Alternative splicing of hepatitis B virus: A novel virus/host interaction altering liver immunity.

BACKGROUND & AIMS: Hepatitis B virus (HBV) RNA can undergo alternative splicing, but the relevance of this post-transcriptional regulation remains elusive. The mechanism of HBV alternative splicing regulation and its impact on liver pathogenesis were investigated. METHODS: HBV RNA-interacting proteins were identified by RNA pull-down, combined with mass spectrometry analysis. HBV splicing regulation was investigated in chemically and surgically induced liver damage, in whole HBV genome transgenic mice and in hepatoma cells. Viral and endogenous gene expression were quantified by quantitative reverse transcription polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay. Resident liver immune cells were studied by fluorescence-activated cell sorting. RESULTS: HBV pregenomic RNA-interacting proteins were identified and 15% were directly related to the splicing machinery. Expression of these splicing factors was modulated in HBV transgenic mice with liver injuries and contributed to an increase of the HBV spliced RNA encoding for HBV splicing-generated protein (HBSP). HBSP transgenic mice with chemically induced liver fibrosis exhibited attenuated hepatic damage. The protective effect of HBSP resulted from a decrease of inflammatory monocyte/macrophage recruitment through downregulation of C-C motif chemokine ligand 2 (CCL2) expression in hepatocytes. In human hepatoma cells, the ability of HBSP to control CCL2 expression was confirmed and maintained in a whole HBV context. Finally, viral spliced RNA detection related to a decrease of CCL2 expression in the livers of HBV chronic carriers underscored this mechanism. CONCLUSION: The microenvironment, modified by liver injury, increased HBSP RNA expression through splicing factor regulation, which in turn controlled hepatocyte chemokine synthesis. This feedback mechanism provides a novel insight into liver immunopathogenesis during HBV infection. Lay summary: Hepatitis B virus persists for decades in the liver of chronically infected patients. Immune escape is one of the main mechanisms developed by this virus to survive. Our study highlights how the crosstalk between virus and liver infected cells may contribute to this immune escape.

Propranolol reduces the accumulation of cytotoxic aggregates in C9orf72-ALS/FTD in vitro models.

Mutations in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The pathogenetic mechanisms linked to this gene are a direct consequence of an aberrant intronic expansion of a GGGGCC hexanucleotide located between the 1a and 1b non-coding exons, which can be transcribed to form cytotoxic RNA foci or even translated into aggregation-prone dipeptide repeat proteins. Importantly, the abnormal length of these repeats affects also the expression levels of C9orf72 itself, which suggests haploinsufficiency as additional pathomechanism. Thus, it appears that both toxic gain of function and loss of function are distinct but still coexistent features contributing to the insurgence of the disease in case of C9orf72 mutations. In this study, we aimed at identifying a strategy to address both aspects of the C9orf72-related pathobiochemistry and provide proof-of-principle information for a better understanding of the mechanisms leading to neuronal loss. By using primary neurons overexpressing toxic poly(GA), the most abundant protein product of the GGGGCC repeats, we found that the antiarrhythmic drug propranolol could efficiently reduce the accumulation of aberrant aggregates and increase the survival of C9orf72-related cultures. Interestingly, the improved catabolism appeared to not depend on major degradative pathways such as autophagy and the proteasome. By analyzing the proteome of poly(GA)-expressing neurons after exposure to propranolol, we found that the drug increased lysosomal degradation through a mechanism directly involving C9orf72 protein, whose levels were increased after treatment. Further confirmation of the beneficial effect of the beta blocker on aggregates' accumulation and survival of hiPSC-derived C9orf72-mutant motoneurons strengthened the finding that addressing both facets of C9orf72 pathology might represent a valid strategy for the treatment of these ALS/FTD cases.

Proteomic profiling of sweat in patients with cystic fibrosis provides new insights into epidermal homoeostasis.

Background: A high proportion of patients with Cystic Fibrosis (CF) also present the rare skin disease aquagenic palmoplantar keratoderma. A possible link between this condition and absence of a functional CF Transmembrane conductance Regulator protein in the sweat acinus and collecting duct remains unknown. Methods: In-depth characterization of sweat proteome profiles was performed in 25 CF patients compared to 12 healthy controls. A 20 µL sweat sample was collected after pilocarpine iontophoresis and liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomic analysis was performed. Results: Sweat proteome profile of CF patients was significantly different from that of healthy subjects with 57 differentially expressed proteins. Cystic Fibrosis sweat proteome was characterized by an increase in 25 proteins including proteases (Kallikrein 7 and 13, Phospholipase B domain containing 1, Cathepsin A L2 and B, Lysosomal Pro-X carboxypeptidase); proinflammatory proteins (Annexin A2, Chitinase-3-like protein 1); cytochrome c and transglutaminases. Thirty-two proteins were downregulated in CF sweat including proteases (Elastase 2), antioxidative protein FAM129 B; membrane-bound transporter SLC6A14 and regulator protein Sodium-hydrogen antiporter 3 regulator 1. Conclusion: This study is the first to report in-depth characterization of endogenous peptides in CF sweat and could help understand the complex physiology of the sweat gland. The proteome profile highlights the unbalanced proteolytic and proinflammatory activity of sweat in CF. These results also suggest a defect in pathways involved in skin barrier integrity in CF patients. Sweat proteome profile could prove to be a useful tool in the context of personalized medicine in CF.