Rituximab Treatment in Hepatitis C Infection: An In Vitro Model to Study the Impact of B Cell Depletion on Virus Infectivity

Hepatitis C virus (HCV) infected patients with vasculitis are often treated with the B-cell-depleting anti-CD20 antibody rituximab. Treatment reduces the cryoglobulins that cause vasculitis, yet it also leads to a transient increase in liver enzymes and HCV genomic RNA in the periphery. The mechanism underlying the increased viral load is unclear and both direct and indirect roles have been proposed for B cells in HCV infection. We previously reported that HCV can associate with B cells and can trans-infect hepatocytes. We established an in vitro assay to study the effect(s) of rituximab on B cell-associated HCV infectivity. Rituximab-mediated lysis of B cells in vitro increases the level of infectious HCV released from B cells. Our results, using a model where virus does not replicate in B cells, recapitulate observations seen in patients and may explain in part the rapid increase in blood HCV RNA observed after rituximab treatment

Nuclear Polarization of Molecular Hydrogen Recombined on a Non-metallic Surface

The nuclear polarization of $\mathrm{H}_2$ molecules formed by recombination of nuclear polarized H atoms on the surface of a storage cell initially coated with a silicon-based polymer has been measured by using the longitudinal double-spin asymmetry in deep-inelastic positron-proton scattering. The molecules are found to have a substantial nuclear polarization, which is evidence that initially polarized atoms retain their nuclear polarization when absorbed on this type of surfac

Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system

Author Manuscript 2010 August 1Hepatitis C virus (HCV), which infects 2–3% of the world population, is a causative agent of chronic hepatitis and the leading indication for liver transplantation1. The ability to propagate HCV in cell culture (HCVcc) is a relatively recent breakthrough and a key tool in the quest for specific antiviral therapeutics. Monitoring HCV infection in culture generally involves bulk population assays, use of genetically modified viruses and/or terminal processing of potentially precious samples. Here we develop a cell-based fluorescent reporter system that allows sensitive distinction of individual HCV-infected cells in live or fixed samples. We demonstrate use of this technology for several previously intractable applications, including live-cell imaging of viral propagation and host response, as well as visualizing infection of primary hepatocyte cultures. Integration of this reporter with modern image-based analysis methods could open new doors for HCV research.New York (State). Dept. of Health (Empire State Stem Cell Fund Contract C023046)United States. Public Health Service (Grant R01 DK56966)National Institutes of Health (U.S.) (Roadmap for Medical Research Grant 1 R01 DK085713-01)Howard Hughes Medical Institute (Investigator

Multiple Interferon Stimulated Genes Synergize with the Zinc Finger Antiviral Protein to Mediate Anti-Alphavirus Activity

The zinc finger antiviral protein (ZAP) is a host factor that mediates inhibition of viruses in the Filoviridae, Retroviridae and Togaviridae families. We previously demonstrated that ZAP blocks replication of Sindbis virus (SINV), the prototype Alphavirus in the Togaviridae family at an early step prior to translation of the incoming genome and that synergy between ZAP and one or more interferon stimulated genes (ISGs) resulted in maximal inhibitory activity. The present study aimed to identify those ISGs that synergize with ZAP to mediate Alphavirus inhibition. Using a library of lentiviruses individually expressing more than 350 ISGs, we screened for inhibitory activity in interferon defective cells with or without ZAP overexpression. Confirmatory tests of the 23 ISGs demonstrating the largest infection reduction in combination with ZAP revealed that 16 were synergistic. Confirmatory tests of all potentially synergistic ISGs revealed 15 additional ISGs with a statistically significant synergistic effect in combination with ZAP. These 31 ISGs are candidates for further mechanistic studies. The number and diversity of the identified ZAP-synergistic ISGs lead us to speculate that ZAP may play an important role in priming the cell for optimal ISG function

