Chemo-small extracellular vesicles released in cisplatin-resistance ovarian cancer cells are regulated by the lysosomal function.

Chemoresistance is a common problem in ovarian cancer (OvCa) treatment, where resistant cells, in response to chemotherapy, secrete small extracellular vesicles (sEVs), known as chemo-sEVs, that transfer resistance to recipient cells. sEVs are formed as intraluminal vesicles (ILVs) within multivesicular endosomes (MVEs), whose trafficking is regulated by Ras-associated binding (RAB) GTPases that mediate sEVs secretion or lysosomal degradation. A decrease in lysosomal function can promote sEVs secretion, but the relationship between MVEs trafficking pathways and sEVs secretion in OvCa chemoresistance is unclear. Here, we show that A2780cis cisplatin (CCDP) resistant OvCa cells had an increased number of MVEs and ILVs structures, higher levels of Endosomal Sorting Complex Required for Transport (ESCRTs) machinery components, and RAB27A compared to A2780 CDDP-sensitive OvCa cells. CDDP promoted the secretion of chemo-sEVs in A2780cis cells, enriched in DNA damage response proteins. A2780cis cells exhibited poor lysosomal function with reduced levels of RAB7, essential in MVEs-Lysosomal trafficking. The silencing of RAB27A in A2780cis cells prevents the Chemo-EVs secretion, reduces its chemoresistance and restores lysosomal function and levels of RAB7, switching them into an A2780-like cellular phenotype. Enhancing lysosomal function with rapamycin reduced chemo-sEVs secretion. Our results suggest that adjusting the balance between secretory MVEs and lysosomal MVEs trafficking could be a promising strategy for overcoming CDDP chemoresistance in OvCa.

Canonical and Divergent N-Terminal HBx Isoform Proteins Unveiled: Characteristics and Roles during HBV Replication.

Hepatitis B virus (HBV) X protein (HBx) is a viral regulatory and multifunctional protein. It is well-known that the canonical HBx reading frame bears two phylogenetically conserved internal in-frame translational initiation codons at Met2 and Met3, thus possibly generating divergent N-terminal smaller isoforms during translation. Here, we demonstrate that the three distinct HBx isoforms are generated from the ectopically expressed HBV HBx gene, named XF (full-length), XM (medium-length), and XS (short-length); they display different subcellular localizations when expressed individually in cultured hepatoma cells. Particularly, the smallest HBx isoform, XS, displayed a predominantly cytoplasmic localization. To study HBx proteins during viral replication, we performed site-directed mutagenesis to target the individual or combinatorial expression of the HBx isoforms within the HBV viral backbone (full viral genome). Our results indicate that of all HBx isoforms, only the smallest HBx isoform, XS, can restore WT levels of HBV replication, and bind to the viral mini chromosome, thereby establishing an active chromatin state, highlighting its crucial activities during HBV replication. Intriguingly, we found that sequences of HBV HBx genotype H are devoid of the conserved Met3 position, and therefore HBV genotype H infection is naturally silent for the expression of the HBx XS isoform. Finally, we found that the HBx XM (medium-length) isoform shares significant sequence similarity with the N-terminus domain of the COMMD8 protein, a member of the copper metabolism MURR1 domain-containing (COMMD) protein family. This novel finding might facilitate studies on the phylogenetic origin of the HBV X protein. The identification and functional characterization of its isoforms will shift the paradigm by changing the concept of HBx from being a unique, canonical, and multifunctional protein toward the occurrence of different HBx isoforms, carrying out different overlapping functions at different subcellular localizations during HBV genome replication. Significantly, our current work unveils new crucial HBV targets to study for potential antiviral research, and human virus pathogenesis.

The histone variant H3.3 regulates the transcription of the hepatitis B virus.

INTRODUCTION AND OBJECTIVES: About 250 million people around the world are chronically infected with the hepatitis B virus (HBV). Those people are at risk of developing hepatocellular carcinoma. The HBV genome is organized as a minichromosome in the infected hepatocyte and is associated with histones and non-histone proteins. In recent years, many groups have investigated the transcriptional regulation of HBV mediated by post-translational modifications on the histones associated with the covalently closed circular DNA (cccDNA). Our aim is to investigate the role of the histone variant H3.3. MATERIALS AND METHODS: An in vitro HBV replication model system based on the transfection of linear HBV genome monomeric molecules was used. We then either ectopically expressed or reduced the levels of H3.3, and of its histone chaperone HIRA. Viral intermediates were quantified and the level of H3K4me3 using Chromatin immunoprecipitation (ChIP) assay was measured. RESULTS: Histone variant H3.3 ectopically expressed in cells assembles into the viral cccDNA, correlating with increasing levels of the active histone mark H3K4me3 and HBV transcription. The opposite results were found upon diminishing H3.3 levels. Furthermore, the assembly of H3.3 into the cccDNA is dependent on the histone chaperone HIRA. Diminishing HIRA levels causes a reduction in the HBV intermediates. CONCLUSIONS: Histone variant H3.3 positively regulates HBV transcription. Importantly, the characterization of the viral chromatin dynamics might allow the discovery of new therapeutic targets to develop drugs for the treatment of chronically-infected HBV patients.