Pharmacological induction of the hypoxia response pathway in Huh7 hepatoma cells limits proliferation but increases resilience under metabolic stress.

The hypoxia response pathway enables adaptation to oxygen deprivation. It is mediated by hypoxia-inducible factors (HIF), which promote metabolic reprogramming, erythropoiesis, angiogenesis and tissue remodeling. This led to the successful development of HIF-inducing drugs for treating anemia and some of these molecules are now in clinic. However, elevated levels of HIFs are frequently associated with tumor growth, poor prognosis, and drug resistance in various cancers, including hepatocellular carcinoma (HCC). Consequently, there are concerns regarding the recommendation of HIF-inducing drugs in certain clinical situations. Here, we analyzed the effects of two HIF-inducing drugs, Molidustat and Roxadustat, in the well-characterized HCC cell line Huh7. These drugs increased HIF-1α and HIF-2α protein levels which both participate in inducing hypoxia response genes such as BNIP3, SERPINE1, LDHA or EPO. Combined transcriptomics, proteomics and metabolomics showed that Molidustat increased the expression of glycolytic enzymes, while the mitochondrial network was fragmented and cellular respiration decreased. This metabolic remodeling was associated with a reduced proliferation and a lower demand for pyrimidine supply, but an increased ability of cells to convert pyruvate to lactate. This was accompanied by a higher resistance to the inhibition of mitochondrial respiration by antimycin A, a phenotype confirmed in Roxadustat-treated Huh7 cells and Molidustat-treated hepatoblastoma cells (Huh6 and HepG2). Overall, this study shows that HIF-inducing drugs increase the metabolic resilience of liver cancer cells to metabolic stressors, arguing for careful monitoring of patients treated with HIF-inducing drugs, especially when they are at risk of liver cancer.

Tethering of hexokinase 2 to mitochondria promotes resistance of liver cancer cells to natural killer cell cytotoxicity.

Hexokinases (HKs) control the first step of glucose catabolism. A switch of expression from liver HK (glucokinase, GCK) to the tumor isoenzyme HK2 is observed in hepatocellular carcinoma progression. Our prior work revealed that HK isoenzyme switch in hepatocytes not only regulates hepatic metabolic functions but also modulates innate immunity and sensitivity to Natural Killer (NK) cell cytotoxicity. This study investigates the impact of HK2 expression and its mitochondrial binding on the resistance of human liver cancer cells to NK-cell-induced cytolysis. We have shown that HK2 expression induces resistance to NK cell cytotoxicity in a process requiring mitochondrial binding of HK2. Neither HK2 nor GCK expression affects target cells' ability to activate NK cells. In contrast, mitochondrial binding of HK2 reduces effector caspase 3/7 activity both at baseline and upon NK-cell activation. Furthermore, HK2 tethering to mitochondria enhances their resistance to cytochrome c release triggered by tBID. These findings indicate that HK2 mitochondrial binding in liver cancer cells is an intrinsic resistance factor to cytolysis and an escape mechanism from immune surveillance.

The PACS-2 protein and trafficking motifs in CCHFV Gn and Gc cytoplasmic domains govern CCHFV assembly.

The Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus that causes high mortality in humans. This enveloped virus harbors two surface glycoproteins (GP), Gn and Gc, that are released by processing of a glycoprotein precursor complex whose maturation takes place in the ER and is completed through the secretion pathway. Here, we characterized the trafficking network exploited by CCHFV GPs during viral assembly, envelopment, and/or egress. We identified membrane trafficking motifs in the cytoplasmic domains (CD) of CCHFV GPs and addressed how they impact these late stages of the viral life cycle using infection and biochemical assays, and confocal microscopy in virus-producing cells. We found that several of the identified CD motifs modulate GP transport through the retrograde trafficking network, impacting envelopment and secretion of infectious particles. Finally, we identified PACS-2 as a crucial host factor contributing to CCHFV GPs trafficking required for assembly and release of viral particles.

Viruses traverse the human proteome through peptide interfaces that can be biomimetically leveraged for drug discovery.

We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.

[Role of cellular metabolism in the control of chronic viral hepatitis]. / Rôle du métabolisme cellulaire dans le contrôle des hépatites virales chroniques.

