Hepatitis C Virus Modulates Raji Cells Via E2-CD81 Engagement And Interferon Alpha Regulates Signaling Pathways In Human Hepatoma Cells

Hepatitis C virus (HCV) is an enveloped, positive-sense RNA virus, which belongs to the Flaviviridae family. The genome encodes one large polyprotein that is processed by viral and cellular proteinases to produce the viral structural proteins (core and glycoproteins E1 and E2) and nonstructural (NS) proteins (p7 through NS5B). Chronic liver disease with HCV infection is an important global health problem that affects 170 million people worldwide.Two heavily N-glycosylated glycoproteins El and E2 are virion-envelope proteins and form heterodimers, which act as the candidate ligands for cellular receptors.Hepatitis C virus modulates activation, survival and immunological phenotype of Raji cells via E2-CD81 engagementHuman CD81 is considered to be a cellular receptor for HCV, based on its ability to bind E2 protein. In fact, the infectivity of HCV pseudoparticle (HCVpp), cell culture produced HCV (HCVcc), and plasma-derived HCV all depend on CD81 expression on the host cell surface.CD81 is a widely distributed cell-surface tetraspanin that participates in different molecular complexes on various cell types, including hepatocytes, B lymphocytes, T lymphocytes and natural killer cells. It has been proposed that HCV exploits CD81 not only to invade hepatocytes but also to modulate the immune system. Indeed, cross-linking of CD81 by HCV E2 protein leads to co-stimulation of human T cells or inhibition of human NK cells in vitro.On B cell, CD81 is known to form B cell costimulatory complex with CD 19, CD21,and interferon-inducible Leu-13 (CD225) proteins. This complex reduces the threshold for B cell activation via the B cell receptor by bridging antigen specific recognition and CD21-mediated complement recognition.HCV infection is associated with B-cell regulatory control disturbance, such as mixed cryoglobulinemia (MC) and non-Hodgkin's lymphoma (NHL).HCV E2 protein is suggested as a regulator in HCV induced B-cell pathophysiology. It has been reported that engagement of CD81 on human B cells by a combination of HCV E2 protein and anti-CD81 mAb leads to the proliferation of the naive B cells, and E2-CD81 interaction induces protein tyrosine phosphorylation and hypermutation of the immunoglobulin genes in B cells. Although such effects of E2 on B cells maybe involved in the development of B-cell pathophysiology, more precise mechanisms need to be explored.The role of B-cell immunity in resolution of HCV infection is less well defined. Recently, it was confirmed that neutralizing antibodies, mainly targeting to E2 protein, play a crucial role in prevention and possibly recovery from viral infections. However, neutralizing antibodies showed relatively low titers and delayed appearance during HCV infection. The chimpanzee is the only available experimental system for HCV infection, the majority of infected chimpanzees developed a low titer neutralizing antibodies response late in disease, which failed to associate with viral clearance.The reasons for this remain to be addressed.In this report, HCV E2 protein and HCVcc were used to engage CD81 on Raji cell surface, we demonstrate that E2-CD81 engagement triggers phosphorylation of IκBα, up-regulates anti-apoptosis Bcl-2 family proteins, enhances Raji cells protection from Fas-mediated death. Moreover, E2-CD81 signal up-regulates CD81 and costimulatory molecules CD80 and CD86, and down-regulates complement receptor CD21.These results maybe helpful for.better understanding of the mechanisms involved in HCV associated B cell lymphoproliferative disorders and delayed appearance of neutralizing antibody.1.CD81 RNA interference,HCV E2 plasmid construction and protein expressionHuman CD81 siRNA expression plasmid pGCsi-U6-CD81 siRNA5 was used as template to amplify the siRNA expression cassette by PCR, then the siRNA expression cassette was inserted into lentivirus vector pLenti6. HEK 293 T cells were co-transfected with mixed plasmids, including vesicular stomatitis virus (VSV) glycoprotein expression plasmid, HIV gag/pol (pLP1)plasmid, HIV rev (pLP2) plasmid and transfer vector pLenti6 vectors containing CD81 siRNA expression cassette.48 h later, the culture supernatants containing lentiviral pseudoparticles were harvested and filtered through 0.45-μm-pore-size membranes. Raji cells were transduced overnight with the packaged lentivirus containing CD81 siRNA expression cassette or mock lentivirus, three days later, the cells were infected with the lentivirus once more, and then the CD81 expression on cell surface was assayed using a FACS Calibur instrument.DNA sequence encoding carboxyl terminal truncated E2 protein (aa 364-661 in HCV polyprotein) of strain H77, genotype la and A mutant version E2-W529/A, in which the 529th aa tryptophan was replaced by alanine, were inserted into pCI-neo plasmid. The expression plasmids were transfected into 293T cells respectively.72 h after transfection, the supernatant was removed, and cells were lysed. The