A novel system for efficient gene transfer into primary human hepatocytes via cell-permeable hepatitis B virus-like particle.

Protein transduction domains (PTDs) have been used to deliver a variety of biologically active cargo across cellular membranes. However the potential of PTDs to mediate transport of nanoparticular structures into the cytoplasm bypassing the endosomal compartment remains unclear. Cell-permeable virus-like particles (VLPs) harboring a marker gene based on hepatitis B virus nucleocaspids were established. Cell permeability was achieved by fusion with translocation motif (TLM)-PTD. Electron and confocal microscopy revealed that these VLPs translocate as complete particles across the plasma membrane and transverse the cytoplasm toward the nucleus. Inhibition of endocytosis did not affect translocation of these VLPs into the cytoplasm. Based on these particles, a gene transfer system was developed. To this end the particles were loaded with DNA-encoding small hepatitis B virus surface antigen (SHBs) or green fluorescence protein (eGFP) that served as marker genes. Although the DNA-packaging efficiency was very low, applying the appropriate number of VLPs to primary human hepatocytes a gene transfer efficiency of approximately 95% was observed. In conclusion, the TLM-PTD has the potential to mediate efficient transfer of assembled particles and its cargo, nucleic acids, into primary human hepatocytes. This provides the basis for development of novel transducible therapeutic or diagnostic particles.

Accumulation of 8-hydroxy-2'-deoxyguanosine adducts in HBx recombinant HepG2 cells and HBx transgenic mice.

BACKGROUND/AIMS: Transgenic mice overexpressing hepatitis B x protein (HBx) show an increased susceptibility to mutations if exposed to mutagens. Also involved in HBx signalling, reactive oxygen intermediates (ROI) can induce DNA adducts such as 8-hydroxy-2'-deoxyguanosine that can in turn lead to G/T transversion mutations. Therefore, we investigated whether HBx expression increases the level of the mutational precursor 8-hydroxy-2'-deoxyguanosine in hepatocellular DNA. METHODS: 8-hydroxy-2'-deoxyguanosine concentrations of DNA hydrolysates of HBx protein expressing HepG2 cells and livers of HBx transgenic mouse lines were determined electrochemically after HPLC fractionation. RESULTS: 8-hydroxy-2'-deoxyguanosine concentrations in genomic DNA of HBx protein expressing cell lines correlated with the factor of transactivation. The 8-hydroxy-2'-deoxyguanosine levels were reduced after incubation of HBx recombinant cell lines with 0.1 or 1 mM of the antioxidant N-acetylcysteine. Hepatic 8-hydroxy-2'-deoxyguanosine concentrations in DNA of old transgenic mice were significantly, i.e. twofold, (p < 0.01) increased as compared to those of old nontransgenic or young transgenic controls and of control mice expressing a second HBV transactivator (MHBs(t76)). CONCLUSION: HBx expression results in elevated DNA adduct levels. This could reflect a direct inhibitory interaction of HBx with cellular repair mechanisms. Alternatively, this may be an effect of an increased generation of reactive oxygen intermediates through HBx.

Integrity of c-Raf-1/MEK signal transduction cascade is essential for hepatitis B virus gene expression.

The genome of hepatitis B virus (HBV) encodes two transcriptional activators: the HBx protein and the PreS2-activator large surface protein (LHBs). Both proteins trigger activation of c-Raf-1/MEK kinase cascade. In case of HBx this can be mediated by a PKC-independent and Ras-dependent mechanism, in case of LHBs activation is PKC-dependent and does not require Ras. Selective destruction of either LHBs- or of HBx-specific activation does not result in significant decrease of viral production from transfected HepG2 cells. Simultaneous inhibition of LHBs- and HBx-dependent activation by blocking signaling steps common to both activators, using trans dominant negative c-Raf-1- or MEK-specific inhibitors, abolished HBV gene expression. In accordance with this no HBV propagation was observed after transfection of a mutated HBV genome defective for HBx- and PreS2-activator function. A detailed analysis revealed that the observed inhibition of HBV- propagation is because of a significant reduction of HBV-specific RNA resulting in an inhibition of the de novo synthesis of viral compounds (viral proteins and nucleic acid) and not by blocking secretion or assembly of the virus. Based on these results we conclude that transcriptional-activator function, mediated by the c-Raf-1/MEK signaling cascade, is essential for HBV gene expression.

