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GB Virus C Particles Inhibit T Cell Activation via Envelope E2 Protein-Mediated Inhibition of TCR Signaling
Abstract
Viruses enter into complex interactions within human hosts, leading to facilitation or suppression of each other's replication. Upon coinfection, GB virus C (GBV-C) suppresses HIV-1 replication in vivo and in vitro, and GBV-C coinfection is associated with prolonged survival in HIV-infected people. GBV-C is a lymphotropic virus capable of persistent infection. GBV-C infection is associated with reduced T cell activation in HIV-infected humans, and immune activation is a critical component of HIV disease pathogenesis. We demonstrate that serum GBV-C particles inhibited activation of primary human T cells. T cell activation inhibition was mediated by the envelope glycoprotein E2, because expression of E2 inhibited TCR-mediated activation of Lck. The region on the E2 protein was characterized and revealed a highly conserved peptide motif sufficient to inhibit TCR-mediated signaling. The E2 region contained a predicted Lck substrate site, and substitution of an alanine or histidine for the tyrosine reversed TCR-signaling inhibition. GBV-C E2 protein and a synthetic peptide representing the inhibitory amino acid sequence were phosphorylated by Lck in vitro. The synthetic peptide also inhibited TCR-mediated activation of primary human CD4(+) and CD8(+) T cells. Extracellular microvesicles from GBV-C E2-expressing cells contained E2 protein and inhibited TCR signaling in bystander T cells not expressing E2. Thus, GBV-C reduced global T cell activation via competition between its envelope protein E2 and Lck following TCR engagement. This novel inhibitory mechanism of T cell activation may provide new approaches for HIV and immunoactivation therapy.
... Identification of how LAVs regulate immune function may provide insights into viral pathogenesis and identify novel approaches to ameliorate or treat inflammatory-based diseases. We previously found that several viruses within the Flaviviridae, specifically human Pegivirus (HPgV), hepatitis C virus (HCV) and yellow fever virus (YFV) infections modulate immunity by inhibiting T cell activation in vitro [22][23][24][25][26]. Three virus-specific mechanisms were identified that result in reduced signaling through the T cell receptor [24][25][26]. ...
... PBMCs were either used fresh or were stored in liquid nitrogen as described [32]. For TCR stimulation studies, vaccine recipient PBMCs stored in liquid nitrogen (1×10 6 cells/ml) were incubated with plate-bound anti-CD3 (100ng/ml, OKT3 clone, eBioscience) and IL-2 released into cell culture supernatants was quantified 16 hours post-stimulation using human IL-2 ELISA kit (BD Biosciences) as previously described and according to the manufacturer's instructions [23,24]. Each experiment was performed in triplicate. ...
... PTPRE, Lck and phosphorylated active site (Y394) LCK were measured either in vaccine recipient PBMCs or in healthy donor PBMCs before and 10 minutes following TCR stimulation with anti-CD3 as described [23,24,26]. Proteins were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (BIORAD). ...
Background:
Live attenuated (LA) vaccines alter immune functions and are associated with beneficial outcomes. We previously demonstrated that yellow fever virus vaccine (LA-YF-Vax) dampens T cell receptor (TCR) signaling in vitro via an RNA-based mechanism. We examined subjects before and after LA-YF-Vax to assess TCR-mediated functions in vivo.
Methods:
Sera and peripheral blood mononuclear cells (PBMCs) were obtained before and after LA-YF-Vax (+/-additional vaccines) or quadrivalent influenza vaccine (QIV). TCR-mediated activation was determined by IL-2 release or phosphorylation of the lymphocyte-specific-Src-kinase. TCR-regulating phosphatase (PTPRE) expression was also measured.
Results:
Compared to pre-vaccination, LA-YF-Vax recipient PBMCs demonstrated transient reduction in IL-2 release following TCR-stimulation and PTPRE levels, unlike QIV control subjects. YFV was detected in (8/14) following LA-YF-Vax. Following incubation of healthy donor PBMCs in serum-derived extracellular vesicles (EVs) prepared from LA-YF-Vax recipients, TCR signaling and PTPRE levels were reduced post-vaccination, even in subjects without detectable YFV RNA.
Conclusions:
LA-YF-Vax reduces TCR functions and PTPRE levels following vaccination. EVs from serum recapitulated this effect in healthy cells. This likely contributes to the reduced immunogenicity for heterologous vaccines following LA-YF-Vax administration. Identification of specific immune mechanisms related to vaccines should contribute to understanding of the "off target", beneficial effects of live vaccines.
... Incubation of HPgV-1 with healthy donor peripheral blood mononuclear cells (PBMCs) or culture of PBMCs from HPgV-1 viremic individuals results in low level release of viral RNA into culture supernatant fluids, and T cell and NK cell functions are altered in vitro and in vivo (see below) (111)(112)(113)(114)(115)(119)(120)(121). The virus cannot be passaged for more than a few cycles in cell culture, and thus circulating PBMCs do not appear highly permissive for infection (94,113). ...
... Examining this in vitro, recombinant HPgV-1 E2 protein reduced IL-2 receptor mediated STAT5 activation following IL-2 receptor stimulation (199). Further, HPgV-1 E2 protein reduced T cell proliferation and cell surface markers of T cell activation following stimulation through the T cell receptor (TCR) (121,199). ...
... A series of CD4+ T cell lines expressing E2 protein or E2 protein deletions identified a 13 amino acid peptide region that was sufficient to reduce TCR-mediated activation (121). HPgV-1 reduced activation of the proximal tyrosine Src kinase (LCK) in the TCR-induced signaling cascade, and the region involved was independent of the peptide region that inhibited HIV replication (121,164) (Figure 4). ...
Two groups identified a novel human flavivirus in the mid-1990s. One group named the virus hepatitis G virus (HGV) and the other named it GB Virus type C (GBV-C). Sequence analyses found these two isolates to be the same virus, and subsequent studies found that the virus does not cause hepatitis despite sharing genome organization with hepatitis C virus. Although HGV/GBV-C infection is common and may cause persistent infection in humans, the virus does not appear to directly cause any other known disease state. Thus, the virus was renamed “human pegivirus 1” (HPgV-1) for “persistent G” virus. HPgV-1 is found primarily in lymphocytes and not hepatocytes, and several studies found HPgV-1 infection associated with prolonged survival in people living with HIV. Co-infection of human lymphocytes with HPgV-1 and HIV inhibits HIV replication. Although three viral proteins directly inhibit HIV replication in vitro, the major effects of HPgV-1 leading to reduced HIV-related mortality appear to result from a global reduction in immune activation. HPgV-1 specifically interferes with T cell receptor signaling (TCR) by reducing proximal activation of the lymphocyte specific Src kinase LCK. Although TCR signaling is reduced, T cell activation is not abolished and with sufficient stimulus, T cell functions are enabled. Consequently, HPgV-1 is not associated with immune suppression. The HPgV-1 immunomodulatory effects are associated with beneficial outcomes in other diseases including Ebola virus infection and possibly graft-versus-host-disease following stem cell transplantation. Better understanding of HPgV-1 immune escape and mechanisms of inflammation may identify novel therapies for immune-based diseases.
... Inhibition of Src-family of kinases (SFKs) blunts the proximal TCR-mediated signaling pathways [20,21]. Activation of T cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin (P/I) activates the distal TCR signaling pathways mediated by the protein kinase C (PKC) and NFAT1, and bypasses the requirement of SFKs [4,35]. To assess the role of active SFKs on distal TCR signaling, Jurkat T cells were incubated with SFKs-specific inhibitor PP2 or the DMSO control [36]. ...
... Lck phosphorylation was not detected in cells treated with PP2 for longer than 1 hour, even at low (2.5 μg/ml) concentrations, suggesting complete Lck inhibition. Consistent with the previous observations [4,35], P/I stimulation bypassed the proximal TCR signaling as only anti-CD3 but not P/I stimulation activated Lck (Y394), ZAP-70 (Y319) and LAT (Y226) (Fig 1D). Following stimulation of distal TCR-mediated signaling with P/I, T cell activation was measured by assessing CD25 surface expression. ...
... As noted, stimulation of distal TCR-mediated signaling by P/I bypasses the proximal TCR-signaling ( Fig 1C) and selectively activates the nuclear factor of activated T cells (NFAT) and the protein kinase C (PKC) pathways [4,35]. NFAT proteins are essential transcription factors required for the activation of T cells, and inhibition of NFAT1 inhibits T cell activation and effector function [32]. ...
T cell signaling is required for activation of both natural and therapeutic T cells including chimeric antigen receptor (CAR) T cells. Identification of novel factors and pathways regulating T cell signaling may aid in development of effective T cell therapies. In resting human T cells, the majority of Src-family of tyrosine kinases (SFKs) are inactive due to phosphorylation of a conserved carboxy-terminal tyrosine residue. Recently, a pool of enzymatically active SFKs has been identified in resting T cells; however, the significance of these is incompletely understood. Here, we characterized the role of active SFKs in resting human T cells. Pharmacologic inhibition of active SFKs enhanced distal TCR signaling as measured by IL-2 release and CD25 surface expression following TCR-independent activation. Mechanistically, inhibition of the active pool of SFKs induced nuclear translocation of NFAT1, and enhanced NFAT1-dependent signaling in resting T cells. The negative regulation of NFAT1 signaling was in part mediated by the Src-kinase Lck as human T cells lacking Lck had increased levels of nuclear NFAT1 and demonstrated enhanced NFAT1-dependent gene expression. Inhibition of active SFKs in resting primary human T cells also increased nuclear NFAT1 and enhanced NFAT1-dependent signaling. Finally, the calcineurin inhibitor FK506 and Cyclosporin A reversed the effect of SFKs inhibition on NFAT1. Together, these data identified a novel role of SFKs in preventing aberrant NFAT1 activation in resting T cells, and suggest that maintaining this pool of active SFKs in therapeutic T cells may increase the efficacy of T cell therapies.
... HPgV and HCV dampen T-cell activation in vitro and in vivo at least in part by inhibiting TCR signaling [1,[9][10][11]. Incubation of T cells with HPgV virions, serum-derived vesicles, or recombinant envelope (E2) protein inhibits activation of the lymphocyte-specific Src kinase Lck [9,10]. HCV also reduces proximal TCR signaling using 2 different mechanisms [1,11]. ...
... HPgV and HCV dampen T-cell activation in vitro and in vivo at least in part by inhibiting TCR signaling [1,[9][10][11]. Incubation of T cells with HPgV virions, serum-derived vesicles, or recombinant envelope (E2) protein inhibits activation of the lymphocyte-specific Src kinase Lck [9,10]. HCV also reduces proximal TCR signaling using 2 different mechanisms [1,11]. ...
... Murine mononuclear cells were stimulated with antimurine CD3/CD28 antibody. TCR-mediated activation was determined by measuring human or murine IL-2 release by enzyme-linked immunosorbent assay as described previously [9,10]. Lck and ZAP-70, PTPRE, YFV-ZKV-DENV env proteins were detected using immune blotting as described previously [1]. ...
The Flavivirus genus within the Flaviviridae is comprised of many important human pathogens including Yellow Fever virus (YFV), Dengue virus (DENV) and Zika virus (ZKV), all of which are global public health concerns. Although the related flaviviruses hepatitis C virus and human pegivirus (formerly named GBV-C) interfere with T cell receptor (TCR) signaling by novel RNA and protein based mechanisms, the effect of other flaviviruses on TCR signaling is unknown. Here, we studied the effect of YFV, DENV, and ZKV on TCR signaling. Both YFV and ZKV replicated in human T cells in vitro; however, only YFV inhibited TCR signaling. This effect was mediated at least in part by the YFV envelope (env) protein coding RNA. Deletion mutagenesis studies demonstrated that expression of a short, YFV env RNA motif (vsRNA) was required and sufficient to inhibit TCR signaling. Expression of this vsRNA and YFV infection of T cells reduced the expression of Src-kinase regulatory phosphatase (PTPRE), while ZKV infection did not. YFV infection in mice resulted in impaired TCR signaling and PTPRE expression, with associated reduction in murine response to experimental ovalbumin vaccination. Together, these data suggest that viruses within the flavivirus genus inhibit TCR signaling in a species-dependent manner.
... In HIV-infected humans, persistent HPgV coinfection is associated with reduced T cell activation, proliferation and function (Maidana-Giret et al., 2009;Schwarze-Zander et al., 2010;Stapleton et al., , 2009 suggesting that HPgV-mediated immune modulation may contribute to viral persistence. In vitro, HPgV envelope glycoprotein (E2) inhibits T cell activation by reducing signaling through the IL-2 receptor and the T cell receptor (TCR), with resultant reduction in activation of the lymphocyte specific tyrosine kinase (Lck) (Bhattarai et al., 2013). The effects of HPgV on T cell activation may contribute to the observed protective effect of HPgV co-infection on survival of HIV-and Ebola-infected individuals (Nunnari et al., 2003;Tillmann et al., 2001;Vahidnia et al., 2012;Xiang et al., 2001;Lauck et al., 2015). ...
... The proportion of cells infected with HPgV is low (approximately 1-10 genome copies per 100 NK cells) (Chivero et al., 2014). The majority of serum-derived HPgV RNA is present in gradient fractions containing extracellular vesicles (EV) that have properties of exosomes (Bhattarai et al., 2013;Chivero et al., 2014). It is difficult if not impossible to exclude the presence of virions from EV preparations; however, HPgV RNA-containing particles prepared from gradients enriched for EVs deliver viral RNA to peripheral blood mononuclear cells, including NK cells (Bhattarai et al., 2013;Chivero et al., 2014). ...
... The majority of serum-derived HPgV RNA is present in gradient fractions containing extracellular vesicles (EV) that have properties of exosomes (Bhattarai et al., 2013;Chivero et al., 2014). It is difficult if not impossible to exclude the presence of virions from EV preparations; however, HPgV RNA-containing particles prepared from gradients enriched for EVs deliver viral RNA to peripheral blood mononuclear cells, including NK cells (Bhattarai et al., 2013;Chivero et al., 2014). ...
