Matrix Conditions and KLF2-Dependent Induction of Heme Oxygenase-1 Modulate Inhibition of HCV Replication by Fluvastatin

Background & Aims

HMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication.

Methods

HO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue.

Results

All statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response in vitro. HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease.

Conclusions

Statin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease.     

Xylanase production by Aureobasidium pullulans on globe artichoke stem: Bioprocess optimization,enzyme characterization and application in saccharification of lignocellulosic biomass

Statistical optimization of the factors affecting xylanase production by Aureobasidium pullulans NRRL Y-2311-1 on globe artichoke stem was performed for the first time. The optimization strategies used resulted in almost six-fold enhancement of xylanase production (66.48?U/ml). Biochemical and thermal characterization of the crude xylanase preparation was performed to elucidate its feasibility for different industrial applications. The optimum conditions for xylanase activity were pH 4.0 and 30–50°C. The enzyme was very stable over a wide pH range of 3.0–8.0. The thermal stability studies revealed an inactivation energy of 183?kJ/mol. Thermodynamic parameters (enthalpy, entropy, and Gibbs free energy) for thermal inactivation were also determined. Primary application of the crude xylanase preparation in saccharification of corn cob subjected to different pretreatment techniques has been evaluated. The crude xylanase preparation was very promising for saccharification of corn cob pretreated with aqueous ammonia. The maximum yield of reducing sugar was 357?mg/g dry substrate, which revealed that the crude xylanase from A. pullulans could be a very good alternative in saccharification of lignocellulosic biomass for biological fuel generation. This study also provides a basis for further exploitation of globe artichoke by-products in microbial production of several other industrially significant metabolites.     

Spread of hepatitis B viruses in vitro requires extracellular progeny and may be codetermined by polarized egress

Viruses can spread by different mechanisms: via intracellular particles through cell junctions to neighboring cells or via secreted virions to adjacent or remote cells. The observation of clusters of hepadnavirus-infected cells both in vivo and in primary hepatocytes neither proves the first mechanism nor excludes the second. In order to test which mechanism, if not both, is used by hepatitis B viruses in order to spread, we used primary duck hepatocytes and duck hepatitis B virus (DHBV) as an infection model. If extracellular progeny virus alone determines spreading, neutralizing antisera or drugs blocking virus binding to hepatocytes should abolish secondary infection. In order to test this, we used DHBV envelope-specific neutralizing antisera, as well as suramin, a known inhibitor of infection. Both reagents strongly reduced hepatocellular attachment of viral particles and almost completely abolished primary infection, whereas an ongoing intracellular infection was not affected as long as no progeny virus was released. In contrast, incubation of infected primary hepatocytes with these reagents during release of progeny virus completely prevented secondary infection. Moreover, the combination of electron and immunofluorescence microscopy analyses revealed the residence of viral particles in cytoplasmic vesicles preferentially located near the basolateral membrane of infected hepatocytes. Taken together, these data strongly suggest that hepatitis B viruses mainly spread by secreted, extracellular progeny and point to polarized egress of viral particles into intercellular compartments, which restricts their diffusion and favors transmission of virus to adjacent cells.     

Itinerary of hepatitis B viruses: delineation of restriction points critical for infectious entry

Little is known about cellular determinants essential for human hepatitis B virus infection. Using the duck hepatitis B virus as a model, we first established a sensitive binding assay for both virions and subviral particles and subsequently elucidated the characteristics of the early viral entry steps. The infection itinerary was found to initiate with the attachment of viral particles to a low number of binding sites on hepatocytes (about 10(4) per cell). Virus internalization was fully accomplished in less than 3 h but was then followed by a period of unprecedented length, about 14 h, until completion of nuclear import of the viral genome. Steps subsequent to virus entry depended on both intact microtubules and their dynamic turnover but not on actin cytoskeleton. Notably, cytoplasmic trafficking of viral particles and emergence of nuclear covalently closed circular DNA requires microtubules during entry only at and for specific time periods. Taken together, these data disclose for the first time a series of steps and their kinetics that are essential for the entry of hepatitis B viruses into hepatocytes and are different from those of any other virus reported so far.     

Duck hepatitis B virus requires cholesterol for endosomal escape during virus entry

The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.     

Waypoint based path planner for socially aware robot navigation

Cluster Computing - Social navigation is beneficial for mobile robots in human inhabited areas. In this paper, we focus on smooth path tracking and handling disruptions during plan execution in...     

Nuclear translocation of papillomavirus minor capsid protein L2 requires Hsc70

Minor capsid protein L2 of papillomaviruses plays an essential role in virus assembly by recruiting viral components to PML bodies, the proposed sites of virus morphogenesis. We demonstrate here that the function of L2 in virus assembly requires the chaperone Hsc70. Hsc70 was found dispersed in naturally infected keratinocytes and cultured cells. A dramatic relocation of Hsc70 from the cytoplasm to PML bodies was induced in these cells by L2 expression. Hsc70-L2 complex formation was confirmed by coimmunoprecipitation. The complex was modulated by the cochaperones Hip and Bag-1, which stabilize and destabilize Hsc70-substrate complexes, respectively. Cytoplasmic depletion of Hsc70 caused retention of wild-type and N-terminally truncated L2, but not of C-terminally truncated L2, in the cytoplasm. This retention was partially reversed by overexpression of Hsc70 fused to green fluorescent protein but not by ATPase-negative Hsc70. Hsc70 associated with L1-L2 virus-like particles (VLPs) but not with VLPs composed either of L1 alone or of L1 and C-terminally truncated L2. Moreover, displacement of Hsc70 from L1-L2 VLPs by encapsidation of DNA, generating pseudovirions, was found. These data indicate that Hsc70 transiently associates with viral capsids during the integration of L2, possibly via the L2 C terminus. Completion of virus assembly results in displacement of Hsc70 from virions.