Browsing by Subject "Hepacivirus"
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Item Characterizing B cell Phenotype during chronic HCV infection(2009-06-15) Ayers, Christopher Lee; Karandikar, NitinChronic hepatitis C virus (HCV) infection is characterized by an attenuation of virus-specific T cell responses. The mechanisms leading to T cell attenuation are still not well understood, and likely involve several integrating correlates. We hypothesized that dysfunctions of antigen presenting cells (APC) may contribute to the immunosuppresed phenotype. We also reasoned that direct viral interactions of HCV with immune cells may be responsible for such dysfunction. We employed a strand-specific real time RT-PCR assay and found that virus is frequently associated with B cells (predominantly positive strand was detected). Interestingly, we also found that ex-vivo derived B cells from chronic HCV individuals were better inducers of allogeneic T cell proliferation and this ability correlated with the presence of HCV RNA in those B cells. During such enhanced allostimulation, we also found an increase in the proportion of CD4+CD25+FoxP3+ T cells, which correlated with an increased suppressive capacity thereby demonstrating a paradoxical link between hyperactive B cells and the generation of suppressive T cells. Furthermore, ex-vivo derived chronic HCV B cells had an attenuated response to mitogenic stimulation with associated apoptosis. In an effort to determine direct HCV involvement in immune cell dysfunction, we evaluated the possibility of culture adapted JFH-1 virus to infect PBMC populations. While we found no evidence of viral replication in PBMC, exposure to JFH-1 resulted in vigorous activation of B cells. Interestingly, the B cell activation did not require viable virions, but was dependent upon CD81 availability and the presence of monocytes. We also determined that upon viral exposure, these B cells replicated the hyper-activation of MLR responses found in ex-vivo derived B cells from chronic HCV individuals. In all, our results suggest a novel model wherein HCV-B cell interaction leads to B cell hyper-activation and consequent paradoxical T cell suppression.Item Control of the Interferon Regulatory Factor - 3 Antiviral Pathway by The Hepatitis C Virus Ns3/4a Protease(2007-05-22) Foy, Eileen; Gale, Michael Jr.Hepatitis C Virus (HCV) is a major human pathogen that affects 200 million people worldwide. A majority of people exposed to HCV become chronically infected. In order to persist, the virus must encode mechanisms to subvert host immune defenses. We hypothesized that HCV disrupts critical host intracellular antiviral signaling pathways that culminate in activation of the antiviral response. Using the cell-based replicon system, HCV was found to inhibit the activation of critical intracellular signaling pathways. Our studies identified the NS3/4A protein as an interferon regulatory factor 3 (IRF-3) and NF-KB antagonist and further mapped this activity to the NS3/4A serine protease domain. HCV antagonism of IRF-3 and NF- KB prevents the expression of interferon (IFN) and IFN-stimulated genes required for viral clearance and the generation of a HCV-specific adaptive immune response. Upstream signaling components of IRF-3 were unknown, therefore, the NS3/4A protease was utilized as a tool to elucidate putative upstream signaling components. Characterization of Toll-like receptor 3 (TLR3) in HCV infection indicated that the NS3/4A protease cleaves TRIF, an essential adaptor protein required for TLR3 signaling. However the TLR3 pathway was not found to be essential for generating the intracellular antiviral response. Furthermore, these studies identified the retinoic acid inducible gene-I (RIG-I) as an intracellular viral double-stranded (ds)RNA sensor through molecular cloning and the characterization of a HCV replication permissive cell line defective in IRF-3 activation. The NS3/4A protease disrupted IRF-3 signaling through both the RIG-I pathway as well as the homologous MDA-5 pathway during viral infection. Lastly, these studies identified novel roles for TBK1, TANK and NEMO as signal transduction intermediates in the intracellular dsRNA response pathways. The HCV NS3/4A protease is critical for proper viral polyprotein processing, in addition to antagonism of intracellular antiviral signaling pathways. Therefore, the HCV NS3/4A protease is an ideal target for novel therapeutics. Indeed, the HCV replicon could be effectively eliminated from a persistently infected cell line through the use of an experimental NS3/4A protease inhibitor. These results offer great promise for the effective treatment of chronic HCV infection.Item Hepatitis C Virus Entry into Hepatocytes and Engagement of Innate Immune Defenses(2011-08-26T17:34:10Z) Owen, David Matthew; Gale, Michael Jr.Hepatitis C virus (HCV) infection is a major cause of liver disease and a global health problem with inadequate treatment options. An improved understanding of how HCV exploits and subverts host factors to establish infection should yield potential targets for therapy. This study uses a recently developed cell culture model of HCV infection to examine HCV entry and engagement of innate immune defenses. HCV associates with host apolipoproteins and enters