Human Pegivirus (HPgV, formally GB virus C) infects lymphocytes and NK cells in vivo, and infection is associated with reduced T cell and NK cell activation in HIV-infected individuals. The mechanism by which HPgV inhibits NK cell activation has not been assessed. Following IL-12 stimulation, IFNγ expression was lower in HIV-HPgV co-infected subjects compared to HIV mono-infected subjects (p=0.02). In addition, HPgV positive human sera, extracellular vesicles containing E2 protein, recombinant E2 protein and synthetic E2 peptides containing a predicted Tyk2 interacting motif inhibited NK cell IL-12-mediated IFNγ release. E2 protein also inhibited Tyk2 activation following IL-12 stimulation. In contrast, cytolytic NK cell function was not altered by HPgV. Inhibition of NK cell-induced proinflammatory/antiviral cytokines may contribute to both HPgV persistence and reduced immune activation during HIV-coinfection. Understanding mechanisms by which HPgV alters immune activation may contribute towards novel immunomodulatory therapies to treat HIV and inflammatory diseases.
Published by Elsevier Inc.
... At least three viral proteins inhibit HIV replication in vitro, including the non-structural NS3 and NS5A proteins and the envelope (E2) protein (George et al., 2012;Jung et al., 2007;Xiang et al., 2006Xiang et al., , 2012. Furthermore, HPgV infection is associated with reduced immune activation in HIV-infected individuals, presumably contributing to the observed improvement in HIV clinical outcomes (Bhattarai et al., 2012b(Bhattarai et al., , 2013Maidana-Giret et al., 2009;Stapleton et al., 2009Stapleton et al., , 2012bStapleton et al., , 2013. Recent studies found that the HPgV E2 protein inhibits T-cell receptor (TCR)-and IL-2 receptor (IL2R)-mediated signalling, which probably contributes to the reduction in T-cell activation (Bhattarai et al., 2012b(Bhattarai et al., , 2013Maidana-Giret et al., 2009;Stapleton et al., 2009Stapleton et al., , 2012bStapleton et al., , 2013. ...
... Furthermore, HPgV infection is associated with reduced immune activation in HIV-infected individuals, presumably contributing to the observed improvement in HIV clinical outcomes (Bhattarai et al., 2012b(Bhattarai et al., , 2013Maidana-Giret et al., 2009;Stapleton et al., 2009Stapleton et al., , 2012bStapleton et al., , 2013. Recent studies found that the HPgV E2 protein inhibits T-cell receptor (TCR)-and IL-2 receptor (IL2R)-mediated signalling, which probably contributes to the reduction in T-cell activation (Bhattarai et al., 2012b(Bhattarai et al., , 2013Maidana-Giret et al., 2009;Stapleton et al., 2009Stapleton et al., , 2012bStapleton et al., , 2013. ...
... Recent studies observed a reduction in T-cell activation and proliferation markers in subjects with HPgV/HIV coinfection compared with HIV-monoinfected individuals (Bhattarai et al., 2013;Maidana-Giret et al., 2009;Rydze et al., 2012;Stapleton et al., 2012b), suggesting that HPgV may replicate preferentially in naïve T-cells. In addition, activation markers on NK cells and B-cells are reduced in HPgV/HIV co-infected individuals, raising the possibility that HPgV may alter the activation of additional immune cell types in vivo (Bhattarai et al., 2012b(Bhattarai et al., , 2013Maidana-Giret et al., 2009;Stapleton et al., 2009Stapleton et al., , 2012bStapleton et al., , 2013. To understand better the distribution of HPgV in PBMCs and to study potential mechanisms of HPgV transmission, we examined highly purified blood mononuclear cells for HPgV RNA and characterized sequence diversity within and between serum and cells obtained from infected subjects. ...
Human Pegivirus (HPgV; originally called GB virus C/hepatitis G virus) is an RNA virus within the Pegivirus genus of the Flaviviridae that commonly causes persistent infection. Worldwide, approximately 750 million people are actively infected (viremic) and an estimated 0.75 to 1.5 billion people have evidence of prior HPgV infection. No causal association between HPgV and disease has been identified; however, several studies describe a beneficial relationship between persistent HPgV infection and survival in HIV-infected individuals. The beneficial effect appears to be related to a reduction in host immune activation. HPgV replicates well in vivo (> 1x107 genome copies detected per ml plasma); however, the virus grows poorly in vitro and systems to study this virus are limited. Consequently, mechanisms of viral persistence and host immune modulation remain poorly characterized, and the primary permissive cell type(s) has not yet been identified. HPgV RNA is found in liver, spleen, bone marrow, and peripheral blood mononuclear cells (PBMCs), including T and B lymphocytes, NK cells, and monocytes, although the mechanism of cell-to-cell transmission is unclear. HPgV RNA is also present in serum microvesicles with properties of exosomes. These microvesicles are able to transmit viral RNA to PBMCs in vitro, resulting in productive infection. This review summarizes existing data on HPgV cellular tropism, the effect of HPgV on immune activation in various PBMCs and discusses how this may influence viral persistence. We conclude that an increased understanding of HPgV replication and immune modulation may provide insights into persistent RNA viral infection of humans.
... The host immunological response underlying GBV-C and HIV coinfection that may contribute to reduced HIV replication and CD4 + T-cell loss and, consequently, to better survival are not well characterized. Prior studies found reduced lymphocyte, monocyte, and NK cell markers of activation in patients with HIV and GBV-C coinfection, compared with those with HIV monoinfection, suggesting that GBV-C infection may modulate host inflammation, thus reducing HIV replication and pathogenesis [27][28][29][30][31]. Furthermore, in vitro studies suggest that E2 and virus particles interfere with T-cell activation and proliferation [32][33][34][35]. ...
... An increase of naive T-cell frequency and a decrease of CD4 + and CD8 + T-cell activation has been demonstrated in HIV-infected individuals, irrespective of their ART exposure [27,30]. GBV-C was associated with a reduced basal level of immune activation, T-cell activation, and proliferation markers in vivo in HIV-infected people [33] and in vitro [32]. It is also suggested that GBV-C infection induces non-T-cell immune responses and reduces NK-cell, monocyte, and B-cell activation markers [45]. ...
... On one hand, it would be important to know whether GBV-C has a silent deleterious effect on the host at the cellular level. On the other hand, during acute GBV-C infection, the depletion of activated or proliferating T cells [30], which are potential targets for HIV, along with a decrease in T-cell activation [32], could decrease the targets for HIV infection and reduce the risk of acquisition of HIV infection and/or downstream viral dissemination and replication manifested in set point HIV viral load. ...
Background:
An association between GB virus C (GBV-C) and improved outcomes of human immunodeficiency virus (HIV) infection has been reported in HIV-positive individuals with active GBV-C coinfection. This study provides insights into the immune mechanisms underlying the protective role of GBV-C in HIV-infected patients.
Methods:
The concentrations of 64 cytokines and chemokines were measured in plasma samples obtained from the Viral Activation Transfusion Study cohort before transfusion and longitudinally from 30 patients positive for both HIV and GBV-C (hereafter, "cases") and 30 patients positive for HIV and negative for GBV-C (hereafter, "controls").
Results:
Cases had lower HIV viral loads and higher CD4 T-cell counts than controls after acquisition of GBV-C infection. Most of the modulated cytokines and chemokines were reduced after GBV-C detection, including many proinflammatory cytokines, suggesting an overall antiinflammatory effect of GBV-C in HIV-positive subjects. Most pathways and functions of the measured cytokines were downregulated in cases, except cell death pathways, which were upregulated in various cell subsets in the 3 months after GBV-C detection.
Conclusions:
GBV-C has a protective effect, in part through a competition mechanism leading to decreased inflammation and improved HIV disease outcome in cases. Further studies are necessary to establish whether GBV-C may have deleterious effects on the host at the cellular level, including depleting the cells that are the targets of HIV.
... At least three viral proteins inhibit HIV replication in vitro, including the non-structural NS3 and NS5A proteins and the envelope (E2) protein (George et al., 2012;Jung et al., 2007;Xiang et al., 2006Xiang et al., , 2012. Furthermore, HPgV infection is associated with reduced immune activation in HIV-infected individuals, presumably contributing to the observed improvement in HIV clinical outcomes (Bhattarai et al., 2012b(Bhattarai et al., , 2013Maidana-Giret et al., 2009;Stapleton et al., 2009Stapleton et al., , 2012bStapleton et al., , 2013. Recent studies found that the HPgV E2 protein inhibits T-cell receptor (TCR)-and IL-2 receptor (IL2R)-mediated signalling, which probably contributes to the reduction in T-cell activation (Bhattarai et al., 2012b(Bhattarai et al., , 2013Maidana-Giret et al., 2009;Stapleton et al., 2009Stapleton et al., , 2012bStapleton et al., , 2013. ...
... Furthermore, HPgV infection is associated with reduced immune activation in HIV-infected individuals, presumably contributing to the observed improvement in HIV clinical outcomes (Bhattarai et al., 2012b(Bhattarai et al., , 2013Maidana-Giret et al., 2009;Stapleton et al., 2009Stapleton et al., , 2012bStapleton et al., , 2013. Recent studies found that the HPgV E2 protein inhibits T-cell receptor (TCR)-and IL-2 receptor (IL2R)-mediated signalling, which probably contributes to the reduction in T-cell activation (Bhattarai et al., 2012b(Bhattarai et al., , 2013Maidana-Giret et al., 2009;Stapleton et al., 2009Stapleton et al., , 2012bStapleton et al., , 2013. ...
... Recent studies observed a reduction in T-cell activation and proliferation markers in subjects with HPgV/HIV coinfection compared with HIV-monoinfected individuals (Bhattarai et al., 2013;Maidana-Giret et al., 2009;Rydze et al., 2012;Stapleton et al., 2012b), suggesting that HPgV may replicate preferentially in naïve T-cells. In addition, activation markers on NK cells and B-cells are reduced in HPgV/HIV co-infected individuals, raising the possibility that HPgV may alter the activation of additional immune cell types in vivo (Bhattarai et al., 2012b(Bhattarai et al., , 2013Maidana-Giret et al., 2009;Stapleton et al., 2009Stapleton et al., , 2012bStapleton et al., , 2013. To understand better the distribution of HPgV in PBMCs and to study potential mechanisms of HPgV transmission, we examined highly purified blood mononuclear cells for HPgV RNA and characterized sequence diversity within and between serum and cells obtained from infected subjects. ...
Human Pegivirus (HPgV; previously called GB virus C/hepatitis G virus) has limited pathogenicity despite causing persistent infection, and is associated with prolonged survival in HIV-infected individuals. Although HPgV RNA is found in and produced by T and B lymphocytes, the primary permissive cell type(s) are unknown. We quantified HPgV RNA in highly purified CD4+ and CD8+ T cells, including naïve, central memory, and effector memory populations, and in B cells (CD19+), NK cells (CD56+) cells and monocytes (CD14+) using real time RT-PCR. Single genome sequencing was performed on virus within individual cell types to estimate genetic diversity among cell populations. HPgV RNA was present in CD4+ and CD8+ T lymphocytes (9 of 9 subjects), B lymphocytes (7 of 10), NK cells and monocytes (both 4 of 5). HPgV RNA levels were higher in naïve (CD45RA+) CD4+ cells than in central memory and effector memory cells (p<0.01). HPgV sequences were highly conserved between patients (0.117 ± 0.02 substitutions per site; range 0.58-0.14) and within subjects (0.006 ± 0.003 substitutions per site; range 0.006-0.010). The non-synonymous/synonymous substitution ratio was 0.07 suggesting low selective pressure. CFSE-labeled HPgV RNA-containing particles precipitated by a commercial exosome isolation reagent delivered CSFE to uninfected monocytes, NK cells, T and B lymphocytes, and HPgV RNA was transferred to peripheral blood mononuclear cells with evidence of subsequent viral replication. Thus, HPgV RNA-containing serum particles including microvesicles may contribute to delivery of HPgV to PBMCs in vivo, explaining the apparent broad tropism of this persistent human RNA virus.
... The reason for the lack of hepatic disease presumably relates to its tissue tropism, as HPgV does not appear to replicate in the liver, and based on both ex vivo and in vitro studies it replicates in B and T lymphocytes, including CD4+ and CD8+ lymphocytes, spleen, and bone marrow (Laskus et al, 1998;Berg et al, 1999;Fan et al, 1999;Tucker et al, 2000;Xiang et al, 2000;George et al, 2003George et al, , 2006Jablonska et al, 2013;Kisiel et al, 2013;Chivero et al, 2014;Bailey et al, 2015). Persistent infection among HIVinfected individuals is highly associated with impaired T-cell receptor and interleukin 2 receptor (IL2R) signalling, resulting in inhibition of T cell apoptosis, and impaired T cell activation and proliferation that may contribute to lymphomagenesis (Moenkemeyer et al, 2008;Stapleton et al, 2009Stapleton et al, , 2012Bhattarai et al, 2012Bhattarai et al, , 2013Lanteri et al, 2015). ...
... Additional biological mechanisms that may contribute to a role for HPgV viraemia in lymphomagenesis include the fact that it is a lymphotropic virus that may cause persistent infection in both T and B lymphocytes (Tucker et al, 2000;George et al, 2006;Chivero et al, 2014). In cell lines derived from HIV-infected patients, HPgV reduces Fas-mediated apoptosis (Moenkemeyer et al, 2008) and virus impairs Tcell receptor and IL2R signalling in primary and transformed T cell lines (Bhattarai et al, , 2013 and in patients with HCV infection (Bhattarai et al, 2017), all of which may contribute to subclinical impairment of immune surveillance and thereby increase the risk of developing NHL (Pietersma et al, 2008). While the lack of association of HPgV with risk of CLL might temper this hypothesis, the associations of NHL subtypes with specific types of infections and/or immunosuppression (primary, transplantation, iatrogenic) have been quite heterogeneous (Cerhan et al, 2018). ...
... Polymorphisms were found in E2 motifs possessing important structural and functional elements that play a key role in cell binding, HIV inhibition (Figs. 4 and 5) and formation of antigenic landscape. The latter is especially important given attempts of pegivirus to evade the immune pressure [51]. However HPgV-1 seems to balance between the need for a broader E2 antigenic diversification and constraints imposed by the necessity to preserve important functions. ...
... Therefore, the accidental use of HPgV-1-contaminated cells for transplantation may pose a risk of transporting the virus to the HSC homing sites. Given an inhibiting effect of pegivirus on proliferation (demonstrated recently for differentiating immune cells [51]) the efficacy of HPgV-1-infected stem cells in replacement therapy may be reduced. The consequences may be further aggravated in immunosuppressed patients. ...
