The third component of human complement (C3) plays a central role in innate immune function as its activation is required to trigger classical as well as alternative complement pathways. In this study, we have observed that sera from patients chronically infected with hepatitis C virus (HCV) displayed significantly lower C3 levels than sera from healthy individuals. Liver biopsy specimens from the same patients also exhibited lower C3 mRNA expression than liver tissues from healthy donors. C3 mRNA level was reduced in hepatocytes upon infection with cell culture-grown HCV genotype 1a or 2a in vitro. Further analysis suggested that HCV core protein displayed a weak repression of C3 promoter activity by downregulating the transcription factor farnesoid X receptor (FXR). On the other hand, HCV NS5A protein strongly downregulated C3 promoter activity at the basal level or in the presence of interleukin-1␤ (IL-1␤) as an inducer. In addition, the expression of the transcription factor CAAT/ enhancer binding protein beta (C/EBP-␤), which binds to the IL-1/IL-6 response element in the C3 promoter, was inhibited in liver biopsy specimens. Furthermore, expression of C/EBP-␤ was reduced in hepatocytes infected with cell culture-grown HCV, as well as in hepatocytes transfected with the NS5A genomic region of HCV. Together, these results underscore the role of HCV NS5A protein in impairing innate immune function.

The fourth component of human complement (C4) plays an important role in innate immune function. C4 activity has been observed to be significantly lower in patients with chronic hepatitis C virus (HCV) infections, although the mechanism remains unknown. In this study, we have examined the mechanisms of C4 regulation by HCV. Liver biopsy specimens from patients with chronic HCV infections displayed significantly lower C4 mRNA levels than liver tissue samples from patients with unrelated liver disease. Further, C4 mRNA levels of the two isoforms (C4A and C4B) were significantly reduced in hepatocytes transfected with RNA from HCV genotype 1a or 2a. Subsequently, a significant C4 regulatory role of HCV core or NS5A upon C4 promoter activity was observed. HCV core or NS5A transgenic mice displayed a reduction in C4 mRNA. Gamma interferon (IFN-␥)-induced C4 promoter activation was also impaired in the presence of HCV proteins. We further demonstrated that HCV core reduced the expression of upstream stimulating factor 1 (USF-1), a transcription factor important for basal C4 expression. On the other hand, the expression of interferon regulatory factor 1 (IRF-1), which is important for IFN-␥-induced C4 expression, was inhibited by hepatocytes expressing HCV NS5A. These results underscore the roles of HCV proteins in innate immune regulation in establishing a chronic infection.The complement system is a set of biochemical pathways that helps to clear pathogens from an organism as part of the innate and acquired immunity programs. Activation of the complement system triggers a wide range of cellular responses ranging from apoptosis to opsonization (27). The complement system plays a critical role in the pathogenesis of a variety of chronic human diseases. Viruses have been shown to attenuate complement activation. Recently, the nonstructural protein (NS1) from flaviviruses (dengue fever virus, West Nile virus, and yellow fever virus) has been reported to attenuate complement activation by interacting with several complement components (1,8). Results from another study suggest that the complement system is also involved in the pathogenesis of a variety of liver disorders, including liver injury and repair, fibrosis, viral hepatitis, alcoholic liver disease, and liver ischemia/reperfusion injury (24). The fourth component of the complement system, C4, plays a vital role in mounting a proper immune response against infection (35). Chronic hepatitis C virus (HCV) infection is a leading cause of progressive liver disease. The mechanisms responsible for HCV persistence are not well understood, although the interactions between HCV and host cells appear to play an important role. Dumestre-Perard et al. (11) reported in a study of a large cohort that in patients with chronic HCV infections, the blood level of specific complement components is depleted, and C4 activity is significantly lower. A decrease in specific C4 activity was reported among relapsers compared with sustained responders, before and during therapy, suggesting it...

Our previous studies demonstrated that hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, display distinct reactivity to different cell surface molecules. In this study, we characterized the interaction of E1 and E2 with apolipoproteins in facilitating virus entry. The results suggested a higher neutralization of VSV/HCV E1-G pseudotype infectivity by antibodies to apolipoprotein E (ApoE) than apolipoprotein B (ApoB), with VSV/HCV E2-G pseudotype infectivity remaining largely unaffected. Neutralization of cell culture grown HCV infectivity by antiserum to ApoE, and to a lesser extent by ApoB, further verified their involvement in virus entry. HCV E1, but not E2, displayed binding with ApoE and ApoB by ELISA. Binding of E1 with apolipoproteins were further supported by coimmunoprecipitation from human hepatocytes expressing E1. Rabbit antiserum to a selected E1 ectodomain derived peptide displayed ~50% neutralization of E1-G pseudotype infectivity. Furthermore, E1 ectodomain derived synthetic peptides significantly inhibited the interaction of E1 with both the apolipoproteins. Investigation on the role of LDL-R as a hepatocyte surface receptor for virus entry suggested a significant reduction in E1-G pseudotype plaque numbers (~70%) by inhibiting LDL-R ligand binding activity using human proprotein convertase subtilisin/kexin type 9 (PCSK9) and platelet factor-4 (PF4), while they had a minimal inhibitory effect on E2-G pseudotype. Conclusion Together, the results suggested an association between HCV E1 and apolipoproteins, which may facilitate virus entry through LDL-R into mammalian cells.

