Tohti Amet

CD59 incorporation protects hepatitis C virus against complement-mediated destruction

Several enveloped viruses including human