Kiyoshi Himmelsbach

The moss Physcomitrella patens contains cyclopentenones but no jasmonates: mutations in allene oxide cyclase lead to reduced fertility and altered sporophyte morphology.

• Two cDNAs encoding allene oxide cyclases (PpAOC1, PpAOC2), key enzymes in the formation of jasmonic acid (JA) and its precursor (9S,13S)-12-oxo-phytodienoic acid (cis-(+)-OPDA), were isolated from the moss Physcomitrella patens. • Recombinant PpAOC1 and PpAOC2 show substrate specificity against the allene oxide derived from 13-hydroperoxy linolenic acid (13-HPOTE); PpAOC2 also shows substrate specificity against the allene oxide derived from 12-hydroperoxy arachidonic acid (12-HPETE). • In protonema and gametophores the occurrence of cis-(+)-OPDA, but neither JA nor the isoleucine conjugate of JA nor that of cis-(+)-OPDA was detected. • Targeted knockout mutants for PpAOC1 and for PpAOC2 were generated, while double mutants could not be obtained. The ΔPpAOC1 and ΔPpAOC2 mutants showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis.

Hepatitis C Virus Infection Sensitizes Human Hepatocytes to TRAIL-Induced Apoptosis in a Caspase 9-Dependent Manner

Apoptosis of infected cells represents a key host defense mechanism against viral infections. The impact of apoptosis on the elimination of hepatitis C virus (HCV)-infected cells is poorly understood. The TRAIL has been implicated in the death of liver cells in hepatitis-infected but not in normal liver cells. To determine the impact of TRAIL on apoptosis of virus-infected host cells, we studied TRAIL-induced apoptosis in a tissue culture model system for HCV infection. We demonstrated that HCV infection sensitizes primary human hepatocytes and Huh7.5 hepatoma cells to TRAIL induced apoptosis in a dose- and time-dependent manner. Mapping studies identified the HCV nonstructural proteins as key mediators of sensitization to TRAIL. Using a panel of inhibitors targeting different apoptosis pathways, we demonstrate that sensitization to TRAIL is caspase-9 dependent and mediated in part via the mitochondrial pathway. Sensitization of hepatocytes to TRAIL-induced apoptosis by HCV infection represents a novel antiviral host defense mechanism that may have important implications for the pathogenesis of HCV infection and may contribute to the elimination of virus-infected hepatocytes.

New aspects of an anti-tumour drug: sorafenib efficiently inhibits HCV replication

Background and aims: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and is associated with significant morbidity and mortality. Since there is evidence for an interaction of NS5A with c-Raf we studied whether the c-Raf inhibitor sorafenib affects HCV replication. Methods: HCV replicating HuH7.5 cells were treated with sorafenib and examined for HCV RNA titres by northern blotting or real time polymerase chain reaction (PCR), for core, NS3 and NS5A expression by immunostaining, and for replication by luciferase reporter assays. Results: Here we demonstrate that in cells replicating infectious HCV particles, NS5A recruits c-Raf to the replicon complex resulting in the activation of c-Raf. Therefore, we studied the effect of inhibition of c-Raf on HCV replication using the anti-tumour drug sorafenib that is known to inhibit c-Raf with high specificity. Sorafenib efficiently blocks HCV replication and viral gene expression. In addition, in HCV-replicating cells sorafenib decreased the hyperphosphorylated form of NS5A and resulted in the formation of additional hypophosphorylated forms. Further, sorafenib caused a rapid dissociation of lipid droplets. We provide evidence that the antiviral effect of sorafenib indeed is caused by inhibition of c-Raf. By contrast, inhibition of targets downstream of c-Raf or inhibition of tyrosine kinases by sunitinib did not affect HCV replication. Conclusion: Our data demonstrate that the well-characterised anti-tumour drug sorafenib efficiently blocks HCV replication in vitro. This novel effect of sorafenib should be further explored as an antiviral strategy for patients with chronic HCV infection.

Hepatitis C virus impairs the induction of cytoprotective Nrf2 target genes by delocalization of small Maf proteins

The expression of a variety of cytoprotective genes is regulated by short cis-acting elements in their promoters, called antioxidant response elements (AREs). A central regulator of ARE-mediated gene expression is the NF-E2-related factor 2 (Nrf2). Nrf2/ARE-regulated genes are crucial for the maintenance of cellular integrity. Hepatitis C virus inhibits the induction of ARE-regulated genes, but neither induction nor inhibition of ARE-regulated gene expression affects HCV replication directly. In HCV-replicating cells the core protein triggers the delocalization of sMaf proteins from the nucleus to the replicon complex. Here sMafs bind to NS3. The extranuclear sMaf proteins prevent Nrf2 from entry in the nucleus and thereby inhibit the induction of Nrf2/ARE-regulated genes. This results in the decreased expression of cytoprotective genes. Consistent with this finding, the elimination of ROI is impaired in HCV-replicating cells as demonstrated by elevated protein oxidation or 8-OH-dG formation, reflecting DNA damage. In conclusion, these data identified a novel mechanism of Nrf2 regulation and suggest that the HCV-dependent inhibition of Nrf2/ARE-regulated genes confers to the HCV-associated pathogenesis by elevation of intracellular ROI that affect integrity of the host genome and regenerative processes.

