Jacques Le Seyec

Infection of a human hepatoma cell line by hepatitis B virus.

Among numerous established human hepatoma cell lines, none has been shown susceptible to hepatitis B virus (HBV) infection. We describe here a cell line, called HepaRG, which exhibits hepatocyte-like morphology, expresses specific hepatocyte functions, and supports HBV infection as well as primary cultures of normal human hepatocytes. Differentiation and infectability are maintained only when these cells are cultured in the presence of corticoids and dimethyl sulfoxide. The specificity of this HBV infection model was ascertained by both the neutralization capacity of HBV-envelope protein-specific antibodies and the competition with an envelope-derived peptide. HepaRG cells therefore represent a tool for deciphering the mechanism of HBV entry. Moreover, their close resemblance to normal human hepatocytes makes them suitable for many applications including drug metabolism studies.

Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening

The entry pathway of the hepatitis C virus (HCV), a major human pathogen, into the cell is incompletely defined. To better characterize this viral life cycle stage, we screened a small interfering RNA library dedicated to the membrane trafficking and remodeling with the infection model of Huh‐7.5.1 cells by HCV pseudoparticles (HCVpp). Results showed that the down‐regulation of different factors implied in clathrin‐mediated endocytosis (CME) inhibits HCVpp cell infection. In addition, knockdown of the phosphatidylinositol 4‐kinase type III‐α (PI4KIIIα) prevented infection by HCVpp or by cell‐culture grown JFH‐1‐based HCV. Moreover, the replication activity of an HCV replicon was also affected by the PI4KIIIα knockdown. Additional investigations on the different members of the PI4K family revealed that the presence of PI4KIIIβ in the host cells influenced their susceptibility to HCVpp infection and their capacity to sustain the HCV replication. The PI4KIII involvement during the HCV life cycle seemed to occur by other ways than the control of the CME or of the membranous expression of HCV receptors. Finally, our library screening completed data on the CME‐dependant entry route of HCV and identified 2 kinases, PI4KIIIα and β,as relevant potential therapeutic targets.—Trotard, M., Lepère‐Douard, C., Régeard, M., Piquet‐Pellorce, C., Lavillette, D., Cosset, F.‐L., Gripon, P., Le Seyec, J. Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening. FASEBJ. 23, 3780–3789 (2009). www.fasebj.org

The Hepatitis C Virus NS2 Protein Is an Inhibitor of CIDE-B-induced Apoptosis

Chronic hepatitis C virus (HCV) infection frequently leads to liver cancer. To determine the viral factor(s) potentially involved in viral persistence, we focused our work on NS2, a viral protein of unknown function. To assign a role for NS2, we searched for cellular proteins that interact with NS2. Performing a two-hybrid screen on a human liver cDNA library, we found that NS2 interacted with the liver-specific pro-apoptotic CIDE-B protein. Binding specificity of NS2 for CIDE-B was confirmed by cell-free assays associated with colocalization studies and coprecipitation experiments on human endogenous CIDE-B. CIDE-B, a member of the novel CIDE family of apoptosis-inducing factors, has been reported to show strong cell death-inducing activity in its C-terminal domain. We show that this CIDE-B killing domain is involved in the NS2 interaction. NS2 binding was sufficient to inhibit CIDE-B-induced apoptosis because an NS2 deletion mutant unable to interact with CIDE-B in vitrolost its capacity to interfere with CIDE-B cell death activity. Although it has been reported that CIDE-B-induced apoptosis is characterized by mitochondrial localization, the precise apoptotic mechanism remained unknown. Here, we show that CIDE-B induced cell death in a caspase-dependent manner through cytochromec release from mitochondria. Furthermore, we found that NS2 counteracted the cytochrome c release induced by CIDE-B.In vivo, the CIDE-B protein level was extremely low in adenovirus-infected transgenic mice expressing the HCV polyprotein compared with that in wild-type mice. We suggest that NS2 interferes with the CIDE-B-induced death pathway and participates in HCV strategies to subvert host cell defense.

