Evidence for existence of an apoptosis-inducing BH3-only protein, sayonara, in Drosophila.

Cells need to sense stresses to initiate the execution of the dormant cell death program. Since the discovery of the first BH3-only protein Bad, BH3-only proteins have been recognized as indispensable stress sensors that induce apoptosis. BH3-only proteins have so far not been identified in Drosophila despite their importance in other organisms. Here, we identify the first Drosophila BH3-only protein and name it sayonara. Sayonara induces apoptosis in a BH3 motif-dependent manner and interacts genetically and biochemically with the BCL-2 homologous proteins, Buffy and Debcl. There is a positive feedback loop between Sayonara-mediated caspase activation and autophagy. The BH3 motif of sayonara phylogenetically appeared at the time of the ancestral gene duplication that led to the formation of Buffy and Debcl in the dipteran lineage. To our knowledge, this is the first identification of a bona fide BH3-only protein in Drosophila, thus providing a unique example of how cell death mechanisms can evolve both through time and across taxa.

A fusion peptide in preS1 and the human protein disulfide isomerase ERp57 are involved in hepatitis B virus membrane fusion process.

Cell entry of enveloped viruses relies on the fusion between the viral and plasma or endosomal membranes, through a mechanism that is triggered by a cellular signal. Here we used a combination of computational and experimental approaches to unravel the main determinants of hepatitis B virus (HBV) membrane fusion process. We discovered that ERp57 is a host factor critically involved in triggering HBV fusion and infection. Then, through modeling approaches, we uncovered a putative allosteric cross-strand disulfide (CSD) bond in the HBV S glycoprotein and we demonstrate that its stabilization could prevent membrane fusion. Finally, we identified and characterized a potential fusion peptide in the preS1 domain of the HBV L glycoprotein. These results underscore a membrane fusion mechanism that could be triggered by ERp57, allowing a thiol/disulfide exchange reaction to occur and regulate isomerization of a critical CSD, which ultimately leads to the exposition of the fusion peptide.

Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production.

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.

Database and Bioinformatic Analysis of BCL-2 Family Proteins and BH3-Only Proteins.

BCL-2 proteins correspond to a structurally, functionally, and phylogenetically heterogeneous group of regulators that play crucial roles in the life and death of animal cells. Some of these regulators also represent therapeutic targets in human diseases including cancer. In the omics era, there is great need for easy data retrieval and fast analysis of the molecular players involved in cell death. In this chapter, we present generic and specific computational resources (such as the reference database BCL2DB) as well as bioinformatics tools that can be used to investigate BCL-2 homologs and BH3-only proteins.

How viral genetic variants and genotypes influence disease and treatment outcome of chronic hepatitis B. Time for an individualised approach?

Chronic hepatitis B virus (HBV) infection remains a global problem. Several HBV genotypes exist with different biology and geographical prevalence. Whilst the future aim of HBV treatment remains viral eradication, current treatment strategies aim to suppress the virus and prevent the progression of liver disease. Current strategies also involve identification of patients for treatment, namely those at risk of progressive liver disease. Identification of HBV genotype, HBV mutants and other predictive factors allow for tailoured treatments, and risk-surveillance pathways, such as hepatocellular cancer screening. In the future, these factors may enable stratification not only of treatment decisions, but also of patients at risk of higher relapse rates when current therapies are discontinued. Newer technologies, such as next-generation sequencing, to assess drug-resistant or immune escape variants and quasi-species heterogeneity in patients, may allow for more information-based treatment decisions between the clinician and the patient. This article serves to discuss how HBV genotypes and genetic variants impact not only upon the disease course and outcomes, but also current treatment strategies. Adopting a personalised genotypic approach may play a role in future strategies to combat the disease. Herein, we discuss new technologies that may allow more informed decision-making for response guided therapy in the battle against HBV.

Direct-acting antiviral therapy decreases hepatocellular carcinoma recurrence rate in cirrhotic patients with chronic hepatitis C.

