Final Report Summary - HCV-BOUND PROTEINS (Hepatitis C virus virion-bound proteins: identification in clinical samples and in vitro dissection of their importance in the viral life cycle)
Project’s public website
http://www.crcl.fr/329-R-Parent-Group.crcl.aspx?language=en-GB
HBV and HCV infections concern 400 million and 170 million people worldwide, respectively. Although much progress has been accomplished in the basic understanding of both viruses' life cycles, knowledge is lacking on the composition of the virions and the implication of the biochemical components of these virions in viral functions such as egress and entry.
In this project, we have developed novel cellular tools to enrich knowledge of the virions/target cells interactions through these proteins, and refined the datas potentially explaining how virion infectivity can be modulated by the implication of such virion-bound host proteins, using HCV as a model system. Other publications derived from the necessities or the advances generated by the project are also mentioned
Results
1 – We have identified FIG4 as a PI-modifying enzyme bound to HCV virions, contributing to the increased knowledge about the making up of a final infectious HCV particle. Our proteomic analysis performed on double gradient-purified hepatitis C virus from highly viremic patients identified the Phosphatidylinositol 3,5-bisphosphate 5-phosphatase FIG4 (SAC3/KIAA0274) as part of the viral particles. These results were validated using immunoprecipitation and neutralization experiments. Reduction of FIG4 expression using RNA interference decreased cholesteryl esters (CE) levels concomitantly with intracellular and supernatant viral infectivity without affecting HCV RNA levels. Conversely, forced expression of WT FIG4 increased intracellular CE levels concomitantly with intracellular and supernatant viral infectivity without affecting HCV RNA levels either. In this setting, specific infectivity peak of secreted material shifted towards fractions of lower density. Triglyceride (TG) levels and lipid droplets (LD) parameters remained unaffected. The 3,5-bisphosphate 5-phosphatase active site of FIG4 was found mandatory for these results. No implication of FIG4 in the secretory pathway of the hepatocyte could be found using either FIG4 null mice or in vitro morphometry or functional assays of the ERGIC/Golgi compartments. Because of the documented implication of CE in the making up and infectivity of HCV, these results show that FIG4 binds to HCV and modulates particle formation in a CE-related manner. Under review.
2- We have evidenced that Very-low-density lipoprotein (VLDL)-producing and hepatitis C virus-replicating HepG2 cells secrete no more lipoviroparticles than VLDL-deficient Huh7.5 cells. In the plasma samples of hepatitis C virus (HCV)-infected patients, lipoviroparticles (LVPs), defined as (very-) low-density viral particles immunoprecipitated with anti-β-lipoproteins antibodies are observed. This HCV-lipoprotein association has major implications with respect to our understanding of HCV assembly, secretion, and entry. However, cell culture-grown HCV (HCVcc) virions produced in Huh7 cells, which are deficient for very-low-density lipoprotein (VLDL) secretion, are only associated with and dependent on apolipoprotein E (apoE), not apolipoprotein B (apoB), for assembly and infectivity. In contrast to Huh7, HepG2 cells can be stimulated to produceVLDL by both oleic acid treatment and inhibition of the MEK/extracellular signal-regulated kinase (ERK) pathway but are not permissive for persistent HCV replication. Here, we developed a new HCV cell culture model to study the interaction between HCV and lipoproteins, based on engineered HepG2 cells stably replicating a blasticidin-tagged HCV JFH1 strain (JB). Control Huh7.5-JB as well as HepG2-JB cell lines persistently replicated viral RNA and expressed viral proteins with a subcellular colocalization of double-stranded RNA (dsRNA), core, gpE2, and NS5A compatible with virion assembly. The intracellular RNA replication level was increased in HepG2-JB cells upon dimethyl sulfoxide (DMSO) treatment, MEK/ERK inhibition, and NS5A overexpression to a level similar to that observed in Huh7.5-JB cells. Both cell culture systems produced infectious virions, which were surprisingly biophysically and biochemically similar. They floated at similar densities on gradients, contained mainly apoE but not apoB, and were not neutralized by anti-apoB antibodies. This suggests that there is no correlation between the ability of cells to simultaneously replicate HCV as well as secrete VLDL and their capacity to produce LVPs. (J Virol. 2013 May;87(9):5065-80. doi: 10.1128/JVI.01405-12.)
3 – We have identified that Netrin-1, a potent carcinogenic molecule was hijacked by HCV in order to increase its specific infectivity. The present study investigated the possible interplay between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis in cancer and, in particular, in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV(+) and hepatitis B virus (HBV)(+) liver biopsies, as compared to uninfected samples. Netrin-1 expression correlates with HCV replication levels in the liver tissue of chronically HCV-infected patients. Netrin-1 is upregulated in all histological stages of HCV(+) hepatic lesions (from minimal liver fibrosis to cirrhosis and HCC) compared to histologically-matched HCV(-) tissues. Both cirrhosis and HCV contribute to induce Netrin-1 expression. Netrin-1 expression is reduced upon anti-HCV treatment and parallels changes in viral load. In vitro, HCV increases the levels and translation of the Netrin-1 mRNA. Knockdown and forced-expression experiments identified the dependence receptor UNC5A as a conveyer of the Netrin-1 signal, which however did not affect death of HCV-infected cells. Netrin-1 enhances HCV virion infectivity and promotes virion entry by increasing the activation and decreasing the recycling of the Epidermal Growth Factor Receptor (EGFR). Hence, Netrin-1 is induced by HCV and is involved in HCV persistence through its ability to up-regulate the function of EGFR, a molecule that is dysregulated in HCC. In revision.