Hepatitis C Virus NS2 Protein Is Phosphorylated by the Protein Kinase CK2 and Targeted for Degradation to the Proteasome

ABSTRACT Hepatitis C virus (HCV) nonstructural 2 (NS2) protein is a hydrophobic transmembrane protein, described to be involved in different functions, such as apoptosis inhibition and gene transcription modulation. We investigated here NS2 protein turnover and found that NS2 was rapidly degraded by the proteasome in different cell lines, as in primary human hepatocytes. Since posttranslational modifications can influence protein turnover, we looked for potential phosphoacceptor sites in NS2. Computational sequence analysis in combination with screening of NS2 point mutants revealed that serine residue 168 was critical for degradation. In the quest of a protein kinase for NS2, we identified by sequence analysis that the serine residue 168 was part of a consensus casein kinase 2 (CK2) recognition site (S/TXXE). This motif was highly conserved since it could be found in the NS2 primary consensus sequences from all HCV genotypes. To verify whether CK2 is involved in NS2 phosphorylation, we showed by an in vitro kinase assay that CK2 phosphorylated NS2, as far as this CK2 motif was conserved. Interestingly, NS2 became resistant to protein degradation when the CK2 motif was modified by a single point mutation. Furthermore, inhibition of CK2 activity by curcumin decreased NS2 phosphorylation in vitro and stabilized NS2 expression in HepG2 cells. Finally, we showed in Huh-7.5 replicon cells that NS2, expressed in the context of the HCV polyprotein, was also sensitive to both proteasome-mediated degradation and CK2 inhibitor treatment. We suggest that NS2 is a short-lived protein whose degradation by the proteasome is regulated in a phosphorylation-dependent manner through the protein kinase CK2.

Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.

We investigated the specific role of the mitogen‐activated protein kinase (MAPK) extracellular signal‐regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long‐term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)‐dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF‐dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2‐dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2. Conclusion: Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long‐term survival and cell cycle regulation of hepatocytes. (HEPATOLOGY 2009.)

Target Gene Specificity of USF-1 Is Directed via p38-mediated Phosphorylation-dependent Acetylation

How transcription factors interpret the output from signal transduction pathways to drive distinct programs of gene expression is a key issue that underpins development and disease. The ubiquitously expressed basic-helix-loop-helix leucine zipper upstream stimulating factor-1 binds E-box regulatory elements (CANNTG) to regulate a wide number of gene networks. In particular, USF-1 is a key component of the tanning process. Following UV irradiation, USF-1 is phosphorylated by the p38 stress-activated kinase on threonine 153 and directly up-regulates expression of the POMC, MC1R, TYR, TYRP-1 and DCT genes. However, how phosphorylation on Thr-153 might affect the activity of USF-1 is unclear. Here we show that, in response to DNA damage, oxidative stress and cellular infection USF-1 is acetylated in a phospho-Thr-153-dependent fashion. Phospho-acetylated USF-1 is nuclear and interacts with DNA but displays altered gene regulatory properties. Phospho-acetylated USF-1 is thus proposed to be associated with loss of transcriptional activation properties toward several target genes implicated in pigmentation process and cell cycle regulation. The identification of this critical stress-dependent USF-1 modification gives new insights into understanding USF-1 gene expression modulation associated with cancer development.

The First Transmembrane Domain of the Hepatitis B Virus Large Envelope Protein Is Crucial for Infectivity

ABSTRACT The early steps of the hepatitis B virus (HBV) life cycle are still poorly understood. Indeed, neither the virus receptor at the cell surface nor the mechanism by which nucleocapsids are delivered to the cytosol of infected cells has been identified. Extensive mutagenesis studies in pre-S1, pre-S2, and most of the S domain of envelope proteins revealed the presence of two regions essential for HBV infectivity: the 77 first residues of the pre-S1 domain and a conformational motif in the antigenic loop of the S domain. In addition, at the N-terminal extremity of the S domain, a putative fusion peptide, partially overlapping the first transmembrane (TM1) domain and preceded by a PEST sequence likely containing several proteolytic cleavage sites, was identified. Since no mutational analysis of these two motifs potentially implicated in the fusion process was performed, we decided to investigate the ability of viruses bearing contiguous deletions or substitutions in the putative fusion peptide and PEST sequence to infect HepaRG cells. By introducing the mutations either in the L and M proteins or in the S protein, we demonstrated the following: (i) that in the TM1 domain of the L protein, three hydrophobic clusters of four residues were necessary for infectivity; (ii) that the same clusters were critical for S protein expression; and, finally, (iii) that the PEST sequence was dispensable for both assembly and infection processes.