Hepatitis viruses modify the cellular metabolism of hepatocytes by interacting with specific enzymes such as glucokinase. The metabolic changes induced by viruses can have a direct impact on the innate antiviral response. The complex interactions between viral components, innate immunity, and hepatocyte metabolism explain why chronic hepatitis infections lead to liver inflammation, progressing to cirrhosis, fibrosis, and hepatocellular carcinoma. Metabolic regulators could be used in innovative therapies to deprive viruses of key metabolites and induce an antiviral defense.

Title: Rôle du métabolisme cellulaire dans le contrôle des hépatites virales chroniques. Abstract: Les virus des hépatites modifient le métabolisme cellulaire des hépatocytes en interagissant avec des enzymes spécifiques, telles que la glucokinase. Les changements métaboliques induits par les virus peuvent avoir un impact direct sur la réponse antivirale innée. Les interactions complexes entre les composants viraux, l'immunité innée et le métabolisme des hépatocytes expliquent pourquoi les infections hépatiques chroniques conduisent à l'inflammation du foie, évoluant vers la cirrhose, la fibrose et le carcinome hépatocellulaire. Des régulateurs du métabolisme pourraient être utilisés dans des thérapies innovantes pour priver les virus de métabolites clés et induire une défense antivirale.

Farnesoid X receptor alpha ligands inhibit HDV in vitro replication and virion infectivity.

BACKGROUND AND AIMS: HDV, a satellite of HBV, is responsible for the most severe form of human viral hepatitis, for which curative therapy is still awaited. Both HBV and HDV use the hepatic transporter of bile acids (ie, Na+-taurocholate cotransporting polypeptide) to enter hepatocytes. We have previously shown that ligands of the farnesoid-X-receptor alpha (FXR), a master regulator of bile acids metabolism, inhibit HBV replication. Here we asked whether FXR ligands can also control HDV infection. APPROACH AND RESULTS: In vitro HDV monoinfections or HDV/HBV coinfections and superinfections were performed in differentiated HepaRG cells (dHepaRG) and primary human hepatocytes. Following treatment with FXR ligands, HDV RNAs and antigens were analyzed by RT-qPCR, northern blot, immunofluorescence, and western blot. Virus secretion was studied by RNA quantification in supernatants, and the infectivity of secreted HDV particles was measured by reinfection of naive HuH7.5-Na+-taurocholate cotransporting polypeptide cells. In HDV/HBV superinfection models, a 10-day treatment with FXR ligand GW4064 decreased intracellular HDV RNAs by 60% and 40% in dHepaRG cells and primary human hepatocytes, respectively. Both HDV genomic and antigenomic RNAs were affected by treatment, which also reduced the amount of intracellular delta antigen. This antiviral effect was also observed in HDV monoinfected dHepaRG cells, abolished by FXR loss of function, and reproduced with other FXR ligands. In HBV/HDV coinfected dHepaRG cells, HDV secretion was decreased by 60% and virion-specific infectivity by >95%. CONCLUSIONS: FXR ligands both inhibit directly (ie, independently of anti-HBV activity) and indirectly (ie, dependently of anti-HBV activity) the replication, secretion, and infectivity of HDV. The overall anti-HDV activity was superior to that obtained with interferon-α, highlighting the therapeutic potential of FXR ligands in HDV-infected patients.

What role for cellular metabolism in the control of hepatitis viruses?

Hepatitis B, C and D viruses (HBV, HCV, HDV, respectively) specifically infect human hepatocytes and often establish chronic viral infections of the liver, thus escaping antiviral immunity for years. Like other viruses, hepatitis viruses rely on the cellular machinery to meet their energy and metabolite requirements for replication. Although this was initially considered passive parasitism, studies have shown that hepatitis viruses actively rewire cellular metabolism through molecular interactions with specific enzymes such as glucokinase, the first rate-limiting enzyme of glycolysis. As part of research efforts in the field of immunometabolism, it has also been shown that metabolic changes induced by viruses could have a direct impact on the innate antiviral response. Conversely, detection of viral components by innate immunity receptors not only triggers the activation of the antiviral defense but also induces in-depth metabolic reprogramming that is essential to support immunological functions. Altogether, these complex triangular interactions between viral components, innate immunity and hepatocyte metabolism may explain why chronic hepatitis infections progressively lead to liver inflammation and progression to cirrhosis, fibrosis and hepatocellular carcinoma (HCC). In this manuscript, we first present a global overview of known connections between the innate antiviral response and cellular metabolism. We then report known molecular mechanisms by which hepatitis viruses interfere with cellular metabolism in hepatocytes and discuss potential consequences on the innate immune response. Finally, we present evidence that drugs targeting hepatocyte metabolism could be used as an innovative strategy not only to deprive viruses of key metabolites, but also to restore the innate antiviral response that is necessary to clear infection.