The PreS2 activator MHBs(t) of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice.

The large hepatitis B virus (HBV) surface protein (LHBs) and C-terminally truncated middle size surface proteins (MHBs(t)) form the family of the PreS2 activator proteins of HBV. Their transcriptional activator function is based on the cytoplasmic orientation of the PreS2 domain. MHBs(t) activators are paradigmatic for this class of activators. Here we report that MHBs(t) is protein kinase C (PKC)-dependently phosphorylated at Ser28. The integrity of the phosphorylation site is essential for the activator function. MHBs(t) triggers PKC-dependent activation of c-Raf-1/Erk2 signaling that is a prerequisite for MHBs(t)-dependent activation of AP-1 and NF-kappaB. To analyze the pathophysiological relevance of these data in vivo, transgenic mice were established that produce the PreS2 activator MHBs(t) specifically in the liver. In these mice, a permanent PreS2-dependent specific activation of c-Raf-1/Erk2 signaling was observed, resulting in an increased hepatocyte proliferation rate. In transgenics older than 15 months, an increased incidence of liver tumors occurs. These data suggest that PreS2 activators LHBs and MHBs(t) exert a tumor promoter-like function by activation of key enzymes of proliferation control.

Site-specific mutation of the hepatitis B virus enhancer II B1 element: effect on virus transcription and replication.

The hepatitis B virus (HBV) enhancer II (EII) is highly liver-specific and plays an important role in regulating the transcription of all HBV genes. In this report, mutational analysis on the B1F-binding site in the major functional unit of HBV EII is described. The activity of HBV EII in EII-CAT reporter plasmids was significantly decreased when the sequence of the B1F-binding site in EII was mutated. Furthermore, a single point mutation in the B1 element that aborted the binding of B1F caused a dramatic decrease in viral gene transcription initiated from the HBV core promoter, which resulted in a reduction of the production of the HBV e antigen and pregenomic RNA, the template for viral DNA replication. In conclusion, the interaction of B1F with its target binding sequence in the EII region is crucial for liver-specific transcription and DNA replication of the virus.

A novel negative cis-regulatory element on the hepatitis B virus S-(+)-strand.

Hepatitis B virus (HBV) has a double-stranded DNA genome. The minus-strand contains coding regions for all known HBV proteins and most of the cis-regulatory elements. Little is known about transcription from the S-(+)-strand and its regulation. Thus, the presence of regulatory elements located on the S-(+)-strand was investigated by inserting nt 1038-1783 of HBV in both orientations between the human cytomegalovirus (HCMV) promoter and a luciferase gene. Transfection experiments revealed that the plasmid containing this HBV DNA fragment in an orientation allowing expression from the S-(+)-strand (antisense) led to inhibition of luciferase gene expression compared to the plasmid containing this sequence in an orientation that allows gene expression from the L-(-)-strand (sense). Deletion analyses delimit the sequence essential for the inhibitory effect to a 150 bp region that also carries part of the enhancerII/core promoter complex. However, the possible influence of this regulatory element has been excluded in various experiments. The repressing HBV sequence acts in an orientation- and position-dependent manner; no inhibition was observed when this DNA element was inserted upstream of the HCMV promoter or downstream of the luciferase gene. Northern blot analyses revealed reduced luciferase mRNA steady-state levels in cells transfected with constructs containing the essential HBV sequence in antisense orientation compared to plasmids containing this sequence in sense orientation. Since nuclear run-on experiments showed similar transcription initiation rates with these plasmids, the diminished luciferase mRNA steady-state levels must be due to altered stabilities, suggesting that nt 1783-1638 of HBV encode an RNA-destabilizing element.