BACKGROUND AND AIMS: Arrival of direct-acting antiviral agents against hepatitis C virus with high-sustained virological response rates and very few side effects has drastically changed the management of hepatitis C virus infection. The impact of direct-acting antiviral exposure on hepatocellular carcinoma recurrence after a first remission in patients with advanced fibrosis remains to be clarified. METHODS: 68 consecutive hepatitis C virus patients with a first hepatocellular carcinoma diagnosis and under remission, subsequently treated or not with a direct-acting antiviral combination, were included. Clinical, biological and virological data were collected at first hepatocellular carcinoma diagnosis, at remission and during the surveillance period. RESULTS: All patients were cirrhotic. Median age was 62 years and 76% of patients were male. Twenty-three patients (34%) were treated with direct-acting antivirals and 96% of them achieved sustained virological response. Median time between hepatocellular carcinoma remission and direct-acting antivirals initiation was 7.2 months (IQR: 3.6-13.5; range: 0.3-71.4) and median time between direct-acting antivirals start and hepatocellular carcinoma recurrence was 13.0 months (IQR: 9.2-19.6; range: 3.0-24.7). Recurrence rate was 1.7/100 person-months among treated patients vs 4.2/100 person-months among untreated patients (P=.008). In multivariate survival analysis, the hazard ratio for hepatocellular carcinoma recurrence after direct-acting antivirals exposure was 0.24 (95% confidence interval: 0.10-0.55; P<.001). CONCLUSIONS: Hepatocellular carcinoma recurrence rate was significantly lower among patients treated with direct-acting antivirals compared with untreated patients. Given the potential impact of our observation, large-scale prospective cohort studies are needed to confirm these results.

Redefining the BH3 Death Domain as a 'Short Linear Motif'.

B cell lymphoma-2 (BCL-2)-related proteins control programmed cell death through a complex network of protein-protein interactions mediated by BCL-2 homology 3 (BH3) domains. Given their roles as dynamic linchpins, the discovery of novel BH3-containing proteins has attracted considerable attention. However, without a clearly defined BH3 signature sequence the BCL-2 family has expanded to include a nebulous group of nonhomologous BH3-only proteins, now justified by an intriguing twist. We present evidence that BH3s from both ordered and disordered proteins represent a new class of short linear motifs (SLiMs) or molecular recognition features (MoRFs) and are diverse in their evolutionary histories. The implied corollaries are that BH3s have a broad phylogenetic distribution and could potentially bind to non-BCL-2-like structural domains with distinct functions.

BCL2DB: database of BCL-2 family members and BH3-only proteins.

BCL2DB (http://bcl2db.ibcp.fr) is a database designed to integrate data on BCL-2 family members and BH3-only proteins. These proteins control the mitochondrial apoptotic pathway and probably many other cellular processes as well. This large protein group is formed by a family of pro-apoptotic and anti-apoptotic homologs that have phylogenetic relationships with BCL-2, and by a collection of evolutionarily and structurally unrelated proteins characterized by the presence of a region of local sequence similarity with BCL-2, termed the BH3 motif. BCL2DB is monthly built, thanks to an automated procedure relying on a set of homemade profile HMMs computed from seed reference sequences representative of the various BCL-2 homologs and BH3-only proteins. The BCL2DB entries integrate data from the Ensembl, Ensembl Genomes, European Nucleotide Archive and Protein Data Bank databases and are enriched with specific information like protein classification into orthology groups and distribution of BH motifs along the sequences. The Web interface allows for easy browsing of the site and fast access to data, as well as sequence analysis with generic and specific tools. BCL2DB provides a helpful and powerful tool to both 'BCL-2-ologists' and researchers working in the various fields of physiopathology. Database URL: http://bcl2db.ibcp.fr.

BRCA1-Dependent Translational Regulation in Breast Cancer Cells.

BRCA1 (Breast Cancer 1) has been implicated in a number of cellular processes, including transcription regulation, DNA damage repair and protein ubiquitination. We previously demonstrated that BRCA1 interacts with PABP1 (Poly(A)-Binding Protein 1) and that BRCA1 modulates protein synthesis through this interaction. To identify the mRNAs that are translationally regulated by BRCA1, we used a microarray analysis of polysome-bound mRNAs in BRCA1-depleted and non-depleted MCF7 cells. Our findings show that BRCA1 modifies the translational efficiency of approximately 7% of the mRNAs expressed in these cells. Further analysis revealed that several processes contributing to cell surveillance such as cell cycle arrest, cell death, cellular growth and proliferation, DNA repair and gene expression, are largely enriched for the mRNAs whose translation is impacted by BRCA1. The BRCA1-dependent translation of these species of mRNAs therefore uncovers a novel mechanism through which BRCA1 exerts its onco-suppressive role. In addition, the BRCA1-dependent translation of mRNAs participating in unexpected functions such as cellular movement, nucleic acid metabolism or protein trafficking is indicative of novel functions for BRCA1. Finally, this study contributes to the identification of several markers associated with BRCA1 deficiency and to the discovery of new potential anti-neoplastic therapeutic targets.

