In the present study, we demonstrated that HCV can modulate the activation, survival and immunological phenotype of Raji cells via E2-CD81 engagement, which may be related with B lymphocyte disorders and weak neutralizing antibody response in HCV patients.
It has been proposed that HCV infects B cells, which may lead to clonal B cell expansions. CD81, SR-BI, CLDN1 and OCLN have been proved to be necessary for HCV infection 
. However, B cells in peripheral blood lack necessary HCV entry receptors and do not support HCV replication 
. Our findings showed that the expressions of CD81 and SR-BI were detectable on Raji cells, but CLDN1 and OCLN were undetectable. Three strains of HCVpp with high infectivity to Huh7.5 cells failed to infect Raji cells, and there was no evidence showing HCVcc could infect Raji cells. These data suggest that HCV viral particles rarely infect B cells, at least under experimental conditions in vitro,
although they may be able to bind with B cells via envelope proteins-cellular receptors interaction.
For the costimulatory role of CD81 on B cells, E2-CD81 binding is suggested as a contributory factor in the pathophysiological process leading HCV infection to B-cell clonal expansion 
. But we did not observe obvious enhancement of E2 protein on proliferation of Raji cells and PHB cells under the present conditions. We think it is possible that the amount of E2 immobilized onto the culture plates is not sufficient to enhance the cell proliferation or more time is required to observe the effect of E2 protein on cell proliferation. Complement-binding of CD21/CD19/CD81 acts a role in enhancing protection of human B cells from Fas-mediated apoptosis 
. We found that treatment of Raji cells or PHB cells with CH11 anti-Fas mAb led to significant cell death, and E2 protein efficiently diminished cell death. The mutant E2-W529/A, which fails to bind with CD81, did not protect cells from death. Treatment of CD81-silenced Raji cells with E2 protein also showed no protective effect.
B cells are susceptible to mitochondria- and receptor-initiated death at various stages of peripheral differentiation and during immune responses, which plays an important role in maintaining homeostatic control of B lymphocytes 
. The transcription factor NF-κB enhances cell viability by activating genes that counteract both mitochondria- and receptor-initiated death pathways 
. Bcl-2 family proteins that consist of anti-apoptotic and pro-apoptotic members are important regulators of apoptosis, which may be either death antagonists (e.g. Bcl-2 and Bcl-xL) or death agonists (e.g. Bax, Bad and Bak), the balance between these two types of Bcl-2 family members has been reported to partly control cell fate 
. In the present study, E2-CD81 engagement triggered phosphorylation of IκBα and increased expression of NF-κB and NF-κB target genes Bcl-2 and Bcl-xL. A higher over-expression rate of Bcl-2 was reported in HCV patients with cryoglobulinemia (MC) compared those without MC, with a further increase in patients with non-Hodgkin lymphoma (NHL) 
. Moreover, antiviral treatment led to a decrease in Bcl-2 expression, which may further support the relationship between HCV infection and induction of Bcl-2 over expression 
. A recent report indicated that mature activated B cells in patients with chronic HCV infection are intrinsically resistant to apoptosis, and expression of Bcl-2 in these cells were commonly elevated 
. Our results indicated that E2-CD81 engagement activates transcription factor NF-κB, which then increases the expression of Bcl-2 proteins and in turn enhances the survival of B cells and protects B cells from apoptosis. This possibility is supported by the observation that improvement of mixed cryoglobulinemia and non-Hodgkin lymphoma in chronic HCV patients after interferon therapy, however, no HCV proteins or HCV genome was able to be detected in villous splenic lymphoma cells in these patients prior to treatment 
It is reported that peripheral B cells from the majority of hepatitis C patients expressed elevated levels of B lymphocyte activation markers and a great number of non-specific activation of T cells infiltrated in liver, and the latter is considered an important cause of hepatocyte damage 
. In the present study, both E2 protein and HCVcc conferred Raji cells and PHB cells more activated phenotype by increasing the expressions of CD80, CD86, which are consistent with the observation that E2 promoted Raji cells to secret TNF-α 
. Since activated B cells gain enhanced ability to stimulate T cells, we think E2 binding to CD81 on B cells should be involved in non-specific activation of T cells.
CD21-mediated complement recognition acts as an important role for B cells' response to specific antigens 
. We found that E2 protein and HCVcc significantly decreased CD21 expression on Raji cells and PHB cells. This phenomenon is also consistent with the activation and maturation phenotype of B cells, which display decreased expression of CD21 
. If E2 in deed lowers CD21 expression in vivo
, which would make B cells lose the ability of capturing opsonized antigen-complement C3d complex, and consequently decrease B cells' response to antigen-BCR engagement. Thus, it is possible that E2-CD81 engagement inhibits antibody response to E2 protein. It is interesting that CD81 itself was elevated by E2 or HCVcc treatment, which may act as a positive feedback between E2 engagement and B cells activation, so as to facilitate the establishment of HCV chronic infection and the progress of B-cell disorders. In fact, the expression of CD81 is increased on circulating B cells from HCV infected individuals, and decreased significantly in patients responded to IFN therapy 
Although few existing characterized viral clones that can replicate in vitro have consistently failed to infect human B cells, some groups have detected HCV RNA in other lymphoid cells, including B- and T-lymphocytes, monocytes, and dendritic cells 
. A B-cell line (SB) established from an HCV-infected non-Hodgkin's B-cell lymphoma was reported to produces HCV particles that can further infect B- and T-lymphocytes in vitro 
. These data hint lymphotropism of HCV in natural infection may be possible. The data here strongly suggest that HCV may interfere with B cells independent on HCV replication in cells.
Together, the present study indicates that E2-CD81 engagement plays a role in activating B cells, protecting B cells from activation-induced cell death, and regulating immunological function of B cells. Therefore, the E2-CD81 engagement should be involved in the HCV associated B-cell disorders and insufficient neutralizing antibody response. These findings provide valuable insights into the development of therapeutic strategies against HCV infection and the related B-cell disturbance.