hepatocytes through complex processes involving some combination of CD81, claudin-I, occludin, and scavenger receptor BI. Here I show that HCV forms a complex with very low density lipoprotein (VLDL) within infected hepatocytes and uses this association to support infection through the low density lipoprotein receptor (LDL-R). Blocking experiments demonstrate that beta-VLDL and apolipoprotein E (apoE) can compete with HCV for entry. Knockdown of the LDL-R by treatment with 25-hydroxycholesterol or siRNA ablated ligand uptake and reduced HCV infection of cells, whereas infection was rescued upon cell ectopic LDL-R expression. Analyses of gradient-fractionated HCV demonstrate that apoE is associated with HCV virions exhibiting peak infectivity and dependence upon the LDL-R for cell entry. These results define the LDL-R as a cooperative HCV co-receptor that supports viral entry and infectivity through interaction with apoE ligand present in an infectious HCV/lipoprotein complex comprising the virion. Furthermore, upon entry HCV induces an initial transient activation of interferon regulatory factor-3 (IRF3) which is dependent on retinoic acid inducible gene I (RIG-I) and interferon-beta promoter stimulator-1 (IPS-1). This activation produces an antiviral activity which inhibits HCV entry and replication. HCV NS3/4A protease activity blocks this activation within 48 hours. At later time points post infection HCV activates NF-kappaB in a RIG-I independent manner leading to inflammatory cytokine production. These studies identify 3 potential targets for future HCV therapy: 1) alteration of HCV-lipoprotein interaction to disrupt entry, 2) blockade of NS3/4A protease activity to restore innate antiviral response, and 3) modulation of HCV induced NF-kappaB signaling to downregulate chronic inflammation.Item Hepatitis C Virus NS3/4A Protease and the Intracellular Antiviral Response: Mapping Complex Virus-Host Interactions(2009-06-17) Johnson, Cynthia L.; Gale, Michael J.Virus infection triggers an innate immune response characterized by host cell production of interferon (IFN). Intermediates of viral replication, including dsRNA, initiate a signaling cascade that is amplified within the cell and alerts neighboring cells of viral invaders. Recognition of dsRNA intermediates occurs through retinoic acid inducible gene-I (RIG-I). RIG-I elicits an antiviral state by binding to the IFN-beta Promoter Stimulator-1 (IPS-1) adaptor protein, activating the latent downstream transcription factors IRF-3 and NF-kappaB. These transcription factors bind to the promoter region of effector genes including IFN-beta, producing an antiviral amplification loop within and around the infected cell. This response is critical for immunity to infection. Hepatitis C virus (HCV) is a serious global health problem with 170 million people chronically infected. HCV persistence is linked to viral regulation of innate host defenses by the nonstructural 3/4A protein complex (NS3/4A) cleavage of IPS-1. NS3 structural composition includes an amino-terminal serine protease and a carboxy-terminal RNA helicase. A structure-function analysis of NS3/4A truncation and deletion mutations was conducted. Mutants lacking the helicase domain retained the ability to control RIG-I signaling, but this regulation was abrogated by truncation of the protease domain. Furthermore, treatment of HCV-infected cells with a NS3/4A protease inhibitor prevented IPS-1 proteolysis, restored RIG-I signaling, and decreased viral protein levels. These results indicate that the NS3/4A protease domain alone can target IPS-1 on the mitochondrial membrane. Current dogma holds that NS3/4A is located on the endoplasmic reticulum, thus the mechanism of NS3/4A targeting IPS-1, a mitochondrial membrane protein, remains unexplained. We have shown that NS3/4A distributes on mitochondria independently of the previously identified NS4A membrane localization motif, in a manner dependent on the first twenty amino acids of the NS3 protease domain. The functional domains of IPS-1 that direct the immune response have not been elucidated. We conducted a structure-function study of IPS-1 that revealed distinct processes of IRF-3 and NF-kappaB activation. Mutational analyses further identified areas of IPS-1 critical for mitochondrial localization, dimerization, and uncoupling IRF-3 and NF-kappaB signaling. These findings improve our understanding of IPS-1 function in innate immunity to virus infection.Item Host-Based Mechanisms of Ribavirin Resistance: Implications In Treatment Response of Hepatatis C Virus Infection(2011-02-01T19:35:17Z) Ibarra, Kristie Dawn; Pfeiffer, JulieMany individuals infected with hepatitis C virus (HCV) fail to respond to therapy, resulting in the development of chronic infection and increased risk for fibrosis, cirrhosis, and hepatocellular carcinoma. The current standard of care consists of pegylated interferon and ribavirin (RBV), a nucleoside analog. While RBV improves treatment outcome, and will likely be an important component of therapy with next-generation viral inhibitors, RBV’s mechanism is controversial. Most of RBV’s proposed mechanisms require RBV import into cells. Therefore, we examined whether host-based RBV resistance develops through reduced cellular uptake, analogous to