4 - In order to develop novel hepatocytic culture systems for virion production (ongoing work), we have defined the diversity of hepatocellular carcinoma clones bearing hematopoietic malignancies-related chromosomal translocation. Interpatient heterogeneity of hepatocellular carcinoma has been in-depth addressed. Intrapatient heterogeneity is less known. Four clones were freshly isolated from an Edmondson grade I HCV-associated hepatocellular carcinoma. Biochemical approaches, functional assays and cytogenetics were used. Albumin inducibility was uncoupled from canonical cytokeratin profiles, suggesting pathological combinations of hepatospecific and biliary markers. Poor differentiation and TGFβ's proproliferative effect on all clones were observed. TGFβ, Interferon α and doxorubicin sensitivity levels were found highly heterogeneous. Progenitor and stem cells markers OV6 and EpCAM were mutually exclusively expressed. All clones were CD44+, while none expressed CD90, CD133, or CD117. Three clones displayed a liver progenitor OV6+ phenotype, and were susceptible to hepatocytic differentiation, among which one fibroblastoid clone displayed intrahepatic parenchymal engraftment capability. A fourth clone, the less motile, displayed a cancer stem cell EpCAM+ phenotype, was essentially β-catenin negative, and was as expected devoid of hepatocytic differentiation capability, yet the most sensitive to doxorubicin treatment. Cytogenetics evidenced in all clones a t(12;22)(p11;q11) translocation found in several myelodysplastic syndromes. All clones, that probably derive from EpCAM+ tumor cells, display aberrant E-cadherin cytosolic localization. Because of their diverse pathophysiolocal features, these freshly isolated, low population doubling-defined, HCC clones may provide novel opportunities to tackle HCC heterogeneity in a single patient background for therapy improvement purposes, especially regarding recently developed targeted strategies. (J Cell Biochem. 2014 Apr;115(4):666-77.)
5 - In order to study infectivity dependence of virions to the hepatic microenvironment prior to infection on hepatocytes, we have developed an immortalized human liver endothelial sinusoidal cell line. The endothelium lines blood and lymph vessels and protects underlying tissues against external agents such as viruses, bacteria and parasites. Yet, microbes and particularly viruses have developed sophisticated ways to bypass the endothelium in order to gain access to inner organs. De novo infection of the liver parenchyma by many viruses and notably hepatitis viruses, is thought to occur through recruitment of virions on the sinusoidal endothelial surface and subsequent transfer to the epithelium. Furthermore, the liver endothelium undergoes profound changes with age and in inflammation or infection. However, primary human liver sinusoidal endothelial cells (LSECs) are difficult to obtain due to scarcity of liver resections. Relevant derived cell lines are needed in order to analyze in a standardized fashion the transfer of pathogens across the liver endothelium. By lentiviral transduction with hTERT only, we have immortalized human LSECs isolated from a hereditary hemorrhagic telangiectasia (HHT) patient and established the non-transformed cell line TRP3. TRP3 express mesenchymal, endothelial and liver sinusoidal markers. Functional assessment of TRP3 cells demonstrated a high capacity of endocytosis, tube formation andreactivity to immune stimulation. However, TRP3 displayed few fenestrae and expressed C-type lectins intracellularly. All these findings were confirmed in the original primary LSECs from which TRP3 were derived suggesting that these features were already present in the liver donor. We consider TRP3 as a model to investigate the functionality of the liver endothelium in hepatic inflammation in infection. (Biochem Biophys Res Commun. 2014 Jul 18;450(1):7-12.)
Conclusions
We have evidenced that Very-low-density lipoprotein (VLDL)-producing and hepatitis C virus-replicating HepG2 cells secrete no more lipoviroparticles than VLDL-deficient Huh7.5 cells, which prompted us to develop more physiological, untransformed cell lines for production of HCV virions of adequate density and biochemical composition. Functional results have been obtained on one HCV-bound protein (a PI phosphatase). In order to study infectivity dependence of virions to the hepatic microenvironment prior to infection on hepatocytes, we have developed an immortalized human liver endothelial sinusoidal cell line. This study is ongoing in order to decipher the early interactions of HBV and HCV virions through their proteome with the liver upon primoinfection, one of the main goals of the laboratory (F. Zoulim/D. Durantel).
More globally our research will aim at increasing knowledge on the contribution of virions-bound proteins to the early interactions of hepatotropic virus virions and their microenvironment in the innate immunity field. From a socioeconomic prospective, these results should end up in the advancement of therapeutic opportunities in the field of chronic hepatitis B, as well as liver transplantation in general, through opsonisation of infectious virions using human proteins as targets.
Groups of the civil society potentially interested by this work are virology and hepatology communities.
Project’s public website: http://www.crcl.fr/329-R-Parent-Group.crcl.aspx?language=en-GB