Background
Human pegivirus-1 (HPgV-1) is a member of the Flaviviridae family whose genomic organization and mode of cellular entry is similar to that of hepatitis C virus (HCV). The E2 glycoprotein of HPgV-1 is the principle mediator in the virus-cell interaction and as such harbors most of HPgV-1’s antigenic determinants. HPgV-1 persists in blood cell precursors which are increasingly used for cell therapy.
Methods
We studied HPgV-1 prevalence in a large cohort of females donating fetal tissues for clinical use. PCR was used for screening and estimation of viral load in viremic plasma and fetal samples. Sequence analysis was performed for portions of the 5′-untranslated and E2 regions of HPgV-1 purified from donor plasmas. Sequencing was followed by phylogenetic analysis.
Results
HPgV-1 was revealed in 13.7% of plasmas, 5.0% of fetal tissues, 5.4% of chorions, exceeding the prevalence of HCV in these types of samples. Transmission of HPgV-1 occurred in 25.8% of traceable mother-chorion-fetal tissues triads. For HPgV-1-positive donors, a high viral load in plasma appears to be a prerequisite for transmission. However, about one third of fetal samples acquired infection from non-viremic individuals. Sequencing of 5′-untranslated region placed most HPgV-1 samples to genotype 2a. At the same time, a portion of E2 sequence provided a much weaker support for this grouping apparently due to a higher variability. Polymorphisms were detected in important structural and antigenic motifs of E2.
Conclusion
HPgV-1 is efficiently transmitted to fetus at early embryonic stages. A high variability in E2 may pose a risk of generation of pathogenic subtypes. Although HPgV-1 is considered benign and no longer tested mandatorily in blood banks, the virus may have adversary effects at target niches if delivered with infected graft upon cell transplantation. This argues for the necessity of HPgV-1 testing of cell samples aimed for clinical use.
... The reason for the lack of hepatic disease presumably relates to its tissue tropism, as HPgV does not appear to replicate in the liver, and based on both ex vivo and in vitro studies it replicates in B and T lymphocytes, including CD4+ and CD8+ lymphocytes, spleen, and bone marrow (Laskus et al, 1998;Berg et al, 1999;Fan et al, 1999;Tucker et al, 2000;Xiang et al, 2000;George et al, 2003George et al, , 2006Jablonska et al, 2013;Kisiel et al, 2013;Chivero et al, 2014;Bailey et al, 2015). Persistent infection among HIVinfected individuals is highly associated with impaired T-cell receptor and interleukin 2 receptor (IL2R) signalling, resulting in inhibition of T cell apoptosis, and impaired T cell activation and proliferation that may contribute to lymphomagenesis (Moenkemeyer et al, 2008;Stapleton et al, 2009Stapleton et al, , 2012Bhattarai et al, 2012Bhattarai et al, , 2013Lanteri et al, 2015). ...
... Additional biological mechanisms that may contribute to a role for HPgV viraemia in lymphomagenesis include the fact that it is a lymphotropic virus that may cause persistent infection in both T and B lymphocytes (Tucker et al, 2000;George et al, 2006;Chivero et al, 2014). In cell lines derived from HIV-infected patients, HPgV reduces Fas-mediated apoptosis (Moenkemeyer et al, 2008) and virus impairs Tcell receptor and IL2R signalling in primary and transformed T cell lines (Bhattarai et al, , 2013 and in patients with HCV infection (Bhattarai et al, 2017), all of which may contribute to subclinical impairment of immune surveillance and thereby increase the risk of developing NHL (Pietersma et al, 2008). While the lack of association of HPgV with risk of CLL might temper this hypothesis, the associations of NHL subtypes with specific types of infections and/or immunosuppression (primary, transplantation, iatrogenic) have been quite heterogeneous (Cerhan et al, 2018). ...
... Alternatively, antigen-specific TCR-mediated activation was stimulated using pooled synthetic peptides (20 μg/ml) with sequences derived from human cytomegalovirus, Epstein-Barr virus, and influenza viruses (CEF control peptides, AnaSpec, EGT Group) [62,63]. TCR-mediated signaling was determined 16 hours post-TCR stimulation by measuring IL-2 using ELISA as described [64,65], or by measuring activated Lck protein as described below. Each experiment was performed in three replicate cultures, and in a minimum of three healthy donor PBMCs with consistent results. ...
... Cell lysates were separated by SDS-PAGE gel electrophoresis, transferred to nitrocellulose membranes, and proteins detected by chemiluminescence as described [65,68]. Primary antibodies included phospho-Lck Y394 (R&D Systems), PTPRE (4B2) and GAPDH (Origene), PTPRE (Rabbit; Abcam), or Actin (Sigma). ...
Among human RNA viruses, hepatitis C virus (HCV) is unusual in that it causes persistent infection in the majority of infected people. To establish persistence, HCV evades host innate and adaptive immune responses by multiple mechanisms. Recent studies identified virus genome-derived small RNAs (vsRNAs) in HCV-infected cells; however, their biological significance during human HCV infection is unknown. One such vsRNA arising from the hepatitis C virus (HCV) E2 coding region impairs T cell receptor (TCR) signaling by reducing expression of a Src-kinase regulatory phosphatase (PTPRE) in vitro. Since TCR signaling is a critical first step in T cell activation, differentiation, and effector function, its inhibition may contribute towards HCV persistence in vivo. The effect of HCV infection on PTPRE expression in vivo has not been examined. Here, we found that PTPRE levels were significantly reduced in liver tissue and peripheral blood mononuclear cells (PBMCs) obtained from HCV-infected humans compared to uninfected controls. Loss of PTPRE expression impaired antigen-specific TCR signaling, and curative HCV therapy restored PTPRE expression in PBMCs; restoring antigen-specific TCR signaling defects. The extent of PTPRE expression correlated with the amount of sequence complementarity between the HCV E2 vsRNA and the PTPRE 3’ UTR target sites. Transfection of a hepatocyte cell line with full-length HCV RNA or with synthetic HCV vsRNA duplexes inhibited PTPRE expression, recapitulating the in vivo observation. Together, these data demonstrate that HCV infection reduces PTPRE expression in the liver and PBMCs of infected humans, and suggest that the HCV E2 vsRNA is a novel viral factor that may contribute towards viral persistence.
... In HIV-positive individuals, HPgV viremia has been associated with prolonged survival and a milder HIV disease course, including higher CD4+ T cell counts, lower HIV viral load, and delayed progression to AIDS (reviewed in [18]). Impacts of HPgV infection that potentially contribute to this antagonism include direct antiviral effects; altered expression of cytokines, chemokines and HIV entry receptors; and modulation of host cell signaling pathways [18,19,27]. However, the mechanisms underlying these phenomena are still not fully understood, and no tractable animal models exist to study them. ...
... Persistent HPgV viremia is associated with prolonged survival and improved surrogate markers of disease progression in HIVpositive individuals [18,83,84]. Several potential mechanisms for the apparent protective effects of HPgV viremia have been identified, including direct antiviral effects; altered expression of cytokines, chemokines and HIV entry receptors; and modulation of host cell signaling pathways [18,19,27]. However, a great deal of uncertainty still surrounds HPgV mitigation of HIV pathogenesis, in part because no tractable animal model exists with which to study this phenomenon. ...
Within the Flaviviridae, the recently designated genus Pegivirus has expanded greatly due to new discoveries in bats, horses, and rodents. Here we report the discovery and characterization of three simian pegiviruses (SPgV) that resemble human pegivirus (HPgV) and infect red colobus monkeys (Procolobus tephrosceles), red-tailed guenons (Cercopithecus ascanius) and an olive baboon (Papio anubis). We have designated these viruses SPgVkrc, SPgVkrtg and SPgVkbab, reflecting their host species' common names, which include reference to their location of origin in Kibale National Park, Uganda. SPgVkrc and SPgVkrtg were detected in 47% (28/60) of red colobus and 42% (5/12) red-tailed guenons, respectively, while SPgVkbab infection was observed in 1 of 23 olive baboons tested. Infections were not associated with any apparent disease, despite the generally high viral loads observed for each variant. These viruses were monophyletic and equally divergent from HPgV and pegiviruses previously identified in chimpanzees (SPgVcpz). Overall, the high degree of conservation of genetic features among the novel SPgVs, HPgV and SPgVcpz suggests conservation of function among these closely related viruses. Our study describes the first primate pegiviruses detected in Old World monkeys, expanding the known genetic diversity and host range of pegiviruses and providing insight into the natural history of this genus.
... For instance, the fusion protein of gp41 and gp120 that HIV-1 uses to target various parts of the T cell receptor (TCR) complex can prevent T cells activation (21)(22)(23)(24). Gb virus C (GBV-C) particles of Flaviviridae can inhibit T cells activation via the TCR signaling pathway mediated by the E2 protein (25). Coxsackievirus downregulate T cell kinase induced by interleukin-2 (IL-2) (26). ...
Mycoplasma genitalium is a prokaryotic microorganism that causes urogenital tract infections. M. genitalium protein of adhesion (MgPa) was essential for M. genitalium attachment and subsequent invasion into host cells. Our prior research confirmed that Cyclophilin A (CypA) was the binding receptor for MgPa and MgPa-CypA interaction can lead to the production of inflammatory cytokines. In this study, we revealed that the recombinant MgPa (rMgPa) could inhibit the CaN-NFAT signaling pathway to reduce the level of IFN-γ, IL-2, CD25, and CD69 in Jurkat cells by binding to the CypA receptor. Moreover, rMgPa inhibited the expressions of IFN-γ, IL-2, CD25, and CD69 in primary mouse T cells. Likewise, the expressions of these T cells activation-related molecules in CypA-siRNA-transfected cells and CypA−/− mouse primary T cell was strengthened by rMgPa. These findings showed that rMgPa suppressed T cell activation by downregulating the CypA-CaN-NFAT pathway, and as a result, acted as an immunosuppressive agent.
IMPORTANCE Mycoplasma genitalium is a sexually transmitted bacterium that can co-infect with other infections and causes nongonococcal urethritis in males, cervicitis, pelvic inflammatory disease, premature birth, and ectopic pregnancy in women. The adhesion protein of M. genitalium (MgPa) is the primary virulence factor in the complicated pathogenicity of M. genitalium. This research proved that MgPa could interact with host cell Cyclophilin A (CypA) and prevent T cell activation by inhibiting Calcineurin (CaN) phosphorylation and NFAT nuclear translocation, which clarified the immunosuppression mechanism of M. genitalium to host T cells. Therefore, this study can provide a new idea that CypA can be used for a therapeutic or prophylactic target for M. genitalium infection.
... HPgV-1 co-infection reduces HIV-1-mediated activation of T, B, and/or NK cells, and contributes to the maintenance of balance between T-helper 1 (Th1) cytokines and Th2 cytokines, which delay the development of AIDS [199][200][201][202]. For example, HPgV-1 E2 glycoprotein inhibits T cell activation by reducing TCR-induced IL-2 production and altering IL-2 signaling pathways [152,156,[202][203][204][205]. Furthermore, HPgV-1 infection is associated with an increase of CD4 and CD8 doublenegative T cells (CD4-CD8-CD3+), which also contribute to reduction of immune activation and maintenance of immune homeostasis, further improving the survival of HIV-1 infected individuals [6,184,206]. ...
Human pegivirus (HPgV-1), previously known as GB virus C (GBV-C) or hepatitis G virus (HGV), is a single-stranded positive RNA virus belonging to the genus Pegivirus of the Flaviviridae family. It is transmitted by percutaneous injuries (PIs), contaminated blood and/or blood products, sexual contact, and vertical mother-to-child transmission. It is widely prevalent in general population, especially in high-risk groups. HPgV-1 viremia is typically cleared within the first 1–2 years of infection in most healthy individuals, but may persist for longer periods of time in immunocompromised individuals and/or those co-infected by other viruses. A large body of evidences indicate that HPgV-1 persistent infection has a beneficial clinical effect on many infectious diseases, such as acquired immunodeficiency syndrome (AIDS) and hepatitis C. The beneficial effects seem to be related to a significant reduction of immune activation, and/or the inhabitation of co-infected viruses (e.g. HIV-1). HPgV-1 has a broad cellular tropism for lymphoid and myeloid cells, and preferentially replicates in bone marrow and spleen without cytopathic effect, implying a therapeutic potential. The paper aims to summarize the natural history, prevalence and distribution characteristics, and pathogenesis of HPgV-1, and discuss its association with other human viral diseases, and potential use in therapy as a biovaccine or viral vector.
... GB virus C (GBV-C), formally known as hepatitis G virus (HGV), is a member of the Flaviviridae family. It has been shown that GB virus C particles inhibit T cell activation via envelope E2 protein-mediated inhibition of T-cell receptor (TCR) signaling [28]. These findings suggest that EVs play various functions in the pathogenesis of viral hepatitis as shown in Figure 1. ...
During the progression from hepatitis to fibrosis, cirrhosis, and liver failure, the accumulation of stressed/damaged hepatocyte elements associated with liver inflammation is critical. The causes of hepatocyte injuries include viral hepatitis infections, alcoholic hepatitis, and non-alcoholic fatty liver disease. Hepatocyte-derived extracellular vesicles (Hep-EVs) released from stressed/damaged hepatocytes are partly responsible for liver disease progression and liver damage because they activate non-parenchymal cells and infiltrate inflammatory cells within the liver, which are in turn are an important source of EVs. This cell-to-cell signaling is prevalent during inflammation in many liver diseases. Accordingly, special emphasis should be placed on liquid biopsy methods for the long-term monitoring of chronic liver diseases. In the present review, we have highlighted various aspects of current liquid biopsy research into chronic liver diseases. We have also reviewed recent progress on liquid biopsies that focus on cell-free DNA (cfDNA), long non-coding RNA (lncRNA), and the proteins in EVs as potential diagnostic tools and novel therapeutic targets in patients with viral hepatitis, fatty liver steatosis, and alcoholic liver diseases.
... HHpgV-1 has been found in B and T lymphocytes, among other cells [25,26]. It has also been noted by several groups that persons with HHpgV-1 viremia have lesser amounts of activation of T and B cells, monocytes, and natural killer (NK) cells than persons without viremia [10,[27][28][29][30][31][32]. Ex vivo stimulation of NK cells taken from people with HHpgV-1 infection resulted in less interferon (IFN)-γ production than cells taken from healthy controls [33]. ...
