Comparative characterization of hepatitis B virus surface antigen derived from different hepatitis B virus genotypes.

For human hepatitis B virus eight distinct and two candidate genotypes are described. These genotypes differ with respect to geographic distribution, molecular virology and virus-associated pathogenesis. Comparative analysis of HBV genotypes revealed, with exception of HBV/G that shows impaired HBsAg release, that no fundamental disparities between genotypes exist regarding glycosylation, subcellular distribution, release of HBsAg and formation of subviral particles. However, there are distinctions regarding the proportion of L to M to S HBs proteins detected intra- and extracellularly for different genotypes. 2D electrophoresis revealed different posttranslational modification patterns for LHBs. In light of the relevance of HBsAg as diagnostic marker, detectability of purified recombinant HBsAg of various genotypes by HBsAg-specific detection systems licensed in Europe was investigated, showing similar sensitivities for genotypes included in this analysis. These data indicate that recombinant HBsAg reproducibly purified following a defined protocol might be used as an alternative to reference materials currently established.

Intracellular accumulation of subviral HBsAg particles and diminished Nrf2 activation in HBV genotype G expressing cells lead to an increased ROI level.

BACKGROUND & AIMS: Hepatitis B virus genotype G (HBV/G) is characterized by a lack of HBeAg secretion and very low HBsAg secretion. This study aimed at (1) comparing HBV genotype G and A2 with respect to morphogenesis and release of HBV-derived particles, (2) characterizing factors contributing to HBV/G-associated pathogenesis. METHODS: HBV/G- and HBV/A-expressing hepatoma cells and infected HepaRG cells were analyzed by confocal laser scanning microscopy, Western blot, real-time PCR, density gradient centrifugation, and electron microscopy. Modulation of the transcription factors Nrf2 and AP-1 was analyzed. RESULTS: While the release of viral particles is not affected in HBV/G replicating cells, the secretion of subviral particles is impaired, although they are produced in high amounts. These subviral particles, which display an increased density and a predominantly filamentous morphology, accumulate at the endoplasmic reticulum. The PreS1PreS2 domain of genotype G, which forms aggregates, causes the block of HBsAg-secretion at the ER and leads to decreased transcriptional activator function of LHBs. Intracellular accumulation of HBsAg and impaired induction of the cytoprotective transcription factor Nrf2 lead to an elevated level of ROIs. This results in activation of JNK and as a consequence in Ser-phosphorylation of IRS-1, which is known to impair insulin signaling, a key factor for liver regeneration. CONCLUSIONS: Although competent for release of viral particles, secretion of subviral particles is impaired in HBV/G expressing cells leading to ER-stress. In parallel, HBV-induced Nrf2 activation diminishes, which causes a decrease of the capacity to inactivate ROIs. This might be related to genotype-specific pathogenesis.

Non-secreted clusterin isoforms are translated in rare amounts from distinct human mRNA variants and do not affect Bax-mediated apoptosis or the NF-κB signaling pathway.

Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU), which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s) of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with in vitro mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU1­449, CLU21­449 and CLU34­449, which all reside in the cytosol of unstressed and stressed HEK­293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.