recombinant proteins was assessed with goat anti E2 polyclonal antibodies using Western blotting.2.E2 binding with CHO and Raji cells, E2 coating and cell stimulationThe human CD81 expression plasmid and mock vector were separately transfected into CHO cells.48 h after transfection, the expression of CD81 on CHO cells was assayed by FACS(fluorescence-activated cell sorting).The binding of HCV E2 protein with the transfectant CHO cells and Raji cells were measured by FACS.E2 mAb H53 was diluted to 10μg/ml in carbonate buffer(15 mM Na2CO3,35 mm NaHCO3,pH 9.6) and added to each well of 96 or 24-well plates. The plates were incubated overnight at 4℃, and then washed three times with phosphate-buffered saline (PBS) and saturated for 30 min at 37℃with complete RPMI1640 medium. The cell extract of 293T cells transfected with HCV E2 expression plasmids or mock vector were added to the wells, and plates were incubated for 60 min at 37℃.After further washing with PBS buffer, Raji cells in complete medium were added to the coated plates, followed by incubation for various time periods.3. HCV, pseudoparticle (HCVpp) production and infection293T cells were cotransfected with expression vector encoding the HCV envelope glycoproteins, gag/pol (pLP1),rev (pLP2), and transfer vector encoding the luciferase. HCV envelope expression plasmids encoding El and E2 glycoproteins of genotype la strain H77, genotype lb strain con-1, genotype 2a strain J6 were used. Supernatants containing HCVpp were harvested at 48 h after transfection, filtered through 0.45-μm-pore-size membrane for infection use.Target cells, including Huh7.5,Raji cells were seeded into 96-well plates at a density of 1×104 cells/well and incubated overnight at 37℃.HCVpp supernatants were added 50μl to each well, and incubated for 5 h. The supernatants were removed and the cells were incubated in regular medium for 72 h at 37℃. Cells were washed once with PBS and lysed with 50μl of cell lysis bufferper well. Luciferase activities were quantified.4. Western blottingCell lysates were boiled for 5 min before being separated by sodium dodecylsulfate (SDS)-12.5% polyacrylamide gels electrophoresis(PAGE).Proteins were then electrophoretically transferred onto nitrocellulose membranes.The membrane was probed with kinds of primary antibodies.Then the membrane was incubated with peroxidase-conjugated anti-mouse IgG, anti-goat IgG or anti-rabbit IgG, respectively. Immunoreactivity was visualized with enhanced chemiluminescence.Interferon alpha Regulates Mitogen-activated Protein Kinase and STAT1 Signaling Pathways in Human Hepatoma CellsSignaling events induced by interferon alpha (IFN-a) account for the molecular mechanisms of its antiviral effect. JAK-STAT pathway plays a critical role in the IFN signaling, and other pathways are also implicated in the IFN-mediated antiviral effect. In this study, regulation of mitogen-activated protein kinase (MAPK) and STAT1 signaling pathways was evaluated in human hepatoma Huh7 and HepG2 cells upon IFN-a treatment. The phosphorylation of ERK was specifically up-regulated by IFN-a, whereas IFN-a seemed to be no effect on upstream kinase MEK phosphorylation. The mild increase in p38 MAPK phosphorylation and significant increase in SAPK/JNK phosphorylation were detectable after exposure to IFN-a, and the phosphorylation of downstream target ATF-2 was also increased, indicating differential up-regulation of the MAPK signaling cascades by IFN-a. The difference occurs in the regulation of MAPK pathways by IFN-a between Huh7 and HepG2.The STAT1 phosphorylation was strongly enhanced by IFN-a. These results show that IFN-a up-regulates MAPK and STAT1 signaling pathways in human hepatoma cells, and may provide information for understanding the IFN signaling.Summary:1.Both HCV E2 protein and HCVcc could trigger phosphorylation of IκBα, up-regulate anti-apoptosis Bcl-2 family proteins, and enhance Raji cells protection from Fas-mediated death. Moreover, such stimulatory signals could up-regulate CD81 and costimulatory molecules CD80, CD86, and down-regulates complement receptor CD21.2.Such an effect is depended on the engagement of E2-CD81 on the cell surface because CD81 silenced Raji cells did not respond to both stimulants, and the E2 mutant E2-W529/A, which could not bind with CD81,could not trigger the responses of Raji cells.3.E2-CD81 engagement may be involved in HCV infection associated B cell lymphoproliferative disorders and low levels of neutralizing antibody production.4.IFN-αcauses differential up-regulation of the MAPK signaling cascades.The STAT1 phosphorylation was strongly enhanced by IFN-a and some differences occur in the regulation of MAPK pathways by IFN-a between Huh7 and HepG2 cells.5. These results are helpful for better understanding of the mechanisms involved in HCV associated B cell lymphoproliferative disorders and delayed appearance of neutralizing antibody and may provide information for understanding the IFN signaling. All of these can be used on research of available vaccines or potential anti-virus drugs against HCV infection.