Sandip K. Bose

Hepatitis C Virus Proteins Inhibit C3 Complement Production

ABSTRACT 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-1β (IL-1β) as an inducer. In addition, the expression of the transcription factor CAAT/enhancer binding protein beta (C/EBP-β), 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-β 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.

Hepatitis C virus infection and insulin resistance

Approximately 170 million people worldwide are chronically infected with hepatitis C virus (HCV). Chronic HCV infection is the leading cause for the development of liver fibrosis, cirrhosis, hepatocellular carcinoma (HCC) and is the primary cause for liver transplantation in the western world. Insulin resistance is one of the pathological features in patients with HCV infection and often leads to development of type II diabetes. Insulin resistance plays an important role in the development of various complications associated with HCV infection. Recent evidence indicates that HCV associated insulin resistance may result in hepatic fibrosis, steatosis, HCC and resistance to anti-viral treatment. Thus, HCV associated insulin resistance is a therapeutic target at any stage of HCV infection. HCV modulates normal cellular gene expression and interferes with the insulin signaling pathway. Various mechanisms have been proposed in regard to HCV mediated insulin resistance, involving up regulation of inflammatory cytokines, like tumor necrosis factor-α, phosphorylation of insulin-receptor substrate-1, Akt, up-regulation of gluconeogenic genes like glucose 6 phosphatase, phosphoenolpyruvate carboxykinase 2, and accumulation of lipid droplets. In this review, we summarize the available information on how HCV infection interferes with insulin signaling pathways resulting in insulin resistance.

Hepatitis C Virus Induces Epithelial-Mesenchymal Transition in Primary Human Hepatocytes

ABSTRACT Hepatitis C virus (HCV)-mediated liver disease progression may reflect distinct molecular mechanisms for increased hepatocyte growth and hepatic stellate cell activation. In this study, we have observed that primary human hepatocytes, when infected in vitro with cell culture-grown HCV genotype 1a or 2a, display viral RNA and protein expression. Infected hepatocytes displayed a fibroblast-like shape and an extended life span. To understand the changes at the molecular level, we examined epithelial-mesenchymal transition (EMT) markers. Increased mRNA and protein expression levels of vimentin, snail, slug, and twist and a loss of the epithelial cell marker E-cadherin were observed. Snail and twist, when examined separately, were upregulated in chronically HCV-infected liver biopsy specimens, indicating an onset of an active EMT state in the infected liver. An increased expression level of fibroblast-specific protein 1 (FSP-1) in the infected hepatocytes was also evident, indicating a type 2 EMT state. Infected hepatocytes had significantly increased levels of phosphorylated β-catenin (Ser552) as an EMT mediator, which translocated into the nucleus and activated Akt. The phosphorylation level of β-catenin at Thr41/Ser45 moieties was specifically higher in control than in HCV-infected hepatocytes, implicating an inactivation of β-catenin. Together, these results suggested that primary human hepatocytes infected with cell culture-grown HCV display EMT via the activation of the Akt/β-catenin signaling pathway. This observation may have implications for liver disease progression and therapeutic intervention strategies using inhibitory molecules.

Forkhead Box Transcription Factor Regulation and Lipid Accumulation by Hepatitis C Virus

ABSTRACT We have previously shown that hepatitis C virus (HCV) infection modulates the expression of forkhead box transcription factors, including FoxO1 and FoxA2, which play key roles in gluconeogenesis and β-oxidation of fatty acid, respectively. The aim of the present study was to determine the role of forkhead box transcription factors in modulating lipid metabolism. HCV infection or core protein expression alone in transfected Huh7.5 cells increased expression of sterol regulatory element binding protein 1c (SREBP-1c) and its downstream target, fatty acid synthase (FASN), which are key proteins involved in lipid synthesis. Knockdown of FoxO1 by small interfering RNA in HCV-infected cells significantly decreased SREBP-1c and FASN expression. Further, HCV infection or core protein expression in Huh7.5 cells significantly decreased the expression of medium-chain acyl coenzyme A dehydrogenase (MCAD) and short-chain acyl coenzyme A dehydrogenase (SCAD), involved in the regulation of β-oxidation of fatty acids. Ectopic expression of FoxA2 in HCV-infected cells rescued the expression of MCAD and SCAD. Oil red O and neutral lipid staining indicated that HCV infection significantly increases lipid accumulation compared to that in the mock-infected control. This was further verified by the increased expression of perilipin-2 and decreased activity of hormone-sensitive lipase (HSL) in HCV-infected hepatocytes, implying increased accumulation of neutral lipids. Knockdown of FoxO1 and ectopic expression of FoxA2 significantly decreased HCV replication. Taken together, these results suggest that HCV modulates forkhead box transcription factors which together increase lipid accumulation and promote viral replication. IMPORTANCE Hepatic steatosis is a frequent complication associated with chronic HCV infection. Its presence is a key prognostic indicator associated with the progression to hepatic fibrosis and hepatocellular carcinoma. Several mechanisms have been proposed to account for the development of steatosis and fatty liver during HCV infection. We observed that HCV infection increases expression of both SREBP-1c and FASN. Further investigation suggested that the expression of SREBP-1c and FASN is controlled by the transcription factor FoxO1 during HCV infection. In addition, HCV infection significantly decreased both MCAD and SCAD expression, which is controlled by FoxA2. HCV infection also increased lipid droplet accumulation, increased perilipin-2 expression, and decreased HSL activity. Thus, knockdown of FoxO1 (decreased lipogenesis) and overexpression of FoxA2 (increased β-oxidation) resulted in a significant disruption of the platform and, hence, a decrease in HCV genome replication. Thus, targeting of FoxO1 and FoxA2 might be useful in developing a therapeutic approach against HCV infection.