... The biological mechanisms that may contribute to a role for HPgV viremia in lymphomagenesis include its being a lymphotropic virus that may cause persistent infection in both T and B lymphocytes [24,30,31]. In cell lines derived from HIV-infected patients, HPgV reduces Fas-mediated apoptosis [55] and virus impairs T-cell receptor and interleukin 2 receptor signaling in primary and transformed T-cell lines [56,57] and in patients with HCV infection [58], all of which may contribute to subclinical impairment of immune surveillance. HPgV also influences cytokine and chemokine gene expression in lymphocytes in vitro and in cells obtained from infected individuals, potentially influencing immune response [59][60][61][62]. ...
Background:
Human pegivirus (HPgV) is a single-strand RNA virus belonging to the Flaviviridae. Although no definitive association between HPgV infection and disease has been identified, previous studies have suggested an association of HPgV viremia with risk of lymphomas.
Methods:
We conducted a systematic review and meta-analysis, including 1 cohort study and 14 case-control studies, assessing the association of HPgV viremia with adult lymphomas. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using a random-effects model, overall and by geographic region and lymphoma subtype.
Results:
The overall OR for lymphoma was 2.85 (95% CI, 1.98-4.11), with statistically significantly elevated ORs observed in 8 of 15 studies. There was a small amount of heterogeneity among studies (I2 = 28.9%; Q = 18.27, P = .16), and the funnel plot provided no evidence for publication bias. The strongest association with lymphoma risk was observed for studies from Southern Europe (OR, 5.68 [95% CI, 1.98-16.3]), whereas weaker ORs (with 95% CIs) were observed for studies from North America (2.24 [1.76-2.85]), Northern Europe (2.90 [.45-18.7), and the Middle East (2.51 [.87-7.27]), but all of similar magnitude. Participants with HPgV viremia had statistically significantly increased risks (OR [95% CI]) for developing diffuse large B-cell (3.29 [1.63-6.62]), follicular (3.01 [1.95-4.63]), marginal zone (1.90 [1.13-3.18]), and T-cell (2.11 [1.17-3.89]) lymphomas, while the risk for Hodgkin lymphoma (3.53 [.48-25.9]) and chronic lymphocytic leukemia (1.45 [.45-4.66]) were increased but did not achieve statistical significance.
Conclusions:
This meta-analysis supports a positive association of HPgV viremia with lymphoma risk, overall and for the major lymphoma subtypes.
... Extracellular vesicles (EVs) play an essential role in mediating communication among diverse cell types and tissues, including the CNS (Cocucci et al. 2009;Thery et al. 2009;Gupta and Pulliam 2014). Release kinetics of EVs, including their number, size distribution as well as their cargo, has been reported to be altered during various pathologies (Piccin et al. 2007;Shi et al. 2014) including cancer (Rabinowits et al. 2009;Taylor and Gercel-Taylor 2011), neurological diseases (Gupta and Pulliam 2014) and viral infections (Bhattarai et al. 2013;Narayanan et al. 2013). Elegant studies have also demonstrated that HIV-infected cells release EVs containing HIV proteins (Gag and Nef) and RNA (the trans-activation response element [TAR]) (Booth et al. 2006;Campbell et al. 2008;Shelton et al. 2012;Narayanan et al. 2013;Rahimian and He 2016b;Sami Saribas et al. 2017) and that these viral components can be transferred to neighboring/distant cells leading to functional impairment of the recipient cells. ...
Although combination antiretroviral therapy (cART) has improved the health of millions of those living with HIV-1 (Human Immunodeficiency Virus, Type 1), the penetration into the central nervous system (CNS) of many such therapies is limited, thereby resulting in residual neurocognitive impairment commonly referred to as NeuroHIV. Additionally, while cART has successfully suppressed peripheral viremia, cytotoxicity associated with the presence of viral Transactivator of transcription (Tat) protein in tissues such as the brain, remains a significant concern. Our previous study has demonstrated that both HIV-1 Tat as well as opiates such as morphine, can directly induce synaptic alterations via independent pathways. Herein, we demonstrate that exposure of astrocytes to HIV-1 protein Tat mediates the induction and release of extracellular vesicle (EV) microRNA-7 (miR-7) that is taken up by neurons, leading in turn, to downregulation of neuronal neuroligin 2 (NLGN2) and ultimately to synaptic alterations. More importantly, we report that these impairments could be reversed by pretreatment of neurons with a neurotrophic factor platelet-derived growth factor-CC (PDGF-CC).
Graphical Abstract
... 46 Extracellular vesicles from cell culture supernatant of cells infected with GBV-C or serum from individuals with GB virus C (GBV-C) infection, a human virus of the Flaviviridae family related to HCV, were shown to inhibit T cell receptor signalling that was associated with the GBV-C envelope 2 protein found in EVs. 48 The GBV found in the EVs was functional and transmitted viral RNA to peripheral blood mononuclear cell (PBMCs) in vitro resulting in productive infection. 49 These observations highlight the importance of EVs in viral hepatitis infection and transmission. ...
Extracellular vesicles (EVs) are membranous vesicles originating from different cells in the liver. The pathophysiological role of EVs is increasingly recognized in liver diseases, including alcoholic liver disease, NAFLD, viral hepatitis and hepatocellular carcinoma. EVs, via their cargo, can provide communication between different cell types in the liver and between organs. EVs are explored as biomarkers of disease and could also represent therapeutic targets and vehicles for treatment delivery. Here, we review advances in understanding the role of EVs in liver diseases and discuss their utility in biomarker discovery and therapeutics.
... In recent years it has become clear that EVs, which were initially considered to be "platelet dust" 3 , constitute an important physiological system of cell-cell communication. Together with two other systems of intercellular communication, namely cell-cell contact interactions and released soluble molecules, EVs coordinate normal physiology and are altered in various pathologies 4,5 including viral infections 6,7 . ...
Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.
... This region (aa 603-619) is highly conserved and the Y613 is conserved in more than 600 isolates representing all HCV genotypes (http://www.hcv.lanl.gov). Previous studies found that a conserved tyrosine in the related human Pegivirus (HPgV) is required TCR-signaling inhibition, and mutation of this residue restores TCR signaling [35]. ...
T cell receptor (TCR) signaling is required for T-cell activation, proliferation, differentiation, and effector function. Hepatitis C virus (HCV) infection is associated with impaired T-cell function leading to persistent viremia, delayed and inconsistent antibody responses, and mild immune dysfunction. Although multiple factors appear to contribute to T-cell dysfunction, a role for HCV particles in this process has not been identified. Here, we show that incubation of primary human CD4+ and CD8+ T-cells with HCV RNA-containing serum, HCV-RNA containing extracellular vesicles (EVs), cell culture derived HCV particles (HCVcc) and HCV envelope pseudotyped retrovirus particles (HCVpp) inhibited TCR-mediated signaling. Since HCVpp’s contain only E1 and E2, we examined the effect of HCV E2 on TCR signaling pathways. HCV E2 expression recapitulated HCV particle-induced TCR inhibition. A highly conserved, 51 nucleotide (nt) RNA sequence was sufficient to inhibit TCR signaling. Cells expressing the HCV E2 coding RNA contained a short, virus-derived RNA predicted to be a Dicer substrate, which targeted a phosphatase involved in Src-kinase signaling (PTPRE). T-cells and hepatocytes containing HCV E2 RNA had reduced PTPRE protein levels. Mutation of 6 nts abolished the predicted Dicer interactions and restored PTPRE expression and proximal TCR signaling. HCV RNA did not inhibit distal TCR signaling induced by PMA and Ionomycin; however, HCV E2 protein inhibited distal TCR signaling. This inhibition required lymphocyte-specific tyrosine kinase (Lck). Lck phosphorylated HCV E2 at a conserved tyrosine (Y613), and phospho-E2 inhibited nuclear translocation of NFAT. Mutation of Y613 restored distal TCR signaling, even in the context of HCVpps. Thus, HCV particles delivered viral RNA and E2 protein to T-cells, and these inhibited proximal and distal TCR signaling respectively. These effects of HCV particles likely aid in establishing infection and contribute to viral persistence.
... If GBV-C infection attenuates EBOV pathogenesis, it is possible that this occurs through modulation of the host immune response. In the context of HIV infection, GBV-C has been associated with a reduced production of proinflammatory cytokines and a reduction in T-cell activation in vivo and in vitro (34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44). Conversely, robust production of proinflammatory cytokines and lymphocyte activation followed by massive T-cell death are thought to play a major role in EBOV pathogenesis and have been associated with poor clinical outcome in retrospective studies (21)(22)(23)(24)(25)(26)(27). ...
In 49 patients with known Ebolavirus (EBOV) outcomes during the ongoing outbreak in Sierra Leone, 13 were co-infected with the immunomodulatory pegivirus GB virus C (GBV-C). 53% of these GBV-C+ patients survived; in contrast, only 22% of GBV-C(-) patients survived. Both survival and GBV-C status were associated with age, with older patients having lower survival rates and intermediate-age patients (21-45 years) having the highest rate of GBV-C infection. Understanding the separate and combined effects of GBV-C and age on EBOV survival could lead to new treatment and prevention strategies, perhaps through age-related pathways of immune activation.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
... Furthermore, GBV-C infection has been associated with decreased apoptosis of T cells which may contribute to the prolonged survival of HIV-infected patients coinfected with GBV-C (14). Recent data showing that GBV-C infection reduces T cell activation and proliferation in HIV-infected individuals suggests that GBV-C infection may impair immune surveillance and immune responsiveness, which may lead to lymphomagenesis (15,45,46). ...
Some retrospective studies suggest an association between infection with GB-virus C (GBV-C) and non-Hodgkin lymphoma (NHL). We evaluated this association prospectively in a nested case-control study within the U.S. Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO). Cases (N=658) and controls (N=1,316) were individually-matched by age, sex, race/ethnicity, timing of study entry and sample selection. Pre-diagnostic PLCO serum samples were tested for GBV-C RNA (as a measure of active infection) and E2 antibody (active or resolved infection). Logistic regression was used to estimate odds ratios (ORs) for the association between GBV-C and NHL overall and NHL subtypes. Twelve cases (1.8%) and 7 controls (0.5%) were GBV-C RNA positive. GBV-C RNA positivity was associated with NHL overall (OR=3.43, 95% CI=1.35-8.71) and, based on small numbers, diffuse large B-cell lymphoma (OR=5.31, 95% CI=1.54-18.36). The association with NHL persisted when the interval between testing and selection was greater than 4 years (OR=6.00, 95% CI= 1.21- 29.73). In contrast, E2 antibody-positivity was not associated with NHL risk (OR=1.08, 95% CI=0.74-1.58). Our study demonstrates that GBV-C infection precedes development of NHL. GBV-C infection may play an etiologic role in a small proportion of NHL cases, perhaps by causing chronic immune stimulation or impaired immunosurveillance.
... Inhibiting the type I interferon response may also allow increased replication of HPgV (Lau et al., 1999). This discrepancy may be resolved by considering that increased HPgV viral levels could in turn enhance HIV-inhibitory effects mediated by the HPgV NS5A (Chang et al., 2007; Rydze et al., 2012; Xiang et al., 2008 Xiang et al., , 2006 Xiang et al., , 2009) and E2 proteins (Bhattarai et al., 2013; Jung et al., 2007; Koedel et al., 2011; McLinden et al., 2013; Mohr et al., 2010; Timmons et al., 2013; Xiang et al., 2012). Type I interferons also promote survival of activated T cells (Gelman et al., 2004; Marrack et al., 1999) which are the primary host cell for HIV (Stevenson et al., 1990), and HPgV infection is associated with reduced expression of T cell activation markers in HIV infected people (Stapleton et al., 2012). ...
We previously found that human pegivirus (HPgV; formerly GBV-C) NS3 protease activity inhibits Human Immunodeficiency Virus (HIV) replication in a CD4+ T cell line. Given the protease׳s similarity to the Hepatitis C virus (HCV) NS3 protease, we characterized HPgV protease activity and asked whether it affects the type I interferon response or is inhibited by HCV protease antagonists. We characterized the activity of proteases with mutations in the catalytic triad and demonstrated that the HCV protease inhibitors Telaprevir, Boceprevir, and Danoprevir do not affect HPgV protease activity. HPgV NS3 protease cleaved MAVS but not TRIF, and it inhibited interferon responses sufficiently to enhance growth of an interferon-sensitive virus. Therefore, HPgV׳s inhibition of the interferon response could help promote HPgV persistence, which is associated with clinical benefits in HIV-infected patients. Our results also imply that HCV protease inhibitors should not interfere with the beneficial effects of HPgV in HPgV/HCV/HIV infected patients.
Introduction
Human pegivirus-1 (HPgV-1) is a so-called commensal virus for which no known associated organ disease has been found to date. Yet, it affects immune-reconstitution as previously studied in the HIV population, in whom active co-infection with HPgV-1 can modulate T and NK cell activation and differentiation leading to a protective effect against the evolution of the disease. Little is known on the effect of HPgV-1 on immune-reconstitution in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients, a patient population in which we and others have previously reported high prevalence of HPgV-1 replication. The aim of this study was to compare the immune reconstitution after allo-HSCT among HPgV-1-viremic and HPgV-1-non-viremic patients.
Methods
Within a cohort study of 40 allo-HSCT patients, 20 allo-HSCT recipients positive in plasma sample for HPgV-1 by rRT-PCR during the first year (1, 3, 6, 12 months) after transplantation were matched with 20 allo-HSCT recipients negative for HPgV-1. T and NK cell reconstitution was monitored by flow cytometry in peripheral blood samples from allo-HSCT recipients at the same time points.
Results
We observed no significant difference in the absolute number and subsets proportions of CD4 and CD8 T cells between patient groups at any analysed timepoint. We observed a significantly higher absolute number of NK cells at 3 months among HPgV-1-viremic patients. Immunophenotypic analysis showed a significantly higher proportion of CD56 bright NK cells mirrored by a reduced percentage of CD56 dim NK cells in HPgV-1-positive patients during the first 6 months after allo-HSCT. At 6 months post-allo-HSCT, NK cell phenotype significantly differed depending on HPgV-1, HPgV-1-viremic patients displaying NK cells with lower CD16 and CD57 expression compared with HPgV-1-negative patients. In accordance with their less differentiated phenotype, we detected a significantly reduced expression of granzyme B in NK cells in HPgV-1-viremic patients at 6 months.