Evolution of Bcl-2 homology motifs: homology versus homoplasy.

Bcl-2 family proteins regulate apoptosis in animals. This protein family includes several homologous proteins and a collection of other proteins lacking sequence similarity except for a Bcl-2 homology (BH)3 motif. Thus, membership in the Bcl-2 family requires only one of the four BH motifs. On this basis, a growing number of diverse BH3-only proteins are being reported. Although compelling cell biological and biophysical evidence validates many BH3-only proteins, claims of significant BH3 sequence similarity are often unfounded. Computational and phylogenetic analyses suggest that only some BH3 motifs arose by divergent evolution from a common ancestor (homology), whereas others arose by convergent evolution or random coincidence (homoplasy), challenging current assumptions about which proteins constitute the extended Bcl-2 family.

Mutations that alter use of hepatitis C virus cell entry factors mediate escape from neutralizing antibodies.

BACKGROUND & AIMS: The development of vaccines and other strategies to prevent hepatitis C virus (HCV) infection is limited by rapid viral evasion. HCV entry is the first step of infection; this process involves several viral and host factors and is targeted by host-neutralizing responses. Although the roles of host factors in HCV entry have been well characterized, their involvement in evasion of immune responses is poorly understood. We used acute infection of liver graft as a model to investigate the molecular mechanisms of viral evasion. METHODS: We studied factors that contribute to evasion of host immune responses using patient-derived antibodies, HCV pseudoparticles, and cell culture-derived HCV that express viral envelopes from patients who have undergone liver transplantation. These viruses were used to infect hepatoma cell lines that express different levels of HCV entry factors. RESULTS: By using reverse genetic analyses, we identified altered use of host-cell entry factors as a mechanism by which HCV evades host immune responses. Mutations that alter use of the CD81 receptor also allowed the virus to escape neutralizing antibodies. Kinetic studies showed that these mutations affect virus-antibody interactions during postbinding steps of the HCV entry process. Functional studies with a large panel of patient-derived antibodies showed that this mechanism mediates viral escape, leading to persistent infection in general. CONCLUSIONS: We identified a mechanism by which HCV evades host immune responses, in which use of cell entry factors evolves with escape from neutralizing antibodies. These findings advance our understanding of the pathogenesis of HCV infection and might be used to develop antiviral strategies and vaccines.

Transcriptional slippage prompts recoding in alternate reading frames in the hepatitis C virus (HCV) core sequence from strain HCV-1.

Since the first report of frameshifting in HCV-1, its sequence has been the paradigm for examining the mechanism that directs alternative translation of the hepatitis C virus (HCV) genome. The region encoding the core protein from this strain contains a cluster of 10 adenines at codons 8-11, which is thought to direct programmed ribosomal frameshifting (PRF), but formal evidence for this process has not been established unequivocally. To identify the mechanisms of frameshifting, this study used a bicistronic dual luciferase reporter system in a coupled transcription/translation in vitro assay. This approach revealed +1 as well as -1 frameshifting, whereas point mutations, selectively introduced between codons 8 and 11, demonstrated that PRF did not readily account for frameshifting in strain HCV-1. Sequence analysis of cDNAs derived from RNA transcribed by T7 RNA polymerase in the dual luciferase reporter system, as well as in both a subgenomic replicon and an infectious clone derived from strain JFH1, identified additions and deletions of adenines between codons 8 and 11 due to transcriptional slippage (TS). Moreover, RNA isolated from cells infected with virus generated by JFH1 containing the A-rich tract also contained heterogeneity in the adenine sequence, strongly suggesting TS by the NS5B viral polymerase. These findings have important implications for insight into frameshifting events in HCV-1 and demonstrate for the first time the involvement of transcriptional slippage in this recoding event.