Targeting the liver X receptor with dendrogenin A differentiates tumour cells to secrete immunogenic exosome-enriched vesicles.

Tumour cells are characterized by having lost their differentiation state. They constitutively secrete small extracellular vesicles (sEV) called exosomes when they come from late endosomes. Dendrogenin A (DDA) is an endogenous tumour suppressor cholesterol-derived metabolite. It is a new class of ligand of the nuclear Liver X receptors (LXR) which regulate cholesterol homeostasis and immunity. We hypothesized that DDA, which induces tumour cell differentiation, inhibition of tumour growth and immune cell infiltration into tumours, could functionally modify sEV secreted by tumour cells. Here, we have shown that DDA differentiates tumour cells by acting on the LXRß. This results in an increased production of sEV (DDA-sEV) which includes exosomes. The DDA-sEV secreted from DDA-treated cells were characterized for their content and activity in comparison to sEV secreted from control cells (C-sEV). DDA-sEV were enriched, relatively to C-sEV, in several proteins and lipids such as differentiation antigens, "eat-me" signals, lipidated LC3 and the endosomal phospholipid bis(monoacylglycero)phosphate, which stimulates dendritic cell maturation and a Th1 T lymphocyte polarization. Moreover, DDA-sEV inhibited the growth of tumours implanted into immunocompetent mice compared to control conditions. This study reveals a pharmacological control through a nuclear receptor of exosome-enriched tumour sEV secretion, composition and immune function. Targeting the LXR may be a novel way to reprogram tumour cells and sEV to stimulate immunity against cancer.

Domain 2 of Hepatitis C Virus Protein NS5A Activates Glucokinase and Induces Lipogenesis in Hepatocytes.

Hepatitis C virus (HCV) relies on cellular lipid metabolism for its replication, and actively modulates lipogenesis and lipid trafficking in infected hepatocytes. This translates into an intracellular accumulation of triglycerides leading to liver steatosis, cirrhosis and hepatocellular carcinoma, which are hallmarks of HCV pathogenesis. While the interaction of HCV with hepatocyte metabolic pathways is patent, how viral proteins are able to redirect central carbon metabolism towards lipogenesis is unclear. Here, we report that the HCV protein NS5A activates the glucokinase (GCK) isoenzyme of hexokinases through its D2 domain (NS5A-D2). GCK is the first rate-limiting enzyme of glycolysis in normal hepatocytes whose expression is replaced by the hexokinase 2 (HK2) isoenzyme in hepatocellular carcinoma cell lines. We took advantage of a unique cellular model specifically engineered to re-express GCK instead of HK2 in the Huh7 cell line to evaluate the consequences of NS5A-D2 expression on central carbon and lipid metabolism. NS5A-D2 increased glucose consumption but decreased glycogen storage. This was accompanied by an altered mitochondrial respiration, an accumulation of intracellular triglycerides and an increased production of very-low density lipoproteins. Altogether, our results show that NS5A-D2 can reprogram central carbon metabolism towards a more energetic and glycolytic phenotype compatible with HCV needs for replication.

Instability of the NS1 Glycoprotein from La Reunion 2018 Dengue 2 Virus (Cosmopolitan-1 Genotype) in Huh7 Cells Is Due to Lysine Residues on Positions 272 and 324.