Discussion
Our study shows that HPgV-1-viremic allo-HSCT recipients displayed an impaired NK cell, but not T cell, immune-reconstitution compared with HPgV-1-non-viremic patients, revealing for the first time a potential association between replication of the non-pathogenic HPgV-1 virus and immunomodulation after allo-HSCT.
Background
Morphine is used extensively in the clinical setting owing to its beneficial effects, such as pain relief; its therapeutic utility is limited as the prolonged use of morphine often results in tolerance and addiction. Astrocytes in the brain are a direct target of morphine action and play an essential role in the development of morphine tolerance. Primary cilia and the cilia-mediated sonic hedgehog (SHH) signaling pathways have been shown to play a role in drug resistance and morphine tolerance, respectively. Extracellular vesicles (EVs) play important roles as cargo-carrying vesicles mediating communication among cells and tissues.
Methods
C57/B6 mice were administered morphine for 8 days to develop tolerance, which was determined using the tail-flick and hot plate assays. EVs were separated from astrocyte-conditioned media using either size exclusion chromatography or ultracentrifugation approaches followed by characterization of EVs using nanoparticle tracking analysis for EV size distribution and number; western blotting for EV markers and electron microscopy for EV morphology. Astrocytes were treated with EVs for 24h followed by assessing primary cilia by fluorescent immunostaining for primary cilia markers (ARL13B and Acetylated Tubulin).
Results
Morphine tolerant mice exhibited an increase in primary cilia length and percentage of ciliated astrocytes. The levels of SHH protein were upregulated in morphine-stimulated astrocyte-derived EVs (ADEVs). SHH on morphine-ADEVs activated SHH signaling in astrocytes through primary cilia. Our in vivo study demonstrated inhibition of either EV release or primary cilia prevents morphine tolerance in mice.
Conclusions
EV-mediated primary ciliogenesis contributes to the development of morphine tolerance.
The human pegivirus type 1 (HPgV-1)—as known as hepatitis G virus and GB virus C—is a common single-stranded RNA flavivirus. Because few studies have demonstrated an association between HPgV-1 infection and disease, screening for HPgV-1 is not performed routinely. Nonetheless, a beneficial impact of HPgV-1 infection on HIV disease progression has been reported in multiple studies. Given the burden of HIV in Asia and the complex interactions between viral co-infections and the host, we provide a comprehensive overview of the existing data from Asia on HPgV-1 infection, including the prevalence and circulating genotypes in all Asian countries with data reported. This review highlights the research conducted thus far and emphasizes the need for additional studies on HPgV-1 across the Asian continent.
Viruses have developed a spectrum of ways to modify cellular pathways to hijack the cell machinery for the synthesis of their nucleic acid and proteins. Similarly, they use intracellular vesicular mechanisms of trafficking for their assembly and eventual release, with a number of viruses acquiring their envelope from internal or plasma cell membranes. There is an increasing number of reports on viral exploitation of cell secretome pathways to avoid recognition and stimulation of the immune response. Extracellular vesicles (EV) containing viral particles have been shown to shield viruses after exiting the host cell, in some cases challenging the boundaries between viral groups traditionally characterised as enveloped and non-enveloped. Apart from viral particles, EV can spread the virus also carrying viral genome and can modify the target cells through their cargo of virus-coded miRNAs and proteins as well as selectively packaged cellular mRNAs, miRNAs, proteins and lipids, differing in composition and quantities from the cell of origin.
GB virus type C (GBV-C) glycoprotein E2 protein disrupts HIV-1 assembly and release by inhibiting Gag plasma membrane targeting, however the mechanism by which the GBV-C E2 inhibits Gag trafficking remains unclear. In the present study, we identified ADP-ribosylation factor 1 (ARF1) contributed to the inhibitory effect of GBV-C E2 on HIV-1 Gag membrane targeting. Expression of GBV-C E2 decreased ARF1 expression in a proteasomal degradation-dependent manner. The restoration of ARF1 expression rescued the HIV-1 Gag processing and membrane targeting defect imposed by GBV-C E2. In addition, GBV-C E2 expression also altered Golgi morphology and suppressed protein traffic through the secretory pathway, which are all consistent with a phenotype of disrupting the function of ARF1 protein. Thus, our results indicate that GBV-C E2 inhibits HIV-1 assembly and release by decreasing ARF1, and may provide insights regarding GBV-C E2’s potential for a new therapeutic approach for treating HIV-1
It is becoming increasingly clear that immunoactivation, which evolved as a system of host defense against pathogens, can become dysregulated and promote the pathogenesis of diverse diseases with both known and unknown etiologies (e.g., AIDS, age-related macular degeneration, cancer) as well as aging. Immunoactivation seems to be a "common denominator" or general mechanism of pathogenesis, and may explain the association and similarities in pathology among otherwise unrelated human diseases. Identification of general mechanisms of immunoactivation may lead to the development of new therapeutic strategies applicable to many diseases even before detailed knowledge of specific etiology and pathogenesis may be available.
Copyright © 2015 Elsevier Inc. All rights reserved.
Extracellular vesicles (EVs) are important in normal physiology and are altered in various pathologies. EVs produced by different cells are antigenically different. Since the majority of EVs are too small for routine flow cytometry, EV composition is studied predominantly in bulk, thus not addressing their antigenic heterogeneity. Here, we describe a nanoparticle-based technique for analyzing antigens on single nano-sized EVs. The technique consists of immuno-capturing of EVs with 15-nm magnetic nanoparticles, staining captured EVs with antibodies against their antigens, and separating them from unbound EVs and free antibodies in a magnetic field, followed by flow analysis. This technique allows us to characterize EVs populations according to their antigenic distribution, including minor EV fractions. We demonstrated that the individual blood EVs carry different sets of antigens, none being ubiquitous, and quantified their distribution. The physiological significance of antigenically different EVs and their correlation with different pathologies can now be directly addressed.
Copyright © 2014. Published by Elsevier Inc.
Hepatitis C virus (HCV) and GB virus type C (GBV-C) are associated with impaired T cell function despite the fact that HCV replicates in hepatocytes and GBV-C in a small proportion of lymphocytes. Recently, we showed that HCV and GBV-C E2-envelope proteins reduce T cell activation via the T cell receptor (TCR) by competing for phosphorylation with a critical kinase in the TCR signaling cascade (Lck). E2 interfered with TCR signaling in E2 expressing cells and in bystander cells. The bystander effect was mediated by virus particles and extracellular microvesicular particles (exosomes). Multiple kinase substrate sites are predicted to reside on viral structural proteins and based on bioinformatic predictions, many RNA virus pathogens may interfere with TCR signaling via a similar mechanism. Identification of T cell inhibitory effects of virus structural proteins may provide novel approaches to enhance the immunogenicity and memory of viral vaccines.
This work reports on the use of an internal electrostatic field to facilitate charge separation at inorganic-organic interfaces, analogous to those in hybrid solar cells. Systematic charge transfer studies show that the donor-acceptor charge transfer rate is highly sensitive to the direction of the internal electric field.
GBV-C infection is associated with prolonged survival and with reduced T cell activation in HIV-infected subjects not receiving combination antiretroviral therapy (cART). The relationship between GBV-C and T cell activation in HIV-infected subjects was examined. HIV-infected subjects on cART with non-detectable HIV viral load (VL) or cART naïve subjects were studied. GBV-C VL and HIV VL were determined. Cell surface markers of activation (CD38(+)/HLA-DR(+)), proliferation (Ki-67+), and HIV entry co-receptor expression (CCR5+ and CXCR4+) on total CD4+ and CD8+ T cells, and on naïve, central memory (CM), effector memory (EM), and effector CD4+ and CD8+ subpopulations were measured by flow cytometry. In subjects with suppressed HIV VL, GBV-C was consistently associated with reduced activation in naïve, CM, EM, and effector CD4+ cells. GBV-C was associated with reduced CD4+ and CD8+ T cell surface expression of activation and proliferation markers, independent of HIV VL classification. GBV-C was also associated with higher proportions of naïve CD4+ and CD8+ T cells, and with lower proportions of EM CD4+ and CD8+ T cells. In conclusion, GBV-C infection was associated with reduced activation of CD4+ and CD8+ T cells in both HIV viremic and HIV RNA suppressed patients. Those with GBV-C infection demonstrated an increased proportion of naive T cells and a reduction in T cell activation and proliferation independent of HIV VL classification, including those with suppressed HIV VL on cART. Since HIV pathogenesis is thought to be accelerated by T cell activation, these results may contribute to prolonged survival among HIV infected individuals co-infected with GBV-C. Furthermore, since cART therapy does not reduce T cell activation to levels seen in HIV-uninfected people, GBV-C infection may be beneficial for HIV-related diseases in those effectively treated with anti-HIV therapy.
Viral nucleic acids often trigger an innate immune response in infected cells. Many viruses, including hepatitis C virus (HCV), have evolved mechanisms to evade intracellular recognition. Nevertheless, HCV-permissive cells can trigger a viral RNA-, TLR7-, and cell-contact-dependent compensatory interferon response in nonpermissive plasmacytoid dendritic cells (pDCs). Here we report that these events are mediated by transfer of HCV-RNA-containing exosomes from infected cells to pDCs. The exosomal viral RNA transfer is dependent on the endosomal sorting complex required for transport (ESCRT) machinery and on Annexin A2, an RNA-binding protein involved in membrane vesicle trafficking, and is suppressed by exosome release inhibitors. Further, purified concentrated HCV-RNA-containing exosomes are sufficient to activate pDCs. Thus, vesicular sequestration and exosomal export of viral RNA may serve both as a viral strategy to evade pathogen sensing within infected cells and as a host strategy to induce an unopposed innate response in replication-nonpermissive bystander cells.
Background:
GB virus C (GBV-C) coinfection is associated with reduced immune activation and a block in CD4(+) T-cell proliferation following interleukin-2 (IL-2) therapy in HIV-infected individuals. We examined peripheral blood mononuclear cells (PBMCs) from HIV-infected subjects with and without GBV-C viraemia to determine if GBV-C correlated with reactivation of latent HIV, T-cell proliferation or T-cell survival following in vitro activation with phytohaemagglutinin A and IL-2 (PHA/IL-2).
Methods:
HIV-infected subjects whose HIV viral load was suppressed on combination antiretroviral therapy (cART) for >6 months were studied. PBMCs were cultured with and without PHA/IL-2 and monitored for HIV reactivation, proliferation and survival. GBV-C viraemia and in vitro replication were detected by real-time RT-PCR. HIV reactivation was determined by measuring HIV p24 antigen in culture supernatants. Proliferation was measured by counting viable cells and survival measured by flow cytometry.
Results:
Of 49 HIV-infected individuals, 26 had GBV-C viraemia. Significantly less HIV reactivation and PBMC proliferation following in vitro activation with PHA/IL-2 was observed in samples from GBV-C viraemic subjects compared with non-viraemic controls. Following 5 weeks in culture, GBV-C replication was associated with preservation of CD4(+) and CD8(+) T-cells compared with non-viraemic controls.
Conclusions:
GBV-C appears to inhibit immune activation and IL-2 signalling pathways, which might contribute to a reduction in reactivation of latent HIV from cellular reservoirs. In addition, GBV-C viraemia was associated with a reduction in activation-induced T-cell death. GBV-C-associated T-cell effects could contribute to the observed protective effect of GBV-C coinfection in HIV-infected individuals.
Viruses must enter host cells to replicate, assemble and propagate. Because of the restricted size of their genomes, viruses have had to evolve efficient ways of exploiting host cell processes to promote their own life cycles and also to escape host immune defence mechanisms. Many viral open reading frames (viORFs) with immune-modulating functions essential for productive viral growth have been identified across a range of viral classes. However, there has been no comprehensive study to identify the host factors with which these viORFs interact for a global perspective of viral perturbation strategies. Here we show that different viral perturbation patterns of the host molecular defence network can be deduced from a mass-spectrometry-based host-factor survey in a defined human cellular system by using 70 innate immune-modulating viORFs from 30 viral species. The 579 host proteins targeted by the viORFs mapped to an unexpectedly large number of signalling pathways and cellular processes, suggesting yet unknown mechanisms of antiviral immunity. We further experimentally verified the targets heterogeneous nuclear ribonucleoprotein U, phosphatidylinositol-3-OH kinase, the WNK (with-no-lysine) kinase family and USP19 (ubiquitin-specific peptidase 19) as vulnerable nodes in the host cellular defence system. Evaluation of the impact of viral immune modulators on the host molecular network revealed perturbation strategies used by individual viruses and by viral classes. Our data are also valuable for the design of broad and specific antiviral therapies.
Angiogenesis plays a crucial role during tumorigenesis and much progress has been recently made in elucidating the role of VEGF and other growth factors in the regulation of angiogenesis. Recently, microRNAs (miRNAs) have been shown to modulate a variety of physiogical and pathological processes. We identified a set of differentially expressed miRNAs in microvascular endothelial cells co-cultured with tumour cells. Unexpectedly, most miRNAs were derived from tumour cells, packaged into microvesicles (MVs), and then directly delivered to endothelial cells. Among these miRNAs, we focused on miR-9 due to the strong morphological changes induced in cultured endothelial cells. We found that exogenous miR-9 effectively reduced SOCS5 levels, leading to activated JAK-STAT pathway. This signalling cascade promoted endothelial cell migration and tumour angiogenesis. Remarkably, administration of anti-miR-9 or JAK inhibitors suppressed MV-induced cell migration in vitro and decreased tumour burden in vivo. Collectively, these observations suggest that tumour-secreted miRNAs participate in intercellular communication and function as a novel pro-angiogenic mechanism.
MicroRNAs (miRNAs) are small noncoding RNAs, 19-24 nucleotides in length, that regulate gene expression and are expressed aberrantly in most types of cancer. MiRNAs also have been detected in the blood of cancer patients and can serve as circulating biomarkers. It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell and can regulate gene expression in the receiving cells by canonical binding to their target messenger RNAs. Here we show that tumor-secreted miR-21 and miR-29a also can function by another mechanism, by binding as ligands to receptors of the Toll-like receptor (TLR) family, murine TLR7 and human TLR8, in immune cells, triggering a TLR-mediated prometastatic inflammatory response that ultimately may lead to tumor growth and metastasis. Thus, by acting as paracrine agonists of TLRs, secreted miRNAs are key regulators of the tumor microenvironment. This mechanism of action of miRNAs is implicated in tumor-immune system communication and is important in tumor growth and spread, thus representing a possible target for cancer treatment.
