RESUMO
Chronic infection with hepatitis C virus (HCV) is one of the main causes of hepatocellular carcinoma. However, the molecular mechanisms linking the infection to cancer development remain poorly understood. Here we used HCV-infected cells and liver biopsies to study how HCV modulates the glutaminolysis pathway, which is known to play an important role in cellular energetics, stress defense, and neoplastic transformation. Transcript levels of glutaminolytic factors were quantified in Huh7.5 cells or primary human hepatocytes infected with the Japanese fulminant hepatitis 1 HCV strain as well as in biopsies of chronic HCV patients. Nutrient deprivation, biochemical analysis, and metabolite quantification were performed with HCV-infected Huh7.5 cells. Furthermore, short hairpin RNA vectors and small molecule inhibitors were used to investigate the dependence of HCV replication on metabolic changes. We show that HCV modulates the transcript levels of key enzymes of glutamine metabolism in vitro and in liver biopsies of chronic HCV patients. Consistently, HCV infection increases glutamine use and dependence. We finally show that inhibiting glutamine metabolism attenuates HCV infection and the oxidative stress associated with HCV infection. CONCLUSION: Our data suggest that HCV establishes glutamine dependence, which is required for viral replication, and, importantly, that glutamine addiction is a hallmark of tumor cells. While HCV induces glutaminolysis to create an environment favorable for viral replication, it predisposes the cell to transformation. Glutaminolytic enzymes may be interesting therapeutic targets for prevention of hepatocarcinogenesis in chronic hepatitis C. (Hepatology 2017;65:789-803).
Assuntos
Glutamina/metabolismo , Hepacivirus/patogenicidade , Hepatócitos/metabolismo , Hepatócitos/virologia , Replicação Viral/genética , Biópsia por Agulha , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Células Cultivadas , Hepacivirus/genética , Hepatite C Crônica/patologia , Hepatite C Crônica/fisiopatologia , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estatísticas não Paramétricas , Transfecção/métodosRESUMO
OBJECTIVE: Inflammation and oxidative stress drive disease progression in chronic hepatitis C (CHC) towards hepatocellular carcinoma. HCV is known to increase intracellular levels of reactive oxygen species (ROS), but how it eliminates ROS is less well known. The role of the ROS scavenger glutathione peroxidase 4 (GPx4), induced by HCV, in the viral life cycle was analysed. DESIGN: The study was performed using a replicative in vitro HCV infection model and liver biopsies derived from two different CHC patient cohorts. RESULTS: A screen for HCV-induced peroxide scavengers identified GPx4 as a host factor required for HCV infection. The physiological role of GPx4 is the elimination of lipid peroxides from membranes or lipoproteins. GPx4-silencing reduced the specific infectivity of HCV by up to 10-fold. Loss of infectivity correlated with 70% reduced fusogenic activity of virions in liposome fusion assays. NS5A was identified as the protein that mediates GPx4 induction in a phosphatidylinositol-3-kinase-dependent manner. Levels of GPx4 mRNA were found increased in vitro and in CHC compared with control liver biopsies. Upon successful viral eradication, GPx4 transcript levels returned to baseline in vitro and also in the liver of patients. CONCLUSIONS: HCV induces oxidative stress but controls it tightly by inducing ROS scavengers. Among these, GPx4 plays an essential role in the HCV life cycle. Modulating oxidative stress in CHC by specifically targeting GPx4 may lower specific infectivity of virions and prevent hepatocarcinogenesis, especially in patients who remain difficult to be treated in the new era of interferon-free regimens.
Assuntos
Glutationa Peroxidase/metabolismo , Hepacivirus/patogenicidade , Hepatite C Crônica/virologia , Peroxidação de Lipídeos , Fígado/virologia , Vírion/patogenicidade , Adulto , Biomarcadores , Biópsia , Estudos de Casos e Controles , Linhagem Celular , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Hepacivirus/metabolismo , Hepatite C Crônica/enzimologia , Hepatite C Crônica/patologia , Humanos , Fígado/enzimologia , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Espécies Reativas de Oxigênio/metabolismo , Vírion/metabolismoRESUMO
Group A rotaviruses, members of the family Reoviridae, are a major cause of infantile acute gastroenteritis. The rotavirus genome consists of 11 dsRNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. It has been shown that some rearranged segments are preferentially encapsidated into viral progenies after serial passages in cell culture. Based on this characteristic, a reverse genetics system was used previously to introduce exogenous segment 7 rearrangements into an infectious rotavirus. This study extends this reverse genetics system to RNA segments 5 and 11. Transfection of exogenous rotavirus rearranged RNA segment 5 or 11 into cells infected with a WT helper rotavirus (bovine strain RF) resulted in subsequent gene rearrangements in the viral progeny. Whilst recombinant viruses were rescued with an exogenous rearranged segment 11, the exogenous segment was modified by a secondary rearrangement. The occurrence of spontaneous rearrangements of WT or exogenous segments is a major hindrance to the use of this reverse genetics approach.