Hepatitis C virus infection upregulates CD55 expression on the hepatocyte surface and promotes association with virus particles

ABSTRACT 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 full-length (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.

HEPATITIS C VIRUS IMPAIRS NATURAL KILLER CELL MEDIATED AUGMENTATION OF COMPLEMENT SYNTHESIS

ABSTRACT Natural killer (NK) cells and the complement system play critical roles in the first line of defense against pathogens. The synthesis of complement components C4 and C3 is transcriptionally downregulated by hepatitis C virus (HCV) core and NS5A proteins, and this negative regulation is apparent in chronically HCV-infected patients. In this study, we have examined the potential contribution of an NK cell line as a model in regulating complement synthesis. Coculture of NK cells (NK3.3) with human hepatoma cells (Huh7.5) expressing HCV core or NS5A protein led to a significant increase in C4 and C3 complement synthesis via enhanced specific transcription factors. Reestablishment of complement protein expression was found to be mediated by direct interaction between NKG2D on NK cells and the hepatocyte protein major histocompatibility complex class I-related chains A and B (MICA/B) and not to be associated with specific cytokine signaling events. On the other hand, C4 and C3 synthesis remained impaired in a coculture of NK cells and Huh7.5 cells infected with cell culture-grown HCV. The association between these two cell types through NKG2D and MICA/B was examined further, with MICA/B expression in HCV-infected hepatocytes found to remain inhibited during coculture. Further experiments revealed that the HCV NS2 and NS5B proteins are responsible for the HCV-associated decrease in MICA/B. These results suggest that HCV disables a key receptor ligand in infected hepatoma cells, thereby inhibiting the ability of infected cells to respond to stimuli from NK cells to positively regulate complement synthesis. IMPORTANCE The complement system contributes to the protection of the host from virus infection. However, the involvement of complement in viral hepatitis has not been well documented. Whether NK cells affect complement component expression in HCV-infected hepatocytes remains unknown. Here, we have shown how HCV subverts the ability of NK cells to positively mediate complement protein expression.

Hepatitis C Virus-Mediated Inhibition of Cathepsin S Increases Invariant-Chain Expression on Hepatocyte Surface

ABSTRACT Hepatocytes are the main source of hepatitis C virus (HCV) replication and contain the maximum viral load in an infected person. Chronic HCV infection is characterized by weak cellular immune responses to viral proteins. Cathepsin S is a lysosomal cysteine protease and controls HLA-DR–antigen complex presentation through the degradation of the invariant chain. In this study, we examined the effect of HCV proteins on cathepsin S expression and found it to be markedly decreased in dendritic cells (DCs) exposed to HCV or in hepatocytes expressing HCV proteins. The downregulation of cathepsin S was mediated by HCV core and NS5A proteins involving inhibition of the transcription factors interferon regulatory factor 1 (IRF-1) and upstream stimulatory factor 1 (USF-1) in gamma interferon (IFN-γ)-treated hepatocytes. Inhibition of cathepsin S by HCV proteins increased cell surface expression of the invariant chain. In addition, hepatocytes stably transfected with HCV core or NS5A inhibited HLA-DR expression. Together, these results suggested that HCV has an inhibitory role on cathepsin S-mediated major histocompatibility complex (MHC) class II maturation, which may contribute to weak immunogenicity of viral antigens in chronically infected humans.

