Publications

Reciprocal regulation of farnesoid X receptor a activity and hepatitis B virus replication in differentiated HepaRG cells and primary human hepatocytes

Hepatitis B virus (HBV) and bile salt metabolism seem tightly connected. HBV enters hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP), the genome of which contains 2 active farnesoid X receptor (FXR) a response elements that participate in HBV transcriptional activity. We investigated in differentiated HepaRG cells and in primary human hepatocytes (PHHs) effects of FXR activation on HBV replication andof infection on the FXRpathway. InHepaRG, FXRagonists (6-ethyl chenodeoxycholic acidandGW4064), butno antagonist, and anFXR-unrelated bile salt inhibited viralmRNA,DNA,andproteinproduction (IC50, 0.1–0.5mM) and reduced covalently closed circular DNA pool size. These effects were independent of the NTCP inhibitor cyclosporine-A, which suggests inhibition occurred at a postentry step. Similar results were obtained in PHH with GW4064. Infection of these cells increased expression of FXRandmodified expression of FXR-regulated genes SHP, APOA1, NTCP, CYP7A1, and CYP8B1 with a more pronounced effect in PHH than in HepaRG. FXR agonists reversed all but one of theHBV-induced FXR gene profile modifications.HBVreplication and FXR regulation seem to be interdependent, and altered bile salt metabolism homeostasis might contribute to the persistence of HBV infection.

Download full version

Virus-host interactomics: new insights and opportunities for antiviral drug discovery

The current therapeutic arsenal against viral infections remains limited, with often poor efficacy and incomplete coverage, and appears inadequate to face the emergence of drug resistance. Our understanding of viral biology and pathophysiology and our ability to develop a more effective antiviral arsenal would greatly benefit from a more comprehensive picture of the events that lead to viral replication and associated symptoms. Towards this goal, the construction of virus-host interactomes is instrumental, mainly relying on the assumption that a viral infection at the cellular level can be viewed as a number of perturbations introduced into the host protein network when viral proteins make new connections and disrupt existing ones. Here, we review advances in interactomic approaches for viral infections, focusing on high-throughput screening (HTS) technologies and on the generation of high-quality datasets. We show how these are already beginning to offer intriguing perspectives in terms of virus-host cell biology and the control of cellular functions, and we conclude by offering a summary of the current situation regarding the potential development of host-oriented antiviral therapeutics.

Download full version

The metabolic sensors FXR, PGC-1, and SIRT1 cooperatively regulate hepatitis B virus transcription

Hepatitis B virus (HBV) genome transcription is highly dependent on liver-enriched, metabolic nuclear receptors (NRs). Among others, NR farnesoid X receptor (FXR ) enhances HBV core promoter activity and pregenomic RNA synthesis. Interestingly, two food-withdrawal-induced FXR modulators, peroxisome proliferator-activated receptor- coactivator 1 (PGC-1 ) and deacetylase SIRT1, have been found to be associated with HBV genomes ex vivo. Whereas PGC-1 induction was shown to increase HBV replication, the effect of SIRT1 on HBV transcription remains unknown. Here, we showed that, in hepatocarcinoma-derived Huh-7 cells, combined activation of FXR by GW4064 and SIRT1 by activator 3 increased HBV core promoter-controlled luciferase expression by 25-fold, compared with a 10-fold increase with GW4064 alone. Using cell lines differentially expressing FXR in overexpression and silencing experiments, we demonstrated that SIRT1 activated the core promoter in an FXR – and PGC-1 -dependent manner. Maximal activation (>150-fold) was observed in FXR – and PGC-1 -overexpressing Huh-7 cells treated with FXR and SIRT1 activators. Similarly, in cells transfected with full-length HBV genomes, maximal induction (3.5-fold) of core promoter-controlled synthesis of 3.5-kb RNA was observed in the same conditions of transfection and treatments. Thus, we identified a subnetwork of metabolic factors regulating HBV replication, strengthening the hypothesis that transcription of HBV and metabolic genes is similarly controlled.