Assuntos
Rearranjo Gênico/genética , RNA Viral/genética , Rotavirus/genética , Proteínas não Estruturais Virais/genética , Animais , Sequência de Bases , Células COS , Linhagem Celular , Galinhas , Chlorocebus aethiops , Rotavirus/patogenicidade , Infecções por Rotavirus/virologia , Análise de Sequência de RNA , TransfecçãoRESUMO
Hepatitis C virus (HCV) is an oncogenic virus that causes chronic liver disease in more than 80% of patients. During the last decade, efficient direct-acting antivirals were introduced into clinical practice. However, clearance of the virus does not reduce the risk of end-stage liver diseases to the level observed in patients who have never been infected. So, investigation of HCV pathogenesis is still warranted. Virus-induced changes in cell metabolism contribute to the development of HCV-associated liver pathologies. Here, we studied the impact of the virus on the metabolism of polyamines and proline as well as on the urea cycle, which plays a crucial role in liver function. It was found that HCV strongly suppresses the expression of arginase, a key enzyme of the urea cycle, leading to the accumulation of arginine, and up-regulates proline oxidase with a concomitant decrease in proline concentrations. The addition of exogenous proline moderately suppressed viral replication. HCV up-regulated transcription but suppressed protein levels of polyamine-metabolizing enzymes. This resulted in a decrease in polyamine content in infected cells. Finally, compounds targeting polyamine metabolism demonstrated pronounced antiviral activity, pointing to spermine and spermidine as compounds affecting HCV replication. These data expand our understanding of HCV's imprint on cell metabolism.
Assuntos
Hepacivirus , Poliaminas , Prolina , Ureia , Replicação Viral , Prolina/metabolismo , Humanos , Hepacivirus/fisiologia , Hepacivirus/efeitos dos fármacos , Poliaminas/metabolismo , Ureia/metabolismo , Ureia/farmacologia , Replicação Viral/efeitos dos fármacos , Arginase/metabolismo , Antivirais/farmacologia , Antivirais/metabolismo , Hepatite C/metabolismo , Hepatite C/virologia , Linhagem Celular Tumoral , Prolina Oxidase/metabolismoRESUMO
Background & Aims: Chronic HCV infection causes cellular stress, fibrosis and predisposes to hepatocarcinogenesis. Mitochondria play key roles in orchestrating stress responses by regulating bioenergetics, inflammation and apoptosis. To better understand the role of mitochondria in the viral life cycle and disease progression of chronic hepatitis C, we studied morphological and functional mitochondrial alterations induced by HCV using productively infected hepatoma cells and patient livers. Methods: Biochemical and imaging assays were used to assess localization of cellular and viral proteins and mitochondrial functions in cell cultures and liver biopsies. Cyclophilin D (CypD) knockout was performed using CRISPR/Cas9 technology. Viral replication was quantified by quantitative reverse-transcription PCR and western blotting. Results: Several HCV proteins were found to associate with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), the points of contact between the ER and mitochondria. Downregulation of CypD, which is known to disrupt MAM integrity, reduced viral replication, suggesting that MAMs play an important role in the viral life cycle. This process was rescued by ectopic CypD expression. Furthermore, HCV proteins were found to associate with voltage dependent anion channel 1 (VDAC1) at MAMs and to reduce VDAC1 protein levels at MAMs in vitro and in patient biopsies. This association did not affect MAM-associated functions in glucose homeostasis and Ca2+ signaling. Conclusions: HCV proteins associate specifically with MAMs and MAMs play an important role in viral replication. The association between viral proteins and MAMs did not impact Ca2+ signaling between the ER and mitochondria or glucose homeostasis. Whether additional functions of MAMs and/or VDAC are impacted by HCV and contribute to the associated pathology remains to be assessed. Impact and implications: Hepatitis C virus infects the liver, where it causes inflammation, cell damage and increases the long-term risk of liver cancer. We show that several HCV proteins interact with mitochondria in liver cells and alter the composition of mitochondrial subdomains. Importantly, HCV requires the architecture of these mitochondrial subdomains to remain intact for efficient viral replication.