More than 2 billion people are infected with Mycobacterium. tuberculosis; however, only 5-10% of those infected will develop active disease. Recent data suggest that containment is controlled locally at the level of the granuloma and that granuloma architecture may differ even within a single infected individual. Formation of a granuloma likely requires exposure to mycobacterial components released from infected macrophages, but the mechanism of their release is still unclear. We hypothesize that exosomes, which are small membrane vesicles containing mycobacterial components released from infected macrophages, could promote cellular recruitment during granuloma formation. In support of this hypothesis, we found that C57BL/6 mouse-derived bone marrow macrophages treated with exosomes released from M. tuberculosis-infected RAW264.7 cells secrete significant levels of chemokines and can induce migration of CFSE-labeled macrophages and splenocytes. Exosomes isolated from the serum of M. bovis bacillus Calmette-Guérin-infected mice could also stimulate macrophage production of chemokines and cytokines ex vivo, but the level and type differed during the course of a 60-d infection. Of interest, the exosome concentration in serum correlated strongly with mouse bacterial load, suggesting some role in immune regulation. Finally, hollow fiber-based experiments indicated that macrophages treated with exosomes released from M. tuberculosis-infected cells could promote macrophage recruitment in vivo. Exosomes injected intranasally could also recruit CD11b(+) cells into the lung. Overall, our study suggests that exosomes may play an important role in recruiting and regulating host cells during an M. tuberculosis infection.
GB virus C (GBV-C) is a lymphotropic human virus discovered in 1995 that is related to hepatitis C virus (HCV). GBV-C infection has not been convincingly associated with any disease; however, several studies found an association between persistent GBV-C infection and improved survival in HIV-positive individuals. GBV-C infection modestly alters T cell homeostasis in vivo through various mechanisms, including modulation of chemokine and cytokine release and receptor expression, and by diminution of T cell activation, proliferation and apoptosis, all of which may contribute to improved HIV clinical outcomes. In vitro studies confirm these clinical observations and demonstrate an anti-HIV replication effect of GBV-C. This review summarizes existing data on potential mechanisms by which GBV-C interferes with HIV, and the research needed to capitalize on this epidemiological observation.
Cells secrete various membrane-enclosed microvesicles from their cell surface (shedding microvesicles) and from internal, endosome-derived membranes (exosomes). Intriguingly, these vesicles have many characteristics in common with enveloped viruses, including biophysical properties, biogenesis, and uptake by cells. Recent discoveries describing the microvesicle-mediated intercellular transfer of functional cellular proteins, RNAs, and mRNAs have revealed additional similarities between viruses and cellular microvesicles. Apparent differences include the complexity of viral entry, temporally regulated viral expression, and self-replication proceeding to infection of new cells. Interestingly, many virally infected cells secrete microvesicles that differ in content from their virion counterparts but may contain various viral proteins and RNAs. For the most part, these particles have not been analyzed for their content or functions during viral infection. However, early studies of microvesicles (L-particles) secreted from herpes simplex virus-infected cells provided the first evidence of microvesicle-mediated intercellular communication. In the case of Epstein-Barr virus, recent evidence suggests that this tumorigenic herpesvirus also utilizes exosomes as a mechanism of cell-to-cell communication through the transfer of signaling competent proteins and functional microRNAs to uninfected cells. This review focuses on aspects of the biology of microvesicles with an emphasis on their potential contributions to viral infection and pathogenesis.
Hepatitis G virus (HGV or GB-C virus) is a newly described virus that is closely related to hepatitis C virus (HCV). Based on sequence analysis and by evaluation of translational initiation codon preferences utilized during in vitro translation, HGV appears to have a truncated or absent core protein at the amino terminus of the HGV polyprotein. Consequently, the biophysical properties of HGV may be very different from those of HCV. To characterize HGV particle types, we evaluated plasma from chronically infected individuals with and without concomitant HCV infection by using sucrose gradient centrifugation, isopycnic banding in cesium chloride, and saline density flotation centrifugation. Similar to HCV, HGV particles included an extremely-low-density virion particle (1.07 to 1.09 g/ml) and a nucleocapsid of approximately 1.18 g/ml. One major difference between the particle types was that HGV was consistently more stable in cesium chloride than HCV. Plasma samples from chronically HGV-infected individuals and controls were assessed by a synthetic peptide-based immunoassay to determine if they contained HGV antibody specific for a conserved region in the coding region upstream of the E1 protein. Chronically HGV-infected individuals contained antibody to the HGV core protein peptide, whereas no binding to a hepatitis A virus peptide control was observed. Competitive inhibition of binding to the HGV peptide confirmed the specificity of the assay. These data indicate that HGV has a nucleocapsid and that at least part of the putative core region of HGV is expressed in vivo.
Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1-infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1-enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti-GBV-C E2 Abs neutralized HIV-1-pseudotyped retrovirus particles but not HIV-1-pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1-neutralizing Abs.
Coinfection with the flavivirus GB virus C (GBV-C) is frequent in patients suffering from HIV type-1 (HIV-1) infection because of shared routes of transmission. GBV-C coinfection has been proposed to exert a beneficial influence on HIV-1 infection. In vitro studies demonstrated down-regulation of C-C chemokine receptor type 5 (CCR5) as a potential mechanism by which GBV-C modulates HIV-1 disease progression. We therefore studied surface expression of the two major HIV-1 coreceptors, CCR5 and CXC chemokine receptor type 4 (CXCR4), on CD4(+) and CD8(+) T-cells in 128 HIV-1-positive patients stratified with respect to their GBV-C status, immune function and highly active antiretroviral therapy (HAART) status in vivo.
GBV-C infection was studied in 128 HIV-1-infected patients by nested reverse transcriptase PCR. Fluorescence-activated cell sorting analysis was used to measure CCR5 and CXCR4 surface expression on CD4(+) and CD8(+) T-cells.
GBV-C RNA replication was detected in 30% (38/128) of patients. In HIV-1-positive patients with advanced immunodeficiency, we found up-regulation of CCR5 surface expression on CD4(+) T-cells; however, in patients with GBV-C coinfection, no up-regulation of CCR5 CD4(+) T-cells was detected. Furthermore, CXCR4 surface expression was reduced in GBV-C-coinfected patients. These findings were independent of HAART status and HIV-1 viral load. HIV-1 coreceptor expression on CD8(+) T-cells was not altered in patients with GBV-C coinfection.
GBV-C coinfection in HIV-1 disease leads to reduced expression of the two major HIV-1 coreceptors, CCR5 and CXCR4, on CD4(+) T-cells in patients at an advanced stage of immunodeficiency, providing a possible molecular explanation for the clinical benefit of GBV-C coinfection in late-stage HIV-1 disease.
Although humans and chimpanzees share >99% identity in alignable protein sequences, they differ surprisingly in the incidence and severity of some common diseases. In general, humans infected with various viruses, such as HIV and hepatitis C virus, appear to develop stronger reactions and long-term complications. Humans also appear to suffer more from other diseases associated with over-reactivity of the adaptive immune system, such as asthma, psoriasis, and rheumatoid arthritis. In this study, we show that human T cells are more reactive than chimpanzee T cells to a wide variety of stimuli, including anti-TCR Abs of multiple isotypes, l-phytohemagglutin, Staphylococcus aureus superantigen, a superagonist anti-CD28 Ab, and in MLRs. We also extend this observation to B cells, again showing a human propensity to react more strongly to stimuli. Finally, we show a relative increase in activation markers and cytokine production in human lymphocytes in response to uridine-rich (viral-like) ssRNA. Thus, humans manifest a generalized lymphocyte over-reactivity relative to chimpanzees, a finding that is correlated with decreased levels of inhibitory sialic acid-recognizing Ig-superfamily lectins (Siglecs; particularly Siglec-5) on human T and B cells. Furthermore, Siglec-5 levels are upregulated by activation in chimpanzee but not human lymphocytes, and human T cell reactivity can be downmodulated by forced expression of Siglec-5. Thus, a key difference in the immune reactivity of chimp and human lymphocytes appears to be related to the differential expression of Siglec-5. Taken together, these data may help explain human propensities for diseases associated with excessive activation of the adaptive immune system.
Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% beta-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change.
Human immunodeficiency virus (HIV) and simian immunodeficiency virus Nef proteins contain a conserved motif with the minimal consensus (PxxP) site for Src homology region 3 (SH3)-mediated protein-protein interactions. Nef PxxP motifs show specific binding to biotinylated SH3 domains of Hck and Lyn, but not to those of other tested Src family kinases or less related proteins. A unique cooperative role of a distant proline is also observed. Endogenous Hck of monocytic U937 cells can be specifically precipitated by matrix-bound HIV-1 Nef, but not by mutant protein lacking PxxP. Intact Nef PxxP motifs are dispensable for Nef-induced CD4 down-regulation, but are required for the higher in vitro replicative potential of Nef+ viruses. Thus, CD4 down-regulation and promotion of viral growth are two distinct functions of Nef, and the latter is mediated via SH3 binding.
The flavivirus GB virus C (GBV-C, also designated hepatitis G virus) was identified in a search for hepatitis viruses, but no disease is currently known to be associated with it. We investigated the relation between coinfection with GBV-C and the long-term outcome in patients infected with the human immunodeficiency virus (HIV).
A total of 197 HIV-positive patients were followed prospectively beginning in 1993 or 1994. Of these patients, 33 (16.8 percent) tested positive for GBV-C RNA, 112 (56.9 percent) had detectable antibodies against the GBV-C envelope protein E2, and 52 (26.4 percent) had no marker of GBV-C infection and were considered unexposed. We assessed the relation between GBV-C infection and the progression of HIV disease. We also tested 169 GBV-C-positive plasma samples with a quantitative branched-chain DNA (bDNA) assay in order to investigate possible correlations between GBV-C viral load and both the CD4+ cell count and the HIV load.
Among the patients who tested positive for GBV-C RNA, survival was significantly longer, and there was a slower progression to the acquired immunodeficiency syndrome (AIDS) (P<0.001 for both comparisons). Survival after the development of AIDS was also better among the GBV-C-positive patients. The association of GBV-C viremia with reduced mortality remained significant in analyses stratified according to age and CD4+ cell count. In an analysis restricted to the years after highly active antiretroviral therapy became available, the presence of GBV-C RNA remained predictive of longer survival (P=0.02). The HIV load was lower in the GBV-C-positive patients than in the GBV-C-negative patients. The GBV-C load correlated inversely with the HIV load (r=-0.33, P<0.001) but did not correlate with the CD4+ cell count.
Coinfection with GBV-C is associated with a reduced mortality rate in HIV-infected patients. GBV-C is not known to cause any disease, but it is possible that its presence leads to an inhibition of HIV replication. However, GBV-C infection could also be a marker for the presence of other factors that lead to a favorable HIV response.
Previous studies have suggested that people with human immunodeficiency virus (HIV) infection who are coinfected with GB virus C (GBV-C, or hepatitis G virus) have delayed progression of HIV disease. GBV-C is related to hepatitis C virus but does not appear to cause liver disease.
We examined the effect of coinfection with GBV-C on the survival of patients with HIV infection. We also evaluated cultures of peripheral-blood mononuclear cells infected with both viruses to determine whether GBV-C infection alters replication in vitro.
Of 362 HIV-infected patients, 144 (39.8 percent) had GBV-C viremia in two tests. Forty-one of the patients with GBV-C viremia (28.5 percent) died during the follow-up period, as compared with 123 of the 218 patients who tested negative for GBV-C RNA (56.4 percent; P<0.001). The mean duration of follow-up for the entire cohort was 4.1 years. In a Cox regression analysis adjusted for HIV treatment, baseline CD4+ T-cell count, age, sex, race, and mode of transmission of HIV, the mortality rate among the 218 HIV-infected patients without GBV-C coinfection was significantly higher than that among the 144 patients with GBV-C coinfection (relative risk, 3.7; 95 percent confidence interval, 2.5 to 5.4). HIV replication, as measured by the detection of p24 antigen in culture supernatants, was reproducibly inhibited in cultures of peripheral-blood mononuclear cells by GBV-C coinfection. Coinfection did not alter the surface expression of HIV cellular receptors on peripheral-blood mononuclear cells, as determined by flow cytometry.
GBV-C infection is common in people with HIV infection and is associated with significantly improved survival.
Scansite identifies short protein sequence motifs that are recognized by modular signaling domains, phosphorylated by protein Ser/Thr- or Tyr-kinases or mediate specific interactions with protein or phospholipid ligands. Each sequence motif is represented as a position-specific scoring matrix (PSSM) based on results from oriented peptide library and phage display experiments. Predicted domain-motif interactions from Scansite can be sequentially combined, allowing segments of biological pathways to be constructed in silico. The current release of Scansite, version 2.0, includes 62 motifs characterizing the binding and/or substrate specificities of many families of Ser/Thr- or Tyr-kinases, SH2, SH3, PDZ, 14-3-3 and PTB domains, together with signature motifs for PtdIns(3,4,5)P(3)-specific PH domains. Scansite 2.0 contains significant improvements to its original interface, including a number of new generalized user features and significantly enhanced performance. Searches of all SWISS-PROT, TrEMBL, Genpept and Ensembl protein database entries are now possible with run times reduced by approximately 60% when compared with Scansite version 1.0. Scansite 2.0 allows restricted searching of species-specific proteins, as well as isoelectric point and molecular weight sorting to facilitate comparison of predictions with results from two-dimensional gel electrophoresis experiments. Support for user-defined motifs has been increased, allowing easier input of user-defined matrices and permitting user-defined motifs to be combined with pre-compiled Scansite motifs for dual motif searching. In addition, a new series of Sequence Match programs for non-quantitative user-defined motifs has been implemented. Scansite is available via the World Wide Web at http://scansite.mit.edu.
GB virus C (GBV-C) infection is common in humans and may persist for decades, although most infected persons clear the virus
and subsequently develop antibodies to the envelope glycoprotein. GBV-C replicates in peripheral blood mononuclear cells (PBMCs)
and CD4+ T lymphocytes in vitro, and depletion of CD4+ T lymphocytes has been proposed as the reason for clearance of GBV-C among persons positive for human immunodeficiency virus.