Chronic hepatitis C virus (HCV) infection isChronic hepatitis C virus (HCV) infection is often associated with insulin resistance and hepatic steatosis (1,5,10,28,38). Insulin resistance is paradoxically associated with a reduced ability for insulin signaling to inhibit glucose production, whereas insulin-stimulated lipogenesis is enhanced in the liver. Insulin regulates gene expression of key enzymes in glucose and lipid metabolism by modulating the activity of specific Forkhead box transcriptional regulators (FoxO1 and FoxA2) in the liver. Insulin binds with receptors, activates Akt, and phosphorylates FoxO1. Akt-catalyzed phosphorylation of FoxO1 impairs its DNA binding ability with a concomitant inhibition of Fox-dependent gene expression (9). Phosphorylated FoxO1 may translocate from the nucleus to the cytoplasm, although the functional relevance of this translocation may not be fully related to localization of the protein (39). In the liver, FoxO1 mediates the expression of genes involved in both glucose and lipid metabolism (3, 24, 32, 34), while FoxA2 promotes lipid metabolism during fasting by triggering expression of the fatty acid oxidation program (41). FoxO1 has three serine/threonine residues that are potential targets for phosphorylation by serine/threonine kinase Akt, one of the downstream targets of phosphatidylinositol 3-kinase (PI3K), and plays an important role in mediating insulin effects. Phosphorylation of Ser 256 at the C-terminal end of the DNA binding domain of FoxO1 is required for effective phosphorylation of Thr 24 and Ser 319 , and phosphorylation at this site can impair DNA binding activity (43). The distribution of FoxO1 in insulin-responsive tissues and its regulation by insulin-stimulated Akt phosphorylation allow FoxO1 to mediate a variety of important metabolic functions (13). On the other hand, insulin inhibits FoxA2 through a mechanism that involves threonine phosphorylation at amino acid position 156 and possibly nuclear exclusion (42). Thus, insulin resistance may be mediated by the modulation of Forkhead box transcription factors, preventing optimal stimulation of the glucose-6-phosphatase (G6P) gene, triglyceride degradation, and fatty acid oxidation.Hepatic glucose output is regulated by the G6P catalytic subunit (G6PC) and phosphoenolpyruvate carboxykinase (PEPCK) rate-limiting enzymes for gluconeogenesis and glucose release. Although several transcription factors have been shown to regulate gluconeogenesis, evidence is accumulating that in vivo shutdown of hepatic glucose output by insulin involves Akt-dependent phosphorylation of FoxO1, which controls the expression of G6P and PEPCK. On the other hand, FoxA2 activates genes involved in hepatic lipid metabolism. Activation of FoxA2 in the liver leads to increased oxidation and secretion of fatty acids in the form of triglycerols (TG). Very-low-density lipoprotein (VLDL) is secreted by hepatocytes in response to de novo synthesis of TG. The secretion and assembly of VLDL-associated triglycerides is regulated at various...

CD55 limits excessive complement activation on the host cell surface by accelerating the decay of C3 convertases. In this study, we observed that hepatitis C virus (HCV) infection of hepatocytes or HCV core protein expression in transfected hepatocytes upregulated CD55 expression at the mRNA and protein levels. Further analysis suggested that the HCV core protein or fulllength (FL) genome enhanced CD55 promoter activity in a luciferase-based assay, which was further augmented in the presence of interleukin-6. Mutation of the CREB or SP-1 binding site on the CD55 promoter impaired HCV core protein-mediated upregulation of CD55. HCV-infected or core protein-transfected Huh7.5 cells displayed greater viability in the presence of CD81 and CD55 antibodies and complement. Biochemical analysis revealed that CD55 was associated with cell culture-grown HCV after purification by sucrose density gradient ultracentrifugation. Consistent with this, a polyclonal antibody to CD55 captured cell culture-grown HCV. Blocking antibodies against CD55 or virus envelope glycoproteins in the presence of normal human serum as a source of complement inhibited HCV infection. The inhibition was enhanced in the presence of both the antibodies and serum complement. Collectively, these results suggest that HCV induces and associates with a negative regulator of the complement pathway, a likely mechanism for immune evasion. The complement system performs a vital effector function in the innate immune system by providing an efficient means for targeting and eliminating infected cells and invading microorganisms, including free viral particles (1-3). Activation of the complement cascade occurs primarily via the classical, alternative, or lectin pathway (2, 4). These three pathways activate C3 via cleavage to C3a and C3b by the C3 convertases. C5 convertases are generated by the association of C3b with the C3 convertases, which in turn cleaves C5 into C5a and C5b. The release of C5b initiates the nonenzymatic process of membrane-attack complex (MAC) formation that then sequentially recruits C6, C7, C8, and C9 proteins (1,3,5). The MAC forms a pore-like structure within the lipid envelope of the pathogen or the membrane of the infected cells that ultimately leads to lysis. In order to avert damage from excessive complement activation and MAC formation, host cells express membrane-bound regulatory proteins to limit these processes (6). Regulators of complement activation (RCA) are expressed on the surfaces of host cells and include CD46, CD55, and CD59 (7-9).Hepatocytes are the primary sites for synthesis of complement components in vivo. During an acute-phase (AP) response, the biosynthesis of these components is increased by the action of AP response-associated cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha (TNF-␣) (10, 11). Hepatocytes may be exposed to high local concentrations of complement components under these conditions and could promote complement-mediated damage (12)(13)(14). Decay-accelerating factor (DAF) or CD55...