The RBP-Jκ Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation

Kaposi's sarcoma-associated herpesvirus (KSHV) is tightly linked to at least two lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). However, the development of KSHV-mediated lymphoproliferative disease is not fully understood. Here, we generated two recombinant KSHV viruses deleted for the first RBP-Jκ binding site (RTA1st) and all three RBP-Jκ binding sites (RTAall) within the RTA promoter. Our results showed that RTA1st and RTAall recombinant viruses possess increased viral latency and a decreased capability for lytic replication in HEK 293 cells, enhancing colony formation and proliferation of infected cells. Furthermore, recombinant RTA1st and RTAall viruses showed greater infectivity in human peripheral blood mononuclear cells (PBMCs) relative to wt KSHV. Interestingly, KSHV BAC36 wt, RTA1st and RTAall recombinant viruses infected both T and B cells and all three viruses efficiently infected T and B cells in a time-dependent manner early after infection. Also, the capability of both RTA1st and RTAall recombinant viruses to infect CD19+ B cells was significantly enhanced. Surprisingly, RTA1st and RTAall recombinant viruses showed greater infectivity for CD3+ T cells up to 7 days. Furthermore, studies in Telomerase-immortalized human umbilical vein endothelial (TIVE) cells infected with KSHV corroborated our data that RTA1st and RTAall recombinant viruses have enhanced ability to persist in latently infected cells with increased proliferation. These recombinant viruses now provide a model to explore early stages of primary infection in human PBMCs and development of KSHV-associated lymphoproliferative diseases

IL28B Genetic Variation Is Associated with Spontaneous Clearance of Hepatitis C Virus, Treatment Response, Serum IL-28B Levels in Chinese Population

<p><b>Background:</b> The interleukin-28B gene (IL28B) locus has been associated with host resistance to hepatitis C virus (HCV) infection and response to PEG-IFN/RBV treatment in western populations. This study was to determine whether this gene variant is also associated with spontaneous clearance of HCV infection, treatment response and IL-28B protein production in Chinese patients.</p> <p><b>Methods:</b> We genotyped IL28B genetic variations (rs12980275, rs8103142, rs8099917 and rs12979860) by pyrosequencing DNA samples from cohorts consisting of 529 subjects with persistent HCV infection, 196 subjects who cleared the infection, 171 healthy individuals and 235 chronic HCV patients underwent IFN/RBV treatment. The expression of IL-28B were measured by ELISA and RT-PCR.</p> <p><b>Results:</b> We found that the four IL28B variants were in complete linkage disequilibrium (r2 = 0.97–0.98). The rs12979860 CC genotype was strongly associated with spontaneously HCV clearance and successful IFN/RBV treatment compared to the CT/TT. IL-28B levels in persistent HCV patients were significantly lower than subjects who spontaneously resolved HCV and healthy controls and were also associated with high levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase). IL-28B levels were also significantly lower in individuals carrying T alleles than CC homozygous.</p> <p><b>Conclusions:</b> Thus, the rs12979860-CC variant upstream of IL28B gene is associated with spontaneous clearance of HCV, susceptible to IFN/RBV treatment and increased IL-28B levels in this Chinese population.</p&gt

Hepatitis C Virus Protects Human B Lymphocytes from Fas-Mediated Apoptosis via E2-CD81 Engagement

HCV infection is often associated with B-cell regulatory control disturbance and delayed appearance of neutralizing antibodies. CD81 is a cellular receptor for HCV and can bind to HCV envelope protein 2 (E2). CD81 also participates to form a B cell costimulatory complex. To investigate whether HCV influences B cell activation and immune function through E2 -CD81 engagement, here, human Burkitt's lymphoma cell line Raji cells and primary human B lymphocytes (PHB) were treated with HCV E2 protein and cell culture produced HCV particles (HCVcc), and then the related cell phenotypes were assayed. The results showed that both E2 and HCVcc triggered phosphorylation of IκBα, enhanced the expression of anti-apoptosis Bcl-2 family proteins, and protected Raji cells and PHB cells from Fas-mediated death. In addition, both E2 protein and HCVcc increased the expression of costimulatory molecules CD80, CD86 and CD81 itself, and decreased the expression of complement receptor CD21. The effects were dependent on E2-CD81 interaction on the cell surface, since CD81-silenced Raji cells did not respond to both treatments; and an E2 mutant that lose the CD81 binding activity, could not trigger the responses of both Raji cells and PHB cells. The effects were not associated with HCV replication in cells, for HCV pseudoparticle (HCVpp) and HCVcc failed to infect Raji cells. Hence, E2-CD81 engagement may contribute to HCV-associated B cell lymphoproliferative disorders and insufficient neutralizing antibody production

Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule

Hepatitis C Virus Core Protein Induces Neuroimmune Activation and Potentiates Human Immunodeficiency Virus-1 Neurotoxicity

BACKGROUND: Hepatitis C virus (HCV) genomes and proteins are present in human brain tissues although the impact of HIV/HCV co-infection on neuropathogenesis remains unclear. Herein, we investigate HCV infectivity and effects on neuronal survival and neuroinflammation in conjunction with HIV infection. METHODOLOGY: Human microglia, astrocyte and neuron cultures were infected with cell culture-derived HCV or exposed to HCV core protein with or without HIV-1 infection or HIV-1 Viral Protein R (Vpr) exposure. Host immune gene expression and cell viability were measured. Patch-clamp studies of human neurons were performed in the presence or absence of HCV core protein. Neurobehavioral performance and neuropathology were examined in HIV-1 Vpr-transgenic mice in which stereotaxic intrastriatal implants of HCV core protein were performed. PRINCIPAL FINDINGS: HCV-encoded RNA as well as HCV core and non-structural 3 (NS3) proteins were detectable in human microglia and astrocytes infected with HCV. HCV core protein exposure induced expression of pro-inflammatory cytokines including interleukin-1β, interleukin-6 and tumor necrosis factor-α in microglia (p<0.05) but not in astrocytes while increased chemokine (e.g. CXCL10 and interleukin-8) expression was observed in both microglia and astrocytes (p<0.05). HCV core protein modulated neuronal membrane currents and reduced both β-III-tubulin and lipidated LC3-II expression (p<0.05). Neurons exposed to supernatants from HCV core-activated microglia exhibited reduced β-III-tubulin expression (p<0.05). HCV core protein neurotoxicity and interleukin-6 induction were potentiated by HIV-1 Vpr protein (p<0.05). HIV-1 Vpr transgenic mice implanted with HCV core protein showed gliosis, reduced neuronal counts together with diminished LC3 immunoreactivity. HCV core-implanted animals displayed neurobehavioral deficits at days 7 and 14 post-implantation (p<0.05). CONCLUSIONS: HCV core protein exposure caused neuronal injury through suppression of neuronal autophagy in addition to neuroimmune activation. The additive neurotoxic effects of HCV- and HIV-encoded proteins highlight extrahepatic mechanisms by which HCV infection worsens the disease course of HIV infection

Productive Hepatitis C Virus Infection of Stem Cell-Derived Hepatocytes Reveals a Critical Transition to Viral Permissiveness during Differentiation

Primary human hepatocytes isolated from patient biopsies represent the most physiologically relevant cell culture model for hepatitis C virus (HCV) infection, but these primary cells are not readily accessible, display individual variability, and are largely refractory to genetic manipulation. Hepatocyte-like cells differentiated from pluripotent stem cells provide an attractive alternative as they not only overcome these shortcomings but can also provide an unlimited source of noncancer cells for both research and cell therapy. Despite its promise, the permissiveness to HCV infection of differentiated human hepatocyte-like cells (DHHs) has not been explored. Here we report a novel infection model based on DHHs derived from human embryonic (hESCs) and induced pluripotent stem cells (iPSCs). DHHs generated in chemically defined media under feeder-free conditions were subjected to infection by both HCV derived in cell culture (HCVcc) and patient-derived virus (HCVser). Pluripotent stem cells and definitive endoderm were not permissive for HCV infection whereas hepatic progenitor cells were persistently infected and secreted infectious particles into culture medium. Permissiveness to infection was correlated with induction of the liver-specific microRNA-122 and modulation of cellular factors that affect HCV replication. RNA interference directed toward essential cellular cofactors in stem cells resulted in HCV-resistant hepatocyte-like cells after differentiation. The ability to infect cultured cells directly with HCV patient serum, to study defined stages of viral permissiveness, and to produce genetically modified cells with desired phenotypes all have broad significance for host-pathogen interactions and cell therapy