We identified GBV-C RNA in purified CD4+ and CD8+ T lymphocytes and CD19+ B lymphocytes removed ex vivo from infected donors and found that GBV-C replicated in vitro in these PBMC subsets, suggesting
that GBV-C is a panlymphotropic virus
GB virus C (GBV-C), which is not known to be pathogenic in humans, replicates in lymphocytes, inhibits the replication of human immunodeficiency virus (HIV) in vitro, and has been associated with a decreased risk of death among HIV-positive persons in some, but not all, studies. Previous studies did not control for differences in the duration of HIV or GBV-C infection.
We evaluated 271 men who were participants in the Multicenter Acquired Immunodeficiency Syndrome Cohort Study for GBV-C viremia (by means of a reverse-transcriptase-polymerase-chain-reaction assay) or E2 antibody (by means of an enzyme-linked immunosorbent assay) 12 to 18 months after seroconversion to positivity for HIV (the early visit); a subgroup of 138 patients was also evaluated 5 to 6 years after HIV seroconversion (the late visit).
GBV-C infection was detected in 85 percent of men with HIV seroconversion on the basis of the presence of E2 antibody (46 percent) or GBV-C RNA (39 percent). Only one man acquired GBV-C viremia between the early and the late visit, but 9 percent of men had clearance of GBV-C RNA between these visits. GBV-C status 12 to 18 months after HIV seroconversion was not significantly associated with survival; however, men without GBV-C RNA 5 to 6 years after HIV seroconversion were 2.78 times as likely to die as men with persistent GBV-C viremia (95 percent confidence interval, 1.34 to 5.76; P=0.006). The poorest prognosis was associated with the loss of GBV-C RNA (relative hazard for death as compared with men with persistent GBV-C RNA, 5.87; P=0.003).
GBV-C viremia was significantly associated with prolonged survival among HIV-positive men 5 to 6 years after HIV seroconversion, but not at 12 to 18 months, and the loss of GBV-C RNA by 5 to 6 years after HIV seroconversion was associated with the poorest prognosis. Understanding the mechanisms of interaction between GBV-C and HIV may provide insight into the progression of HIV disease.
Upon transmission to a new host, HIV targets CCR5+ CD4+ effector memory T cells, resulting in acute, massive depletion of these cells from mucosal effector sites. This depletion does not initially compromise the regenerative capacity of the immune system because naive and most central memory T cells are spared. Here, we discuss evidence suggesting that frequent activation of these spared cells during the chronic phase of HIV infection supplies mucosal tissues with short-lived CCR5+ CD4+ effector cells that prevent life-threatening infections. This immune activation also facilitates continued viral replication, but infection and killing of target T cells by HIV are selective and the impact on effector-cell lifespan is limited. We propose, however, that persistent activation progressively disrupts the functional organization of the immune system, reducing its regenerative capacity and facilitating viral evolution that leads to loss of the exquisite target cell-sparing selectivity of viral replication, ultimately resulting in AIDS.
Although untreated human immunodeficiency virus (HIV)-infected patients maintaining undetectable plasma HIV RNA levels (elite controllers) have high HIV-specific immune responses, it is unclear whether they experience abnormal levels of T cell activation, potentially contributing to immunodeficiency.
We compared percentages of activated (CD38(+)HLA-DR(+)) T cells between 30 elite controllers, 47 HIV-uninfected individuals, 187 HIV-infected individuals with undetectable viremia receiving antiretroviral therapy (antiretroviral therapy suppressed), and 66 untreated HIV-infected individuals with detectable viremia. Because mucosal translocation of bacterial products may contribute to T cell activation in HIV infection, we also measured plasma lipopolysaccharide (LPS) levels.
Although the median CD4(+) cell count in controllers was 727 cells/mm(3), 3 (10%) had CD4(+) cell counts <350 cells/mm(3) and 2 (7%) had acquired immunodeficiency syndrome. Controllers had higher CD4(+) and CD8(+) cell activation levels (P < .001 for both) than HIV-negative subjects and higher CD8(+) cell activation levels than the antiretroviral therapy suppressed (P = .048). In controllers, higher CD4(+) and CD8(+) T cell activation was associated with lower CD4(+) cell counts (P = .009 and P = .047). Controllers had higher LPS levels than HIV-negative subjects (P < .001), and in controllers higher LPS level was associated with higher CD8(+) T cell activation (P = .039).
HIV controllers have abnormally high T cell activation levels, which may contribute to progressive CD4(+) T cell loss even without measurable viremia.
Identification of protein phosphorylation sites with their cognate protein kinases (PKs) is a key step to delineate molecular dynamics and plasticity underlying a variety of cellular processes. Although nearly 10 kinase-specific prediction programs have been developed, numerous PKs have been casually classified into subgroups without a standard rule. For large scale predictions, the false positive rate has also never been addressed. In this work, we adopted a well established rule to classify PKs into a hierarchical structure with four levels, including group, family, subfamily, and single PK. In addition, we developed a simple approach to estimate the theoretically maximal false positive rates. The on-line service and local packages of the GPS (Group-based Prediction System) 2.0 were implemented in Java with the modified version of the Group-based Phosphorylation Scoring algorithm. As the first stand alone software for predicting phosphorylation, GPS 2.0 can predict kinase-specific phosphorylation sites for 408 human PKs in hierarchy. A large scale prediction of more than 13,000 mammalian phosphorylation sites by GPS 2.0 was exhibited with great performance and remarkable accuracy. Using Aurora-B as an example, we also conducted a proteome-wide search and provided systematic prediction of Aurora-B-specific substrates including protein-protein interaction information. Thus, the GPS 2.0 is a useful tool for predicting protein phosphorylation sites and their cognate kinases and is freely available on line.
Background: Progression to AIDS is slower in persons infected with both HIV-1 and GB virus C (GBV-C), also known as hepatitis G virus. Objective: To compare clinical, virologic, and immunologic variables in HIV-1-seropositive patients with and without GBV-C co-infection. Design: Subanalysis of a prospective cohort study. Setting: Institute of Infectious Diseases, University of Catania, Catania, Italy. Patients: 80 asymptomatic HIV-1-seropositive patients. Measurements: GBV-C RNA level; plasma HIV-1 viral load; CD4 + cell counts; and serum levels of interleukin (IL)-2, IL-4, IL-10, and IL-12. Results: At the start of the study, plasma GBV-C RNA was detected in 17 patients (21%). During follow-up, IL-2 and IL-12 levels decreased significantly (P= 0.005 and P=0.01, respectively) and IL-4 and IL-10 levels increased significantly (P = 0.01 and P = 0.004, respectively) in the GBV-C-negative group but did not change substantially in the GBV-C-positive group. Each measured variable differed significantly between GBV-C-positive and GBV-C-negative groups during follow-up (P < 0.001 for IL-12, IL-4, and IL-10; P = 0.002 for IL-2). Conclusion: GB virus C may immunologically interfere with progression of HIV-1 infection to AIDS by maintaining an intact T-helper 1 cytokine profile.
Initiation of ART during acute HIV-1 infection may prevent persistent immune activation. We analyzed longitudinal CD38+HLA-DR+ CD8+ T cell percentages in 31 acutely infected individuals who started early (median 43 days since infection) and successful ART, and maintained viral suppression through 96 weeks. Pre-therapy a median of 72.6% CD8+ T cells were CD38+HLA-DR+, and while this decreased to 15.6% by 96 weeks, it remained substantially higher than seronegative controls (median 8.9%, p=0.008). Shorter time to suppression predicted lower activation at 96 weeks. These results support the hypothesis that very early events in HIV-1 pathogenesis may result in prolonged immune dysfunction.
Double-negative T cells (DNTCs; ie, CD3+CD4–CD8– T cells) play a role in limiting chronic immune activation. GB virus C (GBV-C) infection is associated with reduced T-cell
activation in human immunodeficiency virus (HIV)–infected individuals. T-cell activation and DNTCs were measured in HIV-infected
subjects with a nondetectable HIV load. GBV-C–viremic subjects had significantly reduced CD4+ and CD8+ T-cell activation (P = .003 and .034, respectively) and significantly increased DNTCs (P = .038), compared with nonviremic subjects. GBV-C load correlated with DNTC percentage (P = .004). Thus, GBV-C infection is associated with an increase in DNTCs, which may contribute to reduced immune activation
during HIV infection.
GB virus type C (GBV-C) viremia is associated with reduced CD4+ T cell expansion following IL-2 therapy and with a reduction in T cell activation in HIV-infected individuals. The mechanism(s) by which GBV-C might alter T cell activation or IL-2 signaling have not been studied. In this study, we assess IL-2 release, IL-2R expression, IL-2 signaling, and cell proliferation in tet-off Jurkat cells expressing the GBV-C envelope glycoprotein (E2) following activation through the TCR. TCR activation was induced by incubation in anti-CD3/CD28 Abs. IL-2 release was measured by ELISA, STAT5 phosphorylation was assessed by immunoblot, and IL-2Rα (CD25) expression and cell proliferation were determined by flow cytometry. IL-2 and IL-2Rα steady-state mRNA levels were measured by real-time PCR. GBV-C E2 expression significantly inhibited IL-2 release, CD25 expression, STAT5 phosphorylation, and cellular proliferation in Jurkat cells following activation through the TCR compared with control cell lines. Reducing E2 expression by doxycycline reversed the inhibitory effects observed in the E2-expressing cells. The N-terminal 219 aa of E2 was sufficient to inhibit IL-2 signaling. Addition of purified recombinant GBV-C E2 protein to primary human CD4+ and CD8+ T cells inhibited TCR activation-induced IL-2 release and upregulation of IL-2Rα expression. These data provide evidence that the GBV-C E2 protein may contribute to the block in CD4+ T cell expansion following IL-2 therapy in HIV-infected individuals. Furthermore, the effects of GBV-C on IL-2 and IL-2-signaling pathways may contribute to the reduction in chronic immune activation observed in GBV-C/HIV-coinfected individuals.
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MicroRNAs are fine tuners of diverse biological responses and are expressed in various cell types of the liver. Here we hypothesized that circulating microRNAs (miRNAs) may serve as biomarkers of liver damage and inflammation. We studied miRNA-122, which is abundant in hepatocytes, and miR-155, -146a, and -125b, which regulate inflammation in immune cells in mouse models of alcoholic liver disease (ALD), drug (acetaminophen, APAP)-induced liver injury (DILI), and Toll-like receptor (TLR) 9+4 ligand-induced inflammatory cell-mediated liver damage. We found that serum/plasma miR-122 correlated with alanine aminotransferase (ALT) increases in the liver damage caused by alcohol, APAP, and TLR9 (CpG)+4 (LPS) ligands. MiR-155, a regulator of inflammation, was increased in serum/plasma in alcoholic and inflammatory liver injury. Alcohol failed to increase serum miR-122 in TLR4-deficient and p47phox-deficient mice that were protected from ALD. We found the most robust increase in plasma miR-122 in DILI and it correlated with the highest ALT levels. Consistent with the massive inflammatory cell infiltration in the liver, plasma miR-155 and miR-146a were significantly elevated after CpG+LPS administration. We show for the first time that, depending on the type of liver injury, circulating miRNAs are associated either with the exosome-rich or protein-rich compartments. In ALD and in inflammatory liver injury, serum/plasma miR-122 and miR-155 were predominantly associated with the exosome-rich fraction, whereas in DILI/APAP injury these miRNAs were present in the protein-rich fraction.
Conclusion:
Our results suggest that circulating miRNAs may serve as biomarkers to differentiate between hepatocyte injury and inflammation and the exosome versus protein association of miRNAs may provide further specificity to mechanisms of liver pathology.
GB virus C (GBV-C) infection is associated with prolonged survival in HIV-infected cohorts, and GBV-C E2 protein inhibits HIV entry when added to CD4+ T cells. To further characterize E2 effects on HIV replication, stably transfected Jurkat cell lines expressing GBV-C E2 or control sequences were infected with HIV and replication was measured. HIV replication (all 6 isolates studied) was inhibited in all cell lines expressing a region of 17 amino acids of GBV-C E2, but not in cell lines expressing E2 without this region. In contrast, mumps and yellow fever virus replication was not inhibited by E2 protein expression. Synthetic GBV-C E2 17mer peptides did not inhibit HIV replication unless they were fused to a tat-protein-transduction-domain (TAT) for cellular uptake. These data identify the region of GBV-C E2 protein involved in HIV inhibition, and suggest that this GBV-C E2 peptide must gain entry into the cell to inhibit HIV.
Exactly how ligand binding 'triggers' T cell receptor (TCR) phosphorylation is unclear. It has been proposed that ligand engagement by the TCR somehow activates the Src kinase Lck, which in turn phosphorylates the receptor. Recent data, however, suggest instead that a significant fraction of the Lck in resting T cells is already activated and that the proportion of active Lck does not change during the early stages of T cell activation. We argue that, caveats notwithstanding, these new observations offer support for the 'kinetic-segregation' model of TCR triggering, which involves spatial reorganization of signalling proteins upon ligand binding and requires a fraction of Lck to be active in resting T cells.
In 1967, it was reported that experimental inoculation of serum from a surgeon (G.B.) with acute hepatitis into tamarins resulted in hepatitis. In 1995, two new members of the family Flaviviridae, named GBV-A and GBV-B, were identified in tamarins that developed hepatitis following inoculation with the 11th GB passage. Neither virus infects humans, and a number of GBV-A variants were identified in wild New World monkeys that were captured. Subsequently, a related human virus was identified [named GBV-C or hepatitis G virus (HGV)], and recently a more distantly related virus (named GBV-D) was discovered in bats. Only GBV-B, a second species within the genus Hepacivirus (type species hepatitis C virus), has been shown to cause hepatitis; it causes acute hepatitis in experimentally infected tamarins. The other GB viruses have however not been assigned to a genus within the family Flaviviridae. Based on phylogenetic relationships, genome organization and pathogenic features of the GB viruses, we propose to classify GBV-A-like viruses, GBV-C and GBV-D as members of a fourth genus in the family Flaviviridae, named Pegivirus (pe, persistent; g, GB or G). We also propose renaming 'GB' viruses within the tentative genus Pegivirus to reflect their host origin.