Download full version

Structure homology and interaction redundancy for discovering virus–host protein interactions

Virus–host interactomes are instrumental to understand global perturbations of cellular functions induced by infection and discover new therapies. The construction of such interactomes is, however, technically challenging and time consuming. Here we describe an original method for the prediction of high-confidence interactions between viral and human proteins through a combination of structure and high-quality interactome data. Validation was performed for the NS1 protein of the influenza virus, which led to the identification of new host factors that control viral replication.

Download full version

The Interactomes of Influenza Virus NS1 and NS2 Proteins…

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

Download full version

Viruses and Interactomes in Translation

A decade of high-throughput screenings for intraviral and virus-host protein-protein interactions led to the accumulation of data and to the development of theories on laws governing interactome organization for many viruses. We present here a computational analysis of intraviral protein networks (EBV, FLUAV, HCV, HSV-1, KSHV, SARS-CoV, VACV, and VZV) and virus-host protein networks (DENV, EBV, FLUAV, HCV, and VACV) from up-to-date interaction data, using various mathematical approaches. If intraviral networks seem to behave similarly, they are clearly different from the human interactome. Viral proteins target highly central human proteins, which are precisely the Achilles’ heel of the human interactome. The intrinsic structural disorder is a distinctive feature of viral hubs in virus-host interactomes. Overlaps between virus-host data sets identify a core of human proteins involved in the cellular response to viral infection and in the viral capacity to hijack the cell machinery for viral replication. Host proteins that are strongly targeted by a virus seem to be particularly attractive for other viruses. Such protein-protein interaction networks and their analysis represent a powerful resource from a therapeutic perspective. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.014738, 1–12, 2012.

Download full version

New horizons for antiviral drug discovery from virus-host protein interaction networks

Viruses are recurrent socio economical and health problems each year worldwide. Current drugs are mainly directed against viral components and select resistant strains that urge the need to develop new antiviral therapeutics. High-throughput screening technologies now allow to draw comprehensive genome-wide maps of physical and genetic virus-host interactions. This has been done recently for several viruses such as HIV, HCV, DENV and FLUAV and revealed a wealth of potential antiviral cellular targets. Systems-level analysis of virus-host protein networks and subnetworks begins to uncover several specific points of intervention for a human centered drug development. We present here this new paradigm in antiviral drug discovery together with the first promising antiviral molecules.

Download full version

 

Hepatitis C virus infection protein network

A proteome-wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. A total of 314 protein–protein interactions between HCV and human proteins was identified by yeast two-hybrid and 170 by literature mining. Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HCV are enriched in highly central and interconnected proteins. A global analysis on the basis of functional annotation highlighted the enrichment of cellular pathways targeted by HCV. A network of proteins associated with frequent clinical disorders of chronically infected patients was constructed by connecting the insulin, Jak/STAT and TGFb pathways with cellular proteins targeted by HCV. CORE protein appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins.

Download full version

Transactivation of the Hepatitis B Virus Core…

Hepatitis B virus (HBV) core promoter activity is positively and negatively regulated by nuclear receptors, a superfamily of ligand activated transcription factors, via cis-acting sequences located in the viral genome. In this study, we investigated the role of farnesoid X receptor alpha (FXR) in modulating transcription from the HBV core promoter. FXR is a liver-enriched nuclear receptor activated by bile acids recognizing hormone response elements by forming heterodimers with retinoid X receptor alpha (RXR). Electrophoretic mobility shift assays demonstrated that FXR-RXR heterodimers can bind two motifs on the HBV enhancer II and core promoter regions, presenting high homology to the consensus (AGGTCA) inverted repeat FXR response elements. In transient transfection of the human hepatoma cell line Huh-7, bile acids enhanced the activity of a luciferase reporter containing the HBV enhancer II and core promoter sequences through FXR. Moreover, using a greater-than-genome length HBV construct, we showed that FXR also increased synthesis of the viral pregenomic RNA and DNA replication intermediates. The data strongly suggest that FXR is another member of the nuclear receptor superfamily implicated in the regulation of HBV core promoter activity and that bile acids could play an important role in the natural history of HBV infection.

Download full version