PROMOTION OF CANCER STEM-LIKE CELL PROPERTIES IN HEPATITIS C VIRUS INFECTED HEPATOCYTES

ABSTRACT We have previously reported that hepatitis C virus (HCV) infection of primary human hepatocytes (PHH) induces the epithelial mesenchymal transition (EMT) state and extends hepatocyte life span (S. K. Bose, K. Meyer, A. M. Di Bisceglie, R. B. Ray, and R. Ray, J Virol 86:13621–13628, 2012, http://dx.doi.org/10.1128/JVI.02016-12). These hepatocytes displayed sphere formation on ultralow binding plates and survived for more than 12 weeks. The sphere-forming hepatocytes expressed a number of cancer stem-like cell (CSC) markers, including high levels of the stem cell factor receptor c-Kit. The c-Kit receptor is regarded as one of the CSC markers in hepatocellular carcinoma (HCC). Analysis of c-Kit mRNA displayed a significant increase in the liver biopsy specimens of chronically HCV-infected patients. We also found c-Kit is highly expressed in transformed human hepatocytes (THH) infected in vitro with cell culture-grown HCV genotype 2a. Further studies suggested that HCV core protein significantly upregulates c-Kit expression at the transcriptional level. HCV infection of THH led to a significant increase in the number of spheres displayed on ultralow binding plates and in enhanced EMT and CSC markers and tumor growth in immunodeficient mice. The use of imatinib or dasatinib as a c-Kit inhibitor reduced the level of sphere-forming cells in culture. The sphere-forming cells were sensitive to treatment with sorafenib, a multikinase inhibitor, that is used for HCC treatment. Further, stattic, an inhibitor of the Stat3 molecule, induced sphere-forming cell death. A combination of sorafenib and stattic had a significantly stronger effect, leading to cell death. These results suggested that HCV infection potentiates CSC generation, and selected drugs can be targeted to efficiently inhibit cell growth. IMPORTANCE HCV infection may develop into HCC as an end-stage liver disease. We focused on understanding the mechanism for the risk of HCC from chronic HCV infection and identified targets for treatment. HCV-infected primary and transformed human hepatocytes (PHH or THH) generated CSC. HCV-induced spheres were highly sensitive to cell death from sorafenib and stattic treatment. Thus, our study is highly significant for HCV-associated HCC, with the potential for developing a target-specific strategy for improved therapies.

Correction for Kim et al., “Hepatitis C Virus Impairs Natural Killer Cell-Mediated Augmentation of Complement Synthesis”

Volume 88, no. 5, p. 2564 –2571, 2014, https://doi.org/10.1128/JVI.02988-13. Page 2566, Fig. 1B: The Control blot for the C4 row was a duplicate of the Huh7.5 IL-2 blot for the C4 row in panel A because of an inadvertent error during figure assembly. We have remade Fig. 1B with new C4 and Actin rows with Control and NKCM-treated cells, using a duplicate set of these experiments. Page 2566, Fig. 1A: We have replaced the Huh7.5 NK IL-2 C4 and Actin rows with a new data set generated under similar conditions to avoid confusion with the Huh7.5 NK IL-2 C4 and Actin rows in Fig. 2A, which were used as a control. Figure 1 should appear as shown below.

Association of lipid droplet and hepatitis C virus proteins: insights for virus replication