Many clinical studies have suggested a beneficial effect of GB virus type C (GBV-C) on the course of HIV-1 infection, but the mechanisms involved in such amelioration are not clear. As recent evidence has implicated cellular activation in HIV-1 pathogenesis, we investigated the effect of GBV-C viremia on T-cell activation in early HIV-1 infection.
Forty-eight recently infected HIV-1 patients (23 GBV-C viremic) were evaluated for T-cell counts, expanded immunophenotyping GBV-C RNA detection, and HIV-1 viral load. Nonparametric univariate and multivariate analyses were carried out to identify variables associated with cellular activation, including GBV-C status, HIV-1 viral load, T lymphocyte counts, and CD38 and chemokine (C-C motif) receptor 5 (CCR5) surface expression.
We not only confirmed the positive correlation between HIV-1 viral load and the percentage of T cells positive for CD38(+)CD8(+) but also observed that GBV-C viremic patients had a lower percentage of T cells positive for CD38(+)CD4(+), CD38(+)CD8(+), CCR5(+)CD4(+), and CCR5(+)CD8(+) compared with HIV-1-infected patients who were not GBV-C viremic. In regression models, GBV-C RNA(+) status was associated with a reduction in the CD38 on CD4(+) or CD8(+) T cells and CCR5(+) on CD8(+) T cells, independent of the HIV-1 viral load or CD4(+) and CD8(+) T-cell counts. These results were also supported by the lower expression of CD69 and CD25 in GBV-C viremic patients.
The association between GBV-C replication and lower T-cell activation may be a key mechanism involved in the protection conferred by this virus against HIV-1 disease progression to immunodeficiency in HIV-1-infected patients.
Interleukin-2 (IL-2) is a cytokine with multiple effects on lymphocytes including induction of CD4 T-cell proliferation. IL-2 administration has been shown to increase CD4 cell counts in HIV-infected people receiving antiretroviral therapy. GB virus C (GBV-C) is an apparently nonpathogenic flavivirus that replicates in CD4 T cells and inhibits HIV replication in vitro by mechanisms including downregulation of HIV entry coreceptors (CCR5 and CXCR4) and induction of chemokines (RANTES, MIP-1alpha, MIP-1 beta, and SDF-1). GBV-C replication is significantly inhibited in vitro by activation of primary CD4 cell cultures with IL-2 and phytohemagglutinin. We sought to determine if there is an interaction between GBV-C and IL-2 in vivo.
GBV-C viremia status was characterized in 92 HIV-infected individuals participating in a randomized trial of IL-2 and antiretroviral therapy [AIDS Clinical Trials Group Study (ACTG) 328]. Changes in CD4 cell counts and HIV RNA levels in individuals assigned IL-2 were compared with those in individuals assigned antiretroviral therapy alone.
Individuals lacking GBV-C viremia had a significantly greater rise in CD4 cell count with IL-2, compared with GBV-C viremic individuals (by 511 cells/microl at week 84; interaction P = 0.02): GBV-C viremic individuals assigned IL-2 did not demonstrate a significant increase in CD4 cell count compared with individuals not assigned to receive IL-2 (95% CI for difference -255 to 397 cells/microl).
GBV-C viremia was associated with a block in CD4 cell expansion following IL-2 therapy in the ACTG 328 study, and GBV-C status may be an important factor in IL-2 treatment response.
To study the binding specificity of Src homology 3 (SH3) domains, we have screened a mouse embryonic expression library for peptide fragments that interact with them. Several clones were identified that express fragments of proteins which, through proline-rich binding sites, exhibit differential binding specificity to various SH3 domains. Src-SH3-specific binding uses a sequence of 7 aa of the consensus RPLPXXP, in which the N-terminal arginine is very important. The SH3 domains of the Src-related kinases Fyn, Lyn, and Hck bind to this sequence with the same affinity as that of the Src SH3. In contrast, a quite different proline-rich sequence from the Btk protein kinase binds to the Fyn, Lyn, and Hck SH3 domains, but not to the Src SH3. Specific binding of the Abl SH3 requires a longer, more proline-rich sequence but no arginine. One clone that binds to both Src and Abl SH3 domains through a common site exhibits reversed binding orientation, in that an arginine indispensable for binding to all tested SH3 domains occurs at the C terminus. Another clone contains overlapping yet distinct Src and Abl SH3 binding sites. Binding to the SH3 domains is mediated by a common PXXP amino acid sequence motif present on all ligands, and specificity comes about from other interactions, often ones involving arginine. The rules governing in vivo usage of particular sites by particular SH3 domains are not clear, but one binding orientation may be more specific than another.
The prevalence of GB virus C (GBV-C)/ hepatitis G virus (HGV) RNA and antibodies to the structural E2 protein was investigated in a cohort of HIV-1 infected patients. Of 346 individuals, RNA was detected in 143 and E2 antibodies were detected in 73, for an overall prevalence of 62.4%. Intravenous drug use and homosexuality were identified as major transmission risk factors. GBV-C/HGV RNA prevalence was associated with hepatitis B coinfection, whereas antibodies to E2 were associated with older age and lower CD4+ cell counts. GBV-C/HGV infection was frequent in this group of HIV-infected patients and was associated with older age, lower CD4 + cell counts, and the presence of hepatitis B surface antigen.
Although T cell activation is associated with disease progression in untreated human immunodeficiency virus type 1 (HIV-1)
infection, its significance in antiretroviral-treated patients is unknown. Activated (CD38+HLA-DR+) T cell counts were measured in 99 HIV-infected adults who had maintained a plasma HIV RNA level ⩽1000 copies/mL for a median
of 21 months while receiving antiretroviral therapy. Patients with sustained viral suppression had lower levels of T cell
activation than untreated patients but higher levels than HIV-uninfected control subjects. Persistent T cell activation was
associated with decreased CD4+ T cell gains during therapy. For every 5% increase in the proportion of activated CD8+ T cells, 35 fewer CD4+ T cells/mm3 were gained. Increased T cell activation was associated with shorter duration of viral suppression, hepatitis C virus coinfection,
frequent low-level viremia, and lower nadir CD4+ T cell counts. Interventions that directly target T cell activation or the determinants of activation may prove to be useful
adjuvants to antiretroviral therapy
Progression to AIDS is slower in persons infected with both HIV-1 and GB virus C (GBV-C), also known as hepatitis G virus.
To compare clinical, virologic, and immunologic variables in HIV-1-seropositive patients with and without GBV-C co-infection.
Subanalysis of a prospective cohort study.
Institute of Infectious Diseases, University of Catania, Catania, Italy.
80 asymptomatic HIV-1-seropositive patients.
GBV-C RNA level; plasma HIV-1 viral load; CD4(+) cell counts; and serum levels of interleukin (IL)-2, IL-4, IL-10, and IL-12.
At the start of the study, plasma GBV-C RNA was detected in 17 patients (21%). During follow-up, IL-2 and IL-12 levels decreased significantly (P = 0.005 and P = 0.01, respectively) and IL-4 and IL-10 levels increased significantly (P = 0.01 and P = 0.004, respectively) in the GBV-C-negative group but did not change substantially in the GBV-C-positive group. Each measured variable differed significantly between GBV-C-positive and GBV-C-negative groups during follow-up (P < 0.001 for IL-12, IL-4, and IL-10; P = 0.002 for IL-2).
GB virus C may immunologically interfere with progression of HIV-1 infection to AIDS by maintaining an intact T-helper 1 cytokine profile.
HIV-1 infection is characterized by chronic generalized CD8 and CD4 T cell hyperactivation, the biological effect of which is not understood.
To study the relation between chronic immune activation and CD4 T cell depletion in HIV-1 infection.
Prospective cohort study among participants of the Amsterdam Cohort Studies on HIV-1 infection and AIDS who have a known seroconversion date (n = 102).
CD4 and CD8 T cell activation marker expression was analysed by FACScan before and after seroconversion (1 and 5 years after seroconversion); T cell proliferation and T cell numbers were also measured. Cox proportional hazard analyses were used to study the predictive value of these parameters for progression to AIDS.
Preseroconversion low CD4 T cell numbers or elevated levels of CD4 T cell activation were associated with increased risk for development of AIDS after HIV-1 seroconversion. Progression to AIDS was associated with loss of both CD4 and CD8 naive T cells. The predictive value of CD8 T cell activation was confirmed and, in addition, in the course of infection low CD4 T cell counts and increasing proportions of dividing CD4 T cells, dividing CD8 T cells or elevated CD4 T cell activation marker expression became independent predictors of progression to AIDS.
Increased T cell activation has predictive value for HIV-1 disease progression even before seroconversion. These data support the hypothesis that persistent hyperactivation of the immune system may lead to erosion of the naive T cell pool and CD4 T cell depletion.
Although generalized T-cell activation is an important factor in chronic HIV disease pathogenesis, its role in primary infection remains poorly defined. To investigate the effect of immune activation on T-cell changes in subjects with early HIV infection, and to test the hypothesis that an immunologic activation "set point" is established early in the natural history of HIV disease, a prospective cohort of acutely infected adults was performed. The median density of CD38 molecules on CD4+ and CD8+ T cells was measured longitudinally in 68 antiretroviral-untreated individuals and 83 antiretroviral-treated individuals. At study entry, T-cell activation was positively associated with viremia, with CD8+ T-cell activation levels increasing exponentially at plasma HIV RNA levels more than 10,000 copies/mL. Among untreated patients, the level of CD8+ T-cell activation varied widely among individuals but often remained stable within a given individual. CD8+ T-cell activation and plasma HIV RNA levels over time were independently associated with the rate of CD4+ T-cell loss in untreated individuals. These data indicate that immunologic activation set point is established early in HIV infection, and that this set point determines the rate at which CD4+ T cells are lost over time.
As the human tetraspanin CD81 binds hepatitis C virus (HCV) envelope glycoprotein E2, we addressed the role CD81 may play in cellular trafficking of HCV envelope proteins. Studies on HCV life cycle are complicated by the lack of a robust cell culture system; we therefore transfected mammalian cells with HCV E1-E2 cDNA, with or without human CD81 (huCD81) cDNA. In the absence of huCD81, HCV envelope proteins are almost completely retained in the endoplasmic reticulum. Instead, when huCD81 is present, a fraction of HCV envelope proteins passes through the Golgi apparatus, matures acquiring complex sugars and is found extracellularly associated with exosomes. These are 60-100-nm membrane vesicles enriched in tetraspanins, released into the extracellular milieu by many cell types and having fusogenic activity. We also report that human plasma contains exosomes and that in HCV patients, viral RNA is associated with these circulating vesicles. We propose that the HCV-CD81 complex leaves cells in the form of exosomes, circulates in this form and exploits the fusogenic capabilities of these vesicles to infect cells even in the presence of neutralizing antibodies.
To conduct a meta-analysis to synthesize the evidence regarding the effect of co-infection with GB virus C (GBV-C) on survival of HIV-infected individuals, and to estimate the effect.
A Bayesian meta-analysis was conducted to synthesize evidence from eligible studies. Prospective survival studies of HIV-1-infected individuals, with outcome defined as time from baseline to all-cause death, were included and classified by whether GBV-C status was determined in early or late HIV disease. The primary measure was the hazard ratio (HR) of death for HIV-infected individuals with GBV-C infection versus those without GBV-C infection.
Eleven studies from eight publications met the inclusion criteria. For studies with GBV-C status measured 2 years or less after HIV seroconversion (912 subjects), the combined HR was 0.88 [95% credible interval (CI) 0.30, 1.50]. For studies with GBV-C status measured more than 2 years after HIV seroconversion (1294 subjects), the combined HR was 0.41 (95% CI 0.23, 0.69).
No conclusive evidence was found of an association between survival and GBV-C infection early in HIV disease. However, when GBV-C infection was present later in HIV disease, a significant reduction in the hazard for mortality was observed for those with co-infection. Potential explanations for this difference include a non-proportional benefit of GBV-C over time, possibly related to clearance of GBV-C infection early in HIV disease. The timing of GBV-C infection appears to account for the contradictory results of studies on the effect of GBV-C coinfection on survival of HIV-infected people.
GB virus type C (GBV-C) is an apparently nonpathogenic virus that replicates in T and B lymphocytes and is a common cause of persistent human infection. Among HIV-1-infected individuals, persistent coinfection with GBV-C is associated with prolonged survival, and infection of blood mononuclear cells or CD4+ T cells with GBV-C and HIV in vitro results in significantly reduced HIV-1 replication. To date, the viral protein(s) that lead to HIV inhibition have not been identified. The GBV-C nonstructural phosphoprotein (NS5A) is predicted to have pleotropic effects on cells, including interactions with the IFN-induced dsRNA-activated protein kinase (PKR). We studied GBV-C NS5A to determine whether it is involved in inhibition of HIV replication. GBV-C NS5A protein from an isolate that was cleared by IFN therapy did not inhibit PKR, whereas NS5A from an isolate that was not cleared by IFN-inhibited PKR function in a yeast genetic system. Both of these GBV-C NS5A proteins were expressed in a CD4+ T cell line (Jurkat), and both induced a potent, dose-dependent inhibition of HIV-1 replication, thus the effect was independent of PKR inhibition. NS5A induced the release of the chemokine SDF-1 and decreased surface expression of the HIV coreceptor CXCR4, potentially explaining the HIV inhibition. Deletion mapping of the NS5A protein found that an 85-aa region between amino acids 152 and 237 inhibits HIV-1 replication. Thus, GBV-C NS5A protein alters the cellular milieu necessary for HIV-1 replication and may provide a previously undescribed therapeutic approach for anti-HIV therapy.
GB virus C: the good boy virus? Trends Microbiol
- N Bhattarai
- J T Stapleton
Bhattarai, N., and J. T. Stapleton. 2012. GB virus C: the good boy virus? Trends
Microbiol. 20: 124–130.
The disulfide bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of the molecule
- T Krey
- J Alayer
- C M Kikuti
- A Saulnier
- L Damier-Piolle
- I Petitpas
- D X Johansson
- R G Tawar
- B Baron
- B Robert
Krey, T., J. d'Alayer, C. M. Kikuti, A. Saulnier, L. Damier-Piolle, I. Petitpas,
D. X. Johansson, R. G. Tawar, B. Baron, B. Robert, et al. 2010. The disulfide
bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of
the molecule. PLoS Pathog. 6: e1000762.
Persistent immune activation in HIV-1 infection is associated with progression to AIDS.
- Hazenberg