivatives, which do not inhibit ER α-glucosidases, were less potent but still more effective in this system than IFN-α or ribavirin.

Activation of ERAD Pathway by Human Hepatitis B Virus Modulates Viral and Subviral Particle Production

Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of enveloped DNA viruses. It was previously shown that HBV can induce endoplasmic reticulum (ER) stress and activate the IRE1-XBP1 pathway of the unfolded protein response (UPR), through the expression of the viral regulatory protein X (HBx). However, it remained obscure whether or not this activation had any functional consequences on the target genes of the UPR pathway. Of these targets, the ER degradation-enhancing, mannosidase-like proteins (EDEMs) are thought to play an important role in relieving the ER stress during UPR, by recognizing terminally misfolded glycoproteins and delivering them to the ER-associated degradation (ERAD). In this study, we investigated the role of EDEMs in the HBV life-cycle. We found that synthesis of EDEMs (EDEM1 and its homologues, EDEM2 and EDEM3) is significantly up-regulated in cells with persistent or transient HBV replication. Co-expression of the wild-type HBV envelope proteins with EDEM1 resulted in their massive degradation, a process reversed by EDEM1 silencing. Surprisingly, the autophagy/lysosomes, rather than the proteasome were involved in disposal of the HBV envelope proteins. Importantly, inhibition of the endogenous EDEM1 expression in HBV replicating cells significantly increased secretion of both, enveloped virus and subviral particles. This is the first report showing that HBV activates the ERAD pathway, which, in turn, reduces the amount of envelope proteins, possibly as a mechanism to control the level of virus particles in infected cells and facilitate the establishment of chronic infections.

Cholesterol depletion of hepatoma cells impairs hepatitis B virus envelopment by altering the topology of the large envelope protein.

ABSTRACT Previous reports have shown that cholesterol depletion of the membrane envelope of the hepatitis B virus (HBV) impairs viral infection of target cells. A potential function of this lipid in later steps of the viral life cycle remained controversial, with secretion of virions and subviral particles (SVP) being either inhibited or not affected, depending on the experimental approach employed to decrease the intracellular cholesterol level. This work addressed the role of host cell cholesterol on HBV replication, assembly, and secretion, using an alternative method to inhibition of the enzymes involved in the biosynthesis pathway. Growing HBV-producing cells with lipoprotein-depleted serum (LPDS) resulted in an important reduction of the amount of cholesterol within 24 h of treatment (about 40%). Cell exposure to chlorpromazine, an inhibitor of the clathrin-mediated pathway used by the low-density lipoprotein receptor for endocytosis, also impacted the cholesterol level; however, this level of inhibition was not achievable when the synthesis inhibitor lovastatin was used. HBV secretion was significantly inhibited in cholesterol-depleted cells (by ∼80%), while SVP release remained unaffected. The viral DNA genome accumulated in LPDS-treated cells in a time-dependent manner. Specific immunoprecipitation of nucleocapsids and mature virions revealed an increased amount of naked nucleocapsids, while synthesis of the envelope proteins occurred as normally. Following analysis of the large envelope protein conformation in purified microsomes, we concluded that cholesterol is important in maintaining the dual topology of this polypeptide, which is critical for viral envelopment.

Modulation of the unfolded protein response by the human hepatitis B virus

During productive viral infection the host cell is confronted with synthesis of a vast amount of viral proteins which must be folded, quality controlled, assembled and secreted, perturbing the normal function of the endoplasmic reticulum (ER). To counteract the ER stress, cells activate specific signaling pathways, designated as the unfolded proteins response (UPR), which essentially increase their folding capacity, arrest protein translation, and degrade the excess of misfolded proteins. This cellular defense mechanism may, in turn, affect significantly the virus life-cycle. This review highlights the current understanding of the mechanisms of the ER stress activation by Human Hepatitis B virus (HBV), a deadly pathogen affecting more than 350 million people worldwide. Further discussion addresses the latest discoveries regarding the adaptive strategies developed by HBV to manipulate the UPR for its own benefits, the controversies in the field and future perspectives.

Comparative Proteomics Reveals Novel Components at the Plasma Membrane of Differentiated HepaRG Cells and Different Distribution in Hepatocyte- and Biliary-Like Cells

Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells.

Azo-polysiloxanes as new supports for cell cultures

The paper introduces a new class of materials with azo-polysiloxanic structure bearing the property to generate nano-structured surfaces by laser irradiation. The ability to modulate the optical response of the film, through a modification of the polymer chemical structure, has been investigated. The azo-materials were tested for their ability to support cell adhesion and growth, with very promising results. A future use of these materials as growth support in cell cultures is of great interest, due to an easy, one step-method to generate the surface relief grating and to the possibility to introduce a large range of chemical modifications due to the presence of the chlorobenzyl groups in the polymeric side-chain.