La Reunion island in the South West Indian Ocean is now endemic for dengue following the introduction of dengue virus serotype 2 (DENV-2) cosmopolitan-I genotype in 2017. DENV-2 infection causes a wide spectrum of clinical manifestations ranging from flu-like disease to severe dengue. The nonstructural glycoprotein 1 (NS1) has been identified as playing a key role in dengue disease severity. The intracellular NS1 exists as a homodimer, whereas a fraction is driven towards the plasma membrane or released as a soluble hexameric protein. Here, we characterized the NS1 glycoproteins from clinical isolates DES-14 and RUN-18 that were collected during the DENV-2 epidemics in Tanzania in 2014 and La Reunion island in 2018, respectively. In relation to hepatotropism of the DENV, expression of recombinant DES-14 NS1 and RUN-18 NS1 glycoproteins was compared in human hepatoma Huh7 cells. We observed that RUN-18 NS1 was poorly stable in Huh7 cells compared to DES-14 NS1. The instability of RUN-18 NS1 leading to a low level of NS1 secretion mostly relates to lysine residues on positions 272 and 324. Our data raise the issue of the consequences of a defect in NS1 stability in human hepatocytes in relation to the major role of NS1 in the pathogenesis of the DENV-2 infection.

A hexokinase isoenzyme switch in human liver cancer cells promotes lipogenesis and enhances innate immunity.

During the cancerous transformation of normal hepatocytes into hepatocellular carcinoma (HCC), the enzyme catalyzing the first rate-limiting step of glycolysis, namely the glucokinase (GCK), is replaced by the higher affinity isoenzyme, hexokinase 2 (HK2). Here, we show that in HCC tumors the highest expression level of HK2 is inversely correlated to GCK expression, and is associated to poor prognosis for patient survival. To further explore functional consequences of the GCK-to-HK2 isoenzyme switch occurring during carcinogenesis, HK2 was knocked-out in the HCC cell line Huh7 and replaced by GCK, to generate the Huh7-GCK+/HK2- cell line. HK2 knockdown and GCK expression rewired central carbon metabolism, stimulated mitochondrial respiration and restored essential metabolic functions of normal hepatocytes such as lipogenesis, VLDL secretion, glycogen storage. It also reactivated innate immune responses and sensitivity to natural killer cells, showing that consequences of the HK switch extend beyond metabolic reprogramming.

Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de novo pyrimidine biosynthesis inhibition.

There is an increasing interest in the field of cancer therapy for small compounds targeting pyrimidine biosynthesis, and in particular dihydroorotate dehydrogenase (DHODH), the fourth enzyme of this metabolic pathway. Three available DHODH structures, featuring three different known inhibitors, were used as templates to screen in silico an original chemical library from Erevan University. This process led to the identification of P1788, a compound chemically related to the alkaloid cerpegin, as a new class of pyrimidine biosynthesis inhibitors. In line with previous reports, we investigated the effect of P1788 on the cellular innate immune response. Here we show that pyrimidine depletion by P1788 amplifies cellular response to both type-I and type II interferons, but also induces DNA damage as assessed by γH2AX staining. Moreover, the addition of inhibitors of the DNA damage response led to the suppression of the P1788 stimulatory effects on the interferon pathway. This demonstrates that components of the DNA damage response are bridging the inhibition of pyrimidine biosynthesis by P1788 to the interferon signaling pathway. Altogether, these results provide new insights on the mode of action of novel pyrimidine biosynthesis inhibitors and their development for cancer therapies.

Farnesoid X receptor-α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.

Hepatitis B virus (HBV) infection and bile acid (BA) metabolism are interdependent: infection modifies the expression of the BA nuclear receptor farnesoid X receptor (FXR)-α, and modulation of FXRα activity by ligands alters HBV replication. Mechanisms of HBV control by FXRα remain to be unveiled. FXRα silencing in HBV-infected HepaRG cells decreased the viral covalently closed circular (ccc)DNA pool size and transcriptional activity. Treatment with the FXRα agonist GW4064 inhibited FXRα proviral effect on cccDNA similarly for wild-type and hepatitis B viral X protein (HBx)-deficient virus, whereas agonist-induced inhibition of pregenomic and precore RNA transcription and viral DNA secretion was HBx dependent. These data indicated that FXRα acts as a proviral factor by 2 different mechanisms, which are abolished by FXRα stimulation. Finally, infection of C3H/HeN mice by a recombinant adeno-associated virus-2/8-HBV vector induced a sustained HBV replication in young mice in contrast with the transient decline in adult mice. Four-week GW4064 treatment of infected C3H/HeN mice decreased secretion of HBV DNA and HB surface antigen in adult mice only. These results suggest that the physiologic balance of FXRα expression and activation by bile acid is a key host metabolic pathway in the regulation of HBV infection and that FXRα can be envisioned as a target for HBV treatment.-Mouzannar, K., Fusil, F., Lacombe, B., Ollivier, A., Ménard, C., Lotteau, V., Cosset, F.-L., Ramière, C., André, P. Farnesoid X receptor α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.