Hepatitis B Virus Induces Expression of Antioxidant Response Element-regulated Genes by Activation of Nrf2

The expression of a variety of cytoprotective genes is regulated by short cis-acting elements in their promoters, called antioxidant response elements (AREs). A central regulator of ARE-mediated gene expression is the NF-E2-related factor 2 (Nrf2). Human hepatitis B virus (HBV) induces a strong activation of Nrf2/ARE-regulated genes in vitro and in vivo. This is triggered by the HBV-regulatory proteins (HBx and LHBs) via c-Raf and MEK. The Nrf2/ARE-mediated induction of cytoprotective genes by HBV results in a better protection of HBV-positive cells against oxidative damage as compared with control cells. Furthermore, there is a significantly increased expression of the Nrf2/ARE-regulated proteasomal subunit PSMB5 in HBV-positive cells that is associated with a decreased level of the immunoproteasome subunit PSMB5i. In accordance with this finding, HBV-positive cells display a higher constitutive proteasome activity and a decreased activity of the immunoproteasome as compared with control cells even after interferon α/γ treatment. The HBV-dependent induction of Nrf2/ARE-regulated genes might ensure survival of the infected cell, shape the immune response to HBV, and thereby promote establishment of the infection.

TIP47 plays a crucial role in the life cycle of hepatitis C virus

BACKGROUND & AIMSnHepatitis C virus (HCV) replication/morphogenesis takes place at the membranous web. Viral genome replication occurs in replicon complexes on the cytoplasmic face of the ER whereas HCV assembly is located on the surface of lipid droplets (LDs). This raises the question about targeting of de novo synthesized viral genomes from the replicon complex to LDs and cellular proteins involved in this process such as the LD-associated protein TIP47, also known as cytoplasmic sorting factor.nnnMETHODSnViral replication was studied in HuH7.5 cells using the infectious HCV JHF1 culture system. Proteome analysis was performed by 2D gel electrophoresis and mass spectrometry. Expression of target genes was modulated by siRNA or lentiviral transduction. Confocal microscopy was performed for analysis of subcellular compartments. Protein/protein interactions were studied by co-immunoprecipitations, affinity chromatography, and yeast two-hybrid screens.nnnRESULTSnProteome based analysis revealed that HCV replicating cells contain less TIP47 compared to control cells. However, expression analyses demonstrated an increased TIP47 expression in HCV replicating cells. TIP47 binds to RNA-loaded NS5A. Mapping of the binding domain revealed that NS5A binds to the N-terminal PAT domain of TIP47. Overexpression of TIP47 increases the amount of released viruses, while silencing of TIP47 decreases the amount of released infectious particles. Complete knockdown of TIP47 expression abolishes virus replication.nnnCONCLUSIONSnTIP47 plays an essential role in the HCV life cycle.

TIP47 is associated with the Hepatitis C virus and its interaction with Rab9 is required for release of viral particles

Hepatitis C virus (HCV) morphogenesis and release are closely linked to lipid metabolism. It has been described recently by our group that TIP47 plays an essential role for the targeting of the NS5A-complexed RNA genome from the replicon complex to the lipid droplet. Moreover, apolipoprotein (apo) E was found to be associated with the viral particle. In light of the fact, that TIP47 harbors an apoE like domain and has a high affinity to lipoproteins, the interaction of TIP47 with the viral particle and the potential relevance for the release of the viral particle were investigated. Coimmunoprecipitations and electron microscopy analysis using immunogold labeling revealed that TIP47 binds to the viral particle and stays associated with the released HCV particle. Silencing of the TIP47 binding partner Rab9 by lentiviral transduction abolishes the viral replication. However, destruction of TIP47-Rab9 interactions by deletion/mutation of the Rab9 binding does not abolish the genome replication domain but prevents the release of HCV particles. The binding of these TIP47 mutants to the viral particle is not affected by destruction of the Rab9 binding domain. Moreover, we found that these TIP47 mutants lacking the binding site for Rab9 misdirect the de novo synthesized viral particles to the autophagosomal/lysosomal compartment where the particles are degraded. From this we conclude that the Rab9-complexed TIP47 plays an essential role for the proper release of hepatitis C viral particles.