chemotherapy resistance in some cancers. We examined the effect of host-based RBV resistance on a model RNA virus, poliovirus, HCV replication in cultured hepatoma liver cells, and whether RBV resistance develops in HCV patients. When liver cells permissive for poliovirus or HCV replication were exposed to RBV, resistance developed through reduced activity of the ENT1 nucleoside transporter, and antiviral efficacy was reduced. Importantly, RBV uptake significantly declined in HCV peripheral blood mononuclear cells (PBMCs) following four weeks of therapy. Furthermore, maintenance of RBV uptake correlated with rapid treatment response. Our results uncovered a novel form of antiviral drug resistance, suggesting that host-based RBV resistance develops in HCV patients undergoing therapy and that maintenance of RBV uptake may contribute to rapid viral clearance.Item Identification of Host Factors Required for Hepatitis C virus Infection(2009-01-14) Huang, Hua; Ye, JinHepatitis C virus (HCV), a positive-strand RNA virus, infects more than 170 million people and is the leading cause of liver failure worldwide. A protective vaccine is not yet available. Current interferon-based therapies are only effective in a fraction of patients. Thus, there is an urgent need to develop better therapeutic strategies. However, the intrinsic low fidelity of HCV replication makes it more difficult to develop drugs targeting viral proteins since HCV could quickly generate drug-resistant strains. In this context, drugs targeting host factors that control HCV infection may be more effective therapies in that the potency of drugs is not affected by viral mutations. For this purpose, more knowledge regarding host factors involved in HCV infection is needed. HCV is known to replicate on intracellular membrane vesicles. In searching for host proteins localized in membrane vesicles containing HCV replication complex, a magnetic immuno-isolation procedure was employed. This study identified that Apolipoprotein B, Microsomal Triglyceride Transfer Protein, and Apolipoprotein E, three proteins participated in the assembly of very low-density lipoprotein (VLDL) were enriched in membrane vesicles. It was demonstrated that agents inhibiting VLDL assembly also inhibit the secretion of HCV. These studies raise the possibility for treating HCV infection with agents blocking VLDL secretion. HCV infection is also known to produce reactive oxygen species (ROS), which initiate lipid peroxidation. We found that when HCV-infected cells were exposed to polyunsaturated fatty acids (PUFAs) in the absence of lipid-soluble antioxidants, a dramatic increase in lipid peroxidation caused a reduction in HCV RNA. Because peroxidation of PUFAs only occurs in HCV-infected cells that produce ROS, PUFAs could be used to suppress HCV replication in patients without intolerable toxicity. Different in vitro model systems have been used to study the HCV lifecycle. However, the genotype1 HCV, which is the most difficult strain to treat in clinic, still can not be grown in cultured cells. We found a cell line named HCV Replication Permissive 1 (HRP1) that supports the replication of genotype 1 HCV. Interestingly, HRP1 cells appear to have normal interferon response, suggesting a novel innate antiviral pathway may be disrupted in these cells.Item Inhibition of Cell Proliferation through Regulated Intramembrane Proteolysis of CREB3L1(2012-07-10) Denard, Bray Standard; Ye, JinCREB3L1/OASIS is a cellular transcription factor synthesized as a membrane- bound precursor and activated by regulated intramembrane proteolysis in response to stimuli like ER stress. By comparing gene expression between Huh7 subclones that are permissive for hepatitis C virus (HCV) replication versus the non-permissive parental Huh7 cells, we identified CREB3L1 as a host factor that inhibits proliferation of virus-infected cells. Upon infection with diverse DNA and RNA viruses, including murine gamma-herpesvirus 68, HCV, West Nile virus (WNV), and Sendai virus, CREB3L1 was proteolytically cleaved, allowing its NH2 terminus to enter the nucleus and induce multiple genes encoding inhibitors of the cell cycle to block cell proliferation. Consistent with this, we observed a necessity for CREB3L1 expression to be silenced in proliferating cells that harbor replicons of HCV or WNV. Our results indicate that CREB3L1 may play an important role in limiting virus spread by inhibiting proliferation of virus-infected cells. Doxorubicin is used extensively for chemotherapy of diverse types of cancer, yet the mechanism through which it inhibits proliferation of cancer cells remains unclear. Here we report that doxorubicin stimulates de novo synthesis of ceramide, which in turn activates CREB3L1, a transcription factor synthesized as a membrane-bound precursor. Doxorubicin stimulates proteolytic cleavage of CREB3L1 by Site-1 Protease and Site-2 Protease, allowing the NH2-terminal domain of CREB3L1 to enter the nucleus where it activates transcription of genes encoding inhibitors of the cell cycle, including p21. Knockdown of CREB3L1 mRNA in human hepatoma Huh7 cells and immortalized human fibroblast SV589 cells conferred increased resistance to doxorubicin, whereas overexpression of CREB3L1 in human breast cancer MCF-7 cells markedly enhanced