RESUMO
Virophages can parasitize giant DNA viruses and may provide adaptive anti-giant virus defense in unicellular eukaryotes. Under laboratory conditions, the virophage mavirus integrates into the nuclear genome of the marine flagellate Cafeteria burkhardae and reactivates upon superinfection with the giant virus CroV. In natural systems, however, the prevalence and diversity of host-virophage associations has not been systematically explored. Here, we report dozens of integrated virophages in four globally sampled C. burkhardae strains that constitute up to 2% of their host genomes. These endogenous mavirus-like elements (EMALEs) separated into eight types based on GC-content, nucleotide similarity, and coding potential and carried diverse promoter motifs implicating interactions with different giant viruses. Between host strains, some EMALE insertion loci were conserved indicating ancient integration events, whereas the majority of insertion sites were unique to a given host strain suggesting that EMALEs are active and mobile. Furthermore, we uncovered a unique association between EMALEs and a group of tyrosine recombinase retrotransposons, revealing yet another layer of parasitism in this nested microbial system. Our findings show that virophages are widespread and dynamic in wild Cafeteria populations, supporting their potential role in antiviral defense in protists.
Viruses exist in all ecosystems in vast numbers and infect many organisms. Some of them are harmful but others can protect the organisms they infect. For example, a group of viruses called virophages protect microscopic sea creatures called plankton from deadly infections by so-called giant viruses. In fact, virophages need plankton infected with giant viruses to survive because they use enzymes from the giant viruses to turn on their own genes. A virophage called mavirus integrates its genes into the DNA of a type of plankton called Cafeteria. It lays dormant in the DNA until a giant virus called CroV infects the plankton. This suggests that the mavirus may be a built-in defense against CroV infections and laboratory studies seem to confirm this. But whether wild Cafeteria also use these defenses is unknown. Hackl et al. show that virophages are common in the DNA of wild Cafeteria and that the two appear to have a mutually beneficial relationship. In the experiments, the researchers sequenced the genomes of four Cafeteria populations from the Atlantic and Pacific Oceans and looked for virophages in their DNA. Each of the four Cafeteria genomes contained dozens of virophages, which suggests that virophages are important to these plankton. This included several relatives of the mavirus and seven new virophages. Virophage genes were often interrupted by so called jumping genes, which may take advantage of the virophages the way the virophages use giant viruses to meet their own needs. The experiments show that virophages often co-exist with marine plankton from around the world and these relationships are likely beneficial. In fact, the experiments suggest that the virophages may have played an important role in the evolution of these plankton. Further studies may help scientists learn more about virus ecology and how viruses have shaped the evolution of other creatures.
Assuntos
Genoma/fisiologia , Retroelementos/fisiologia , Estramenópilas/genética , Estramenópilas/virologia , Virófagos/fisiologia , FilogeniaRESUMO
The heterotrophic stramenopile Cafeteria roenbergensis is a globally distributed marine bacterivorous protist. This unicellular flagellate is host to the giant DNA virus CroV and the virophage mavirus. We sequenced the genomes of four cultured C. roenbergensis strains and generated 23.53 Gb of Illumina MiSeq data (99-282 × coverage per strain) and 5.09 Gb of PacBio RSII data (13-45 × coverage). Using the Canu assembler and customized curation procedures, we obtained high-quality draft genome assemblies with a total length of 34-36 Mbp per strain and contig N50 lengths of 148 kbp to 464 kbp. The C. roenbergensis genome has a GC content of ~70%, a repeat content of ~28%, and is predicted to contain approximately 7857-8483 protein-coding genes based on a combination of de novo, homology-based and transcriptome-supported annotation. These first high-quality genome assemblies of a bicosoecid fill an important gap in sequenced stramenopile representatives and enable a more detailed evolutionary analysis of heterotrophic protists.
Assuntos
Genoma , Estramenópilas/genética , Composição de Bases , Anotação de Sequência Molecular , Análise de Sequência de DNA , TranscriptomaRESUMO
The rotavirus (RV) genome consists of 11 double-stranded RNA segments. Sometimes, partial sequence duplication of an RNA segment leads to a rearranged RNA segment. To specify the impact of rearrangement, the replication efficiencies of human RV with rearranged segments 7, 11 or both were compared to these of the homologous human wild-type RV (wt-RV) and of the bovine wt-RV strain RF. As judged by viral growth curves, rotaviruses with a rearranged genome (r-RV) had no selective growth advantage over the homologous wt-RV. In contrast, r-RV were selected over wt-RV during competitive experiments (i.e mixed infections between r-RV and wt-RV followed by serial passages in cell culture). Moreover, when competitive experiments were performed between a human r-RV and the bovine wt-RV strain RF, which had a clear growth advantage, rearranged segments 7, 11 or both always segregated in viral progenies even when performing mixed infections at an MOI ratio of 1 r-RV to 100 wt-RV. Lastly, bovine reassortant viruses that had inherited a rearranged segment 7 from human r-RV were generated. Although substitution of wt by rearranged segment 7 did not result in any growth advantage, the rearranged segment was selected in the viral progenies resulting from mixed infections by bovine reassortant r-RV and wt-RV, even for an MOI ratio of 1 r-RV to 10(7) wt-RV. Lack of selective growth advantage of r-RV over wt-RV in cell culture suggests a mechanism of preferential packaging of the rearranged segments over their standard counterparts in the viral progeny.