Nucleolin interacts with influenza A nucleoprotein and contributes to viral ribonucleoprotein complexes nuclear trafficking and efficient influenza viral replication.

Influenza viruses replicate their single-stranded RNA genomes in the nucleus of infected cells and these replicated genomes (vRNPs) are then exported from the nucleus to the cytoplasm and plasma membrane before budding. To achieve this export, influenza viruses hijack the host cell export machinery. However, the complete mechanisms underlying this hijacking remain not fully understood. We have previously shown that influenza viruses induce a marked alteration of the nucleus during the time-course of infection and notably in the nucleolar compartment. In this study, we discovered that a major nucleolar component, called nucleolin, is required for an efficient export of vRNPs and viral replication. We have notably shown that nucleolin interacts with the viral nucleoprotein (NP) that mainly constitutes vRNPs. Our results suggest that this interaction could allow vRNPs to "catch" the host cell export machinery, a necessary step for viral replication.

Reciprocal regulation of farnesoid X receptor α 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) α 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 and of infection on the FXR pathway. In HepaRG cells, FXR agonists (6-ethyl chenodeoxycholic acid and GW4064), but no antagonist, and an FXR-unrelated bile salt inhibited viral mRNA, DNA, and protein production (IC50, 0.1-0.5 µM) 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 PHHs with GW4064. Infection of these cells increased expression of FXR and modified expression of FXR-regulated genes SHP, APOA1, NTCP, CYP7A1, and CYP8B1 with a more pronounced effect in PHHs than in HepaRG cells. FXR agonists reversed all but one of the HBV-induced FXR gene profile modifications. HBV replication and FXR regulation seem to be interdependent, and altered bile salt metabolism homeostasis might contribute to the persistence of HBV infection.-Radreau, P., Porcherot, M., Ramière, C., Mouzannar, K., Lotteau, V., André, P. Reciprocal regulation of farnesoid X receptor α activity and hepatitis B virus replication in differentiated HepaRG cells and primary human hepatocytes.

Early Steps of Jaagsiekte Sheep Retrovirus-Mediated Cell Transformation Involve the Interaction between Env and the RALBP1 Cellular Protein.

UNLABELLED: Ovine pulmonary adenocarcinoma is a naturally occurring lung cancer in sheep induced by the Jaagsiekte sheep retrovirus (JSRV). Its envelope glycoprotein (Env) carries oncogenic properties, and its expression is sufficient to induce in vitro cell transformation and in vivo lung adenocarcinoma. The identification of cellular partners of the JSRV envelope remains crucial for deciphering mechanisms leading to cell transformation. We initially identified RALBP1 (RalA binding protein 1; also known as RLIP76 or RIP), a cellular protein implicated in the ras pathway, as a partner of JSRV Env by yeast two-hybrid screening and confirmed formation of RALBP1/Env complexes in mammalian cells. Expression of the RALBP1 protein was repressed in tumoral lungs and in tumor-derived alveolar type II cells. Through its inhibition using specific small interfering RNA (siRNA), we showed that RALBP1 was involved in envelope-induced cell transformation and in modulation of the mTOR (mammalian target of rapamycin)/p70S6K pathway by the retroviral envelope. IMPORTANCE: JSRV-induced lung adenocarcinoma is of importance for the sheep industry. While the envelope has been reported as the oncogenic determinant of the virus, the cellular proteins directly interacting with Env are still not known. Our report on the formation of RALBP/Env complexes and the role of this interaction in cell transformation opens up a new hypothesis for the dysregulation observed upon virus infection in sheep.

Control of temporal activation of hepatitis C virus-induced interferon response by domain 2 of nonstructural protein 5A.