the sensitivity of these cells to doxorubicin. These results suggest that measurement of CREB3L1 expression may be a useful biomarker in identifying cancer cells sensitive to doxorubicin. [Keywords: CRE34, HCV, RIP, Doxorubicin, ceramide, myriocin, proliferation, virus, p21, collagen]Item The Role of Interferon Stimlated Genes in Resistance and Immunity to Hepatitis C Virus Infection(2009-01-15) Erickson, Andrea Kaup; Gale, MichaelHepatitis C Virus (HCV) is a global public health issue with 170 million people chronically infected. The only approved treatment for HCV infection is interferon-alpha based therapy, resulting in viral clearance in approximately fifty percent of patients treated. Interferon therapy mimics endogenous type I interferon signaling, which plays a crucial role in the innate antiviral response through the regulation of interferon stimulated genes (ISGs). ISGs encode antiviral effector proteins that limit viral replication and spread. Hundreds of ISGs have been identified in the liver of HCV patients who respond to IFN therapy; however the functions of most these genes are not known. In order to better understand the molecular mechanisms of the IFN antiviral response, studies were initiated to identify and characterize the functions of novel ISGs involved in controlling HCV infection. To determine the most effective IFN for these studies, the antiviral activity and functional effects of three distinct type I IFN subtypes were evaluated. Consensus interferon demonstrated maximal suppression of HCV replication, correlating with enhanced IFN signaling and ISG induction. A functional genomics analysis of IFN treated primary hepatocytes resulted in the identification of eighty-six ISGs differentially induced by consensus interferon. Future evaluations of the role of these genes in HCV outcome, using integrated approaches as described here, will be invaluable in further defining the molecular mechanisms of the innate IFN antiviral response. Although a number of ISGs have recently been reported to function as antiviral proteins in vitro, these studies did not validate the involvement of these genes in innate immunity or disease outcome. In order to evaluate the biological relevance of a genetic variant of the oligoadenylate synthetase 1 (OAS1) gene in resistance to HCV infection, an integrated approach of epidemiology, molecular genetics, and functional biology was used. Genetic and epidemiologic analyses identified a single-base mutation in OAS1 that associates with HCV resistance in individuals with a high-risk for HCV infection. Functional studies of the resulting OAS1 variant demonstrated altered biological activities of the protein, resulting in enhanced suppression of HCV genotype 1a and 2a infectious clones. Furthermore, a recombinant drug form of the OAS1 variant demonstrated broad antiviral activity, suggesting great promise for this protein as a therapeutic for HCV infection.Item Viral and Host Genetic Determinants of Hepatitis C Virus Persistence and Interferon Resistance(2006-05-16) Sumpter, Rhea Myers, Jr.; Gale, Michael Jr.Approximately 170 million people worldwide are chronically infected with hepatitis C virus (HCV), which is an important cause of cirrhosis and hepatocellular carcinoma. HCV replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. The development of the HCV RNA replicon system has allowed the study of persistent HCV RNA replication in tissue culture. We evaluated HCV/IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV genotype 1b subgenomic RNA replicon that differed only at two sites within the NS5A coding region. A replicon with a lysine (K) insertion at HCV codon 2040 (K2040) replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an leucine (L) to serine (S) point mutation at HCV codon 2198 (L2198S) replicated poorly and triggered a cellular response characterized by IFN-! production and low-level interferon-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high passage (HP) variant with 6 additional point mutations throughout the HCV protein-coding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-" treatment and was associated with viral-directed reduction in host cell expression and action of ISG56, an antagonist of HCV RNA translation. We also demonstrated that HCV subgenomic RNA replicons can be used to model the early events of HCV infection. We found that HCV RNA replicons rapidly induce the cellular antiviral response upon their transfection into host Huh7 cells and we determined that intracellular HCV double stranded RNA (dsRNA) is a potent agonist of host dsRNAactivated pathways. A Huh7 derived cell line that is highly permissive for transduction by HCV replicons is specifically defective in the activation of interferon regulatory factor (IRF)-3 by virus infection or HCV dsRNA transfection. We found that a mutation in the caspase recruitment domain (CARD) of the DExH/D-box helicase protein RIG-I, a component of the TLR3-independent intracellular dsRNA-responsive IRF-3 activation pathway, was responsible for this phenotype. Restoration of RIG-I-mediated IRF-3 activation through genetic complementation resulted in decreased permissiveness to HCV RNA replication. These results establish the RIG-I!IRF-3 pathway as a critical determinant of HCV persistence.