The Translocation Motif of Hepatitis B Virus Envelope Proteins Is Dispensable for Infectivity

ABSTRACT The early events of hepatitis B virus (HBV) infection remain unclear. In 2006, Stoeckl et al. proposed a new entry mechanism involving a translocation motif (TLM) present in the pre-S2 domain of envelope proteins (L. Stoeckl, A. Funk, A. Kopitzki, B. Brandenburg, S. Oess, H. Will, H. Sirma, and E. Hildt, Proc. Natl. Acad. Sci. USA 103:6730-6734, 2006). After receptor binding and internalization into the endosomal compartment, this motif would allow the translocation of HBV particles through the endosomal membrane into the cytosol. In this study we have used two different mutated viruses containing a truncated TLM and showed their ability to infect human hepatocytes in primary culture, thus demonstrating the dispensability of the TLM for HBV infectivity.

Inhibition of hepatitis B virus production associated with high levels of intracellular viral DNA intermediates in iron-depleted HepG2.2.15 cells

BACKGROUNDS/AIMS The effects of iron-depletion on hepatitis B virus (HBV) replication were examined in HepG2.2.15 cells. METHODS Proliferating cells were iron-depleted with desferrioxamine (DFO), at 20 or 100 microM for 48 h. Levels of viral mRNAs, cytoplasmic DNA replicative intermediates and virion production were examined. A comparative study was performed with hydroxyurea, a specific inhibitor of ribonucleotide reductase. RESULTS In desferrioxamine treated cells, virion production is dramatically decreased, while viral replicative intermediates accumulate in the cytoplasm. DFO, like hydroxyurea, blocks cell cycle progression in the G1/S transition or S phase with a corresponding 2-fold increase of viral mRNAs. As expected, hydroxyurea leads to a strong reduction of virion production associated with low levels of intracellular replicative intermediates. CONCLUSIONS These results strongly suggest that iron depletion affects the HBV life cycle indirectly through the cell cycle arrest and directly through the inhibition of the viral DNA secretion. They also indicate the need to re-evaluate with caution the iron depletion protocols on HBV infected patients since a decrease of viral markers in the serum following iron-depletion may not reflect a decrease of viral replicative forms, but on the contrary, could be associated with active viral DNA synthesis.

RIPK1 protects from TNF- α -mediated liver damage during hepatitis

Cell death of hepatocytes is a prominent characteristic in the pathogenesis of liver disease, while hepatolysis is a starting point of inflammation in hepatitis and loss of hepatic function. However, the precise molecular mechanisms of hepatocyte cell death, the role of the cytokines of hepatic microenvironment and the involvement of intracellular kinases, remain unclear. Tumor necrosis factor alpha (TNF-α) is a key cytokine involved in cell death or survival pathways and the role of RIPK1 has been associated to the TNF-α-dependent signaling pathway. We took advantage of two different deficient mouse lines, the RIPK1 kinase dead knock-in mice (Ripk1K45A) and the conditional knockout mice lacking RIPK1 only in liver parenchymal cells (Ripk1LPC-KO), to characterize the role of RIPK1 and TNF-α in hepatitis induced by concanavalin A (ConA). Our results show that RIPK1 is dispensable for liver homeostasis under steady-state conditions but in contrast, RIPK1 kinase activity contributes to caspase-independent cell death induction following ConA injection and RIPK1 also serves as a scaffold, protecting hepatocytes from massive apoptotic cell death in this model. In the Ripk1LPC-KO mice challenged with ConA, TNF-α triggers apoptosis, responsible for the observed severe hepatitis. Mechanism potentially involves both TNF-independent canonical NF-κB activation, as well as TNF-dependent, but canonical NF-κB-independent mechanisms. In conclusion, our results suggest that RIPK1 kinase activity is a pertinent therapeutic target to protect liver against excessive cell death in liver diseases.