BACKGROUND & AIMS: Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a multifunctional protein playing a crucial role in diverse steps of the viral replication cycle and perturbing multiple host cell pathways. We showed previously that removal of a region in domain 2 (D2) of NS5A (mutant NS5A(D2Δ)) is dispensable for viral replication in hepatoma cell lines. By using a mouse model and immune-competent cell systems, we studied the role of D2 in controlling the innate immune response. METHODS: In vivo replication competence of NS5A(D2Δ) was studied in transgenic mice with human liver xenografts. Results were validated using primary human hepatocytes (PHHs) and mechanistic analyses were conducted in engineered Huh7 hepatoma cells with reconstituted innate signaling pathways. RESULTS: Although the deletion in NS5A removed most of the interferon (IFN) sensitivity determining-region, mutant NS5A(D2Δ) was as sensitive as the wild type to IFN-α and IFN-λ in vitro, but severely attenuated in vivo. This attenuation could be recapitulated in PHHs and was linked to higher activation of the IFN response, concomitant with reduced viral replication and virus production. Importantly, immune-reconstituted Huh7-derived cell lines revealed a sequential activation of the IFN-response via RIG-I (retinoic acid-inducible gene I) and MDA5 (Myeloma differentiation associated factor 5), respectively, that was significantly higher in the case of the mutant lacking most of NS5A D2. CONCLUSIONS: Our study reveals an important role of NS5A D2 for suppression of the IFN response that is activated by HCV via RIG-I and MDA5 in a sequential manner.

Epstein-Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1.

The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.

Activity of hexokinase is increased by its interaction with hepatitis C virus protein NS5A.

UNLABELLED: The study of cellular central carbon metabolism modulations induced by viruses is an emerging field. Human cytomegalovirus (HCMV), herpes simplex virus (HSV), Kaposi's sarcoma-associated herpesvirus (KSHV), and hepatitis C virus (HCV) have been shown recently to reprogram cell metabolism to support their replication. During HCV infection the global glucidolipidic metabolism of hepatocytes is highly impacted. It was suggested that HCV might modify glucose uptake and glycolysis to increase fatty acids synthesis, but underlying mechanisms have not been completely elucidated. We thus investigated how HCV may modulate glycolysis. We observed that in infected Huh7.5 cells and in subgenomic replicon-positive Huh9.13 cells, glucose consumption as well as lactate secretion was increased. Using protein complementation assays and coimmunoprecipitation, we identified a direct interaction between the HCV NS5A protein and cellular hexokinase 2 (HK2), the first rate-limiting enzyme of glycolysis. NS5A expression was sufficient to enhance glucose consumption and lactate secretion in Huh7.5 cells. Moreover, determination of HK activity in cell homogenates revealed that addition of exogenous NS5A protein, either the full-length protein or its D2 or D3, but not D1, domain, was sufficient to increase enzyme activity. Finally, determination of recombinant HK2 catalytic parameters (V(max) and K(m)) in the presence of NS5A identified this viral protein as an activator of the enzyme. In summary, this study describes a direct interaction between HCV NS5A protein and cellular HK2 which is accompanied by an increase in HK2 activity that might contribute to an increased glycolysis rate during HCV infection. IMPORTANCE: Substantial evidence indicates that viruses reprogram the central carbon metabolism of the cell to support their replication. Nevertheless, precise underlying mechanisms are poorly described. Metabolic pathways are structured as connected enzymatic cascades providing elemental biomolecular blocks necessary for cell life and viral replication. In this study, we observed an increase in glucose consumption and lactate secretion in HCV-infected cells, revealing higher glycolytic activity. We also identified an interaction between the HCV NS5A nonstructural protein and cellular hexokinase 2, the first rate-limiting enzyme of glycolysis. This interaction results in an enhancement of catalytic parameters of the enzyme, which might explain, at least in part, the aerobic glycolysis shift observed in HCV-infected cells.

The metabolic sensors FXRα, PGC-1α, 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 α (FXRα) enhances HBV core promoter activity and pregenomic RNA synthesis. Interestingly, two food-withdrawal-induced FXRα modulators, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and deacetylase SIRT1, have been found to be associated with HBV genomes ex vivo. Whereas PGC-1α 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 FXRα 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 FXRα in overexpression and silencing experiments, we demonstrated that SIRT1 activated the core promoter in an FXRα- and PGC-1α-dependent manner. Maximal activation (>150-fold) was observed in FXRα- and PGC-1α-overexpressing Huh-7 cells treated with FXRα 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.