Localization determines function: N-terminally truncated NS5A fragments accumulate in the nucleus and impair HCV replication

BACKGROUND/AIMSnThe Hepatitis C Virus (HCV) nonstructural protein 5A (NS5A) is an essential part of the ER-localized HCV-replicon complex. Although NS5A harbours a conserved NLS in its C-terminal domain, NS5A is associated with the cytoplasmic face of the ER by an amphipathic helix close to its N-terminus.nnnMETHODSnIntracellular distribution of NS5A in HCV replicating cells was analyzed by confocal microscopy and subcellular fractionation. The effect on HCV replication was analyzed using the JFH-1-based infection/replication system.nnnRESULTSnDuring viral life cycle N-terminally truncated NS5A fragments are caspase-dependent formed that lack the ER-attachment signal and are localized within the nucleus. These N-terminally truncated fragments inhibit HCV replication. If their formation is blocked by inhibition of caspases HCV replication is increased. The C-terminal domain of NS5A binds to c-Raf and thereby localizes it to the replicon complex. This interaction is essential for HCV replication. The N-terminally truncated NS5A fragments are still able to bind c-Raf. However, due to their nuclear localization they withdraw c-Raf from the replicon complex into the nucleus resulting in an impaired HCV replication.nnnCONCLUSIONSnFormation of N-terminally truncated NS5A fragments could represent a mechanism to regulate HCV replication by withdrawal of essential factors from the replicon complex.

Identification of α-taxilin as an essential factor for the life cycle of hepatitis B virus

BACKGROUND & AIMSnα-taxilin was identified as binding partner of syntaxins and is supposed to regulate vesicular trafficking. However, the physiological functions of α-taxilin and its potential relevance for the life cycle of hepatitis B virus (HBV) are still poorly understood.nnnMETHODSnTransfected hepatoma cells, infected primary human hepatocytes, and liver tissue of HBV-infected patients were used to study the expression of α-taxilin. Subcellular localization and colocalization were analyzed by confocal laser scanning microscopy (CLSM). Protein-protein interactions were further investigated by co-immunoprecipitations. Silencing of α-taxilin expression was performed by lentiviral gene transfer.nnnRESULTSnHBV producing cells show a significant higher level of α-taxilin. HBV induces α-taxilin expression, by its regulatory proteins HBx and LHBs via c-Raf. This indicates that α-taxilin is essential for the release of HBV particles. CLSM and co-immunoprecipitations demonstrated that the PreS1PreS2 domain of LHBs interacts with α-taxilin. α-taxilin harbors a YXXL motif that represents a classic late domain. In accordance with this, it was found by co-immunoprecipitations that α-taxilin interacts with the ESCRT I component tsg101. CLSM revealed that a fraction of α-taxilin colocalizes with LHBs and tsg101.nnnCONCLUSIONSnα-taxilin plays an essential role for release of HBV-DNA containing particles. It might act as an adapter that binds, on the one hand, to LHBs and, on the other hand, to tsg101 and thereby helps recruit the ESCRT machinery to the viral envelope proteins.

Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

Deoxynucleotide triphosphates (dNTPs) are essential for efficient hepatitis B virus (HBV) replication. Here, we investigated the influence of the restriction factor SAMHD1, a dNTP hydrolase (dNTPase) and RNase, on HBV replication. We demonstrated that silencing of SAMHD1 in hepatic cells increased HBV replication, while overexpression had the opposite effect. SAMHD1 significantly affected the levels of extracellular viral DNA as well as intracellular reverse transcription products, without affecting HBV RNAs or cccDNA. SAMHD1 mutations that interfere with the dNTPase activity (D137N) or in the catalytic center of the histidine-aspartate (HD) domain (D311A), and a phospho-mimetic mutation (T592E), abrogated the inhibitory activity. In contrast, a mutation diminishing the potential RNase but not dNTPase activity (Q548A) and a mutation disabling phosphorylation (T592A) did not affect antiviral activity. Moreover, HBV restriction by SAMHD1 was rescued by addition of deoxynucleosides. Although HBV infection did not directly affect protein level or phosphorylation of SAMHD1, the virus upregulated intracellular dATPs. Interestingly, SAMHD1 was dephosphorylated, thus in a potentially antiviral-active state, in primary human hepatocytes. Furthermore, SAMHD1 was upregulated by type I and II interferons in hepatic cells. These results suggest that SAMHD1 is a relevant restriction factor for HBV and restricts reverse transcription through its dNTPase activity.