Hepatitis C virus (HCV) infection represents a major worldwide disease burden, with an estimated prevalence of >185 million people (1). Among the various comorbidities associated with chronic HCV infection, hepatic steatosis is a frequent complication, and its presence is a key prognostic indicator associated with progression to hepatic fibrosis and with the development of hepatocellular carcinoma (2). Several mechanisms have been proposed to account for the development of steatosis and fatty liver in the setting of HCV infection (3). HCV infection enhances lipogenesis, reduces secretion of VLDL, attenuates β-oxidation of lipids, and increases virus growth and replication through complex pathways that intersect via modulating host cell lipid metabolism (4). In addition, recent studies have implicated lipid droplet formation and turnover in the life cycle of HCV. Specifically, studies have identified a role for the triglyceride-synthesizing enzyme diacylglycerol acyltransferase 1 (DGAT1) in both the formation of lipid droplets within the bilayer of the endoplasmic reticulum (ER) and also in the formation of infectious virions (5). Those studies demonstrated that DGAT1 (but not DGAT2) physically interacts with components of the nucleocapsid core and in turn recruits the viral replication complex to the newly generated lipid droplets (5). In addition, more-recent studies have demonstrated that this physical interaction between DGAT1 and HCV core components then interferes with triglyceride lipolysis and lipid droplet turnover, an effect that further enhances hepatic steatosis (6). These findings collectively suggest that HCV infection impacts hepatic lipid metabolism over a broad range of pathways, including coopting elements of lipid droplet formation and turnover as well as modulating lipogenesis, FA oxidation, and VLDL secretion. However, despite the obvious public health impact and importance of HCV infection and its role in modulating hepatic lipid metabolism, there is still much to learn about the molecular and biochemical mechanisms by which the structural and nonstructural protein components of the HCV function within these pathways. This relative dearth of information is at least partially explained by the lack of preclinical experimental models of replicating HCV. Accordingly, most of the available information still derives from studies using transfected cell lines that support HCV genome replication, either as subgenomic or full-length constructs. With this background, the article from Tanaka et al. (7) in this issue of The Journal of Lipid Research provides new insights into the role of HCV NS4B (a nonstructural protein of the virus, known to be an integral ER membrane protein) and its association with lipid droplets. The authors demonstrated this association using state-of-the-art confocal imaging and biochemical fractionation of cell lines supporting HCV replication. Although the association of HCV with lipid droplets is well known (Moradpour et al., 1996; Miyanari et al., 2007, cited in the referenced article), the molecular and biochemical mechanisms that promote this interaction remain obscure. Among the key findings of this report is the identification of specific domains and potential clusters of amino acid residues that facilitate the interaction of NS4B with the lipid droplet. The authors found that the N-terminal domain interacts more strongly with lipid droplets compared with the C terminus, with almost 100% of the N-terminal domain demonstrating association compared with ~40% following transfection of the C-terminal domain. In addition, the authors identify a specific amino acid residue within the N-terminal α helix to be responsible for NS4B binding to lipid droplets. Mutating this key tryptophan residue to alanine (W43A) abrogated association of HCV NS4B with lipid droplets. This is an interesting and novel finding with respect to association of NS4B and lipid droplets and adds to our knowledge. Moving forward, it will be important to understand how a single amino acid residue change [both with nonpolar side chains (tryptophan and alanine)] abrogates the association of NS4B with the lipid droplet. Another key aspect of these observations is the identification of lipid droplet binding residues in NS4B that are important for HCV replication. Mutation of specific hydrophobic residue (W50A) resulted in defective virus replication and severely reduced release of infectious virus particles as compared with wild-type. It remains unclear whether the inhibition is at the level of HCV RNA replication or at the virus assembly stage. Additionally, whether mutation in NS4B within HCV affects accumulation of lipid droplets within hepatocytes remains unclear. In the current work, Tanaka et al. (7) used Huh7-derived cell lines supporting HCV RNA replication in order to study aspects of both protein localization and the interaction of NS4B and lipid droplets. They confirmed the results using confocal microscopy, biochemical fractionation of cells, and Western blot analyses. Although the authors found HCV core protein in the lipid droplets associated with the ER membrane, they did not demonstrate core protein in the purified lipid droplets. This observation contrasts with an earlier report (8), demonstrating HCV genotype 3 core protein binding to lipid droplets. In the current report, Tanaka et al. (7) used HCV genotype 1b (clone O) and genotype 2a (clone JFH1) in their experiments, and did not observe binding of the HCV core to lipid droplets. This observation raises the question as to whether the mechanism of HCV interaction with lipid droplets differs with respect to the genotype of the virus. It would be interesting to see if the proposed mechanisms for NS4B and lipid droplet interaction holds true for primary human hepatocytes as a natural host of HCV infection. Overall, this paper from Tanaka et al. (7) provides interesting observations regarding the molecular mechanism of HCV NS4B association with lipid droplets and that expand our understanding of the factors that influence HCV replication. These studies further highlight the importance of NS4B and its interactions with lipid droplets as a druggable target for inhibition of HCV growth across different genotypes, with potential for developing antiviral modalities. This implication is consistent with an earlier report suggesting that disrupting the amphipathic N-terminal helix of NS4B by site-directed mutagenesis abolished HCV RNA replication in a subgenomic replicon system from HCV genotype 1b (9). In addition, other studies demonstrated that residues in the C-terminus of HCV NS4B in HCV genotype 2a subgenomic replicon were critical for viral RNA replication (10). The same group of investigators suggested that the C-terminal domain of NS4B influences production of infectious virus. The present findings from Tanaka et al. (7) suggest that NS4B strongly associates with lipid droplets, principally through its N-terminal amphipathic helix. These findings raise the following questions: a) Is HCV RNA replication dependent upon association with lipid droplet? b) Does the lipid droplet act as a platform for RNA replication and/or virus assembly? c) How and in which step does the mutated NS4B impair HCV growth? d) Do lipid droplets accumulate when cells are infected with HCV harboring a mutated NS4B? Beyond these specific questions, however, there are more-general issues of relevance to our understanding of HCV infection and hepatic lipid metabolism. For example, given the requirement for the association of HCV with lipid droplets and the possible role of NS4B, what is the potential for impacting viral replication with pharmacologic manipulation of hepatic lipogenesis and FA oxidation, strategies that would presumably attenuate lipid droplet formation (11)? Alternatively, one might envision a scenario in which modulating VLDL secretion may alter infectious HCV production and release via apoE/apoB inhibition (12–14), and this approach may warrant testing to determine the effects in vivo. Further experimental observations will be required to more completely test the functional relevance of the current findings, but these studies point to an important intersection in lipid droplet biology and HCV replication.