HCV is a major public health problem, infecting more than 170 million people worldwide. Most cases of HCV infection become persistent and may eventually lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma. Although hepatocytes are the major site of viral replication, a broad clinical spectrum of extrahepatic complications and diseases are associated with chronic HCV infection, including mixed cryoglobulinemia, non-Hodgkins lymphoma, cutaneous vasculitis, glomerulonephritis, neuropathy, and lymphoproliferative disorders. The existence of extrahepatic reservoirs of HCV replication, particularly in PBMCs, remains highly controversial. It is unclear how B cells become dysregulated during the course of chronic HCV infection. In our previous study, we demonstrated that in 7 out of 7 chronic HCV patients, HCV is preferentially associated with CD19+ B cells in PBMC subpopulations. Using in vitro reconstitution experiments by mixing cell-free HCV particles from chronic HCV carriers with PBMCs from healthy blood donors, we found that HCV particles preferentially bind to CD19+ B cells. HCV particles produced from in vitro cell cultures have minimal binding capacity to CD19+ B cells. However, when HCV particles produced in cell culture were pre-incubated with serum samples from HCV recovered patients and then mixed with PBMCs from healthy blood donors, the viral particles become preferentially bound to CD19+ B cells. Initially we thought the serum components which promote the binding of HCV to B cells were induced by HCV infection, such as HCV antigen-specific antibodies. Later, we demonstrated that the same activity can also be found in serum from healthy blood donors without any evidence of previous exposure to HCV. Recently, we have further demonstrated that the association of HCV with CD19+ B cells is mediated by the complement system (Hepatology 2016). In this study, we used in vitro cultured virus and uninfected PBMCs from healthy blood donors to investigate the necessary serum components that activate the binding of HCV to B cells. First, we found that in the absence of exogenous serum factors, there was negligible binding of culture-produced HCV particles to B cells. However, upon pre-incubation of culture- produced HCV particles with serum, HCV binding to B cells was increased by more than two orders of magnitude compared to virus in the absence of serum. The essential serum factors were found to be heat- labile and present in both healthy blood donors and HCV recovered patients, suggesting that complement might be integral to the binding process. Second, the preferential binding activity of HCV to B cells could be blocked by anti-complement C3 antibodies. In experiments with complement-depleted serum and purified complement proteins, we demonstrated that complement proteins C1, C2, C3, and C4 were required to activate such binding activity. Third, using antibodies against cell surface markers, we showed that the binding complex mainly involved CD21 (complement receptor 2), CD19, CD20, and CD81. In human B cells, CD21 is known to form a costimulatory complex with CD19 and CD81. Co-ligation of the B cell antigen receptor (BCR) with this costimulatory complex can lower the threshold required for BCR-mediated B cell activation and proliferation. One of our major goals in this program is to elucidate the pathways through which complement mediated HCV attachment to B cells affects intracellular pathways that foster oncogenesis. For our next phase of study, we will focus on the following projects: 1) To identify signaling pathways associated with HCV binding to CD21; 2) To elucidate the possible biological responses and consequences of HCV binding to B cells using cytokine array assay; and 3) To investigate the potential molecular mechanisms of HCV associated lymphoid-neoplasms using RNA sequencing analysis of B cells derived from chronic HCV patients and matched healthy controls. The complement system plays a central role in innate immune defense and consists of both soluble factors and cell surface receptors that interact to sense and respond to a wide range of invading microorganisms. Activation of complement system has been demonstrated during infection by several enveloped viruses including HIV-1, herpesvirus, Ebola virus, and influenza virus.. However, these viruses have also evolved escape mechanisms to evade the complement system by incorporating host regulators of complement activation into their viral envelopes and, as a result, escape antibody-dependent complement-mediated virus lysis. Complement activation upon HCV infection has been implicated in several studies. In addition, CD55 and CD59, regulators of complement activation, have been shown to be incorporated into HCV particles, which led to resistance to complement-mediated antibody-dependent virolysis, In chronic HCV patients, the level of immune-complexes in blood is often elevated. Since complement receptor 1/CD35 on erythrocytes has been shown to play a key role in transporting complement-opsonized immune-complexes to liver and spleen, the potential roles of this receptor on HCV infection need to be further elucidated. Recently we have completed the manuscript describing the complement-mediated binding of HCV to erythrocytes. To further explore the pathogenesis of HCV-related immune complex disease, we will focus on the following projects: 1) To determine the levels of complement-activated immune complexes (IC) in plasma samples from chronic HCV patients and compare to those from matched healthy controls with a minimum of 30 individuals from each group; 2) To investigate the genetic polymorphism of complement receptor 1 (CR1/CD35) gene associated with CR1 expression on erythrocytes from both groups of individuals, and how this CR1 expression level correlates with IC level; 3) To identify the potential molecular signatures of B cells from chronic HCV patients with elevated levels of complement-activated IC in plasma samples using RNA- seq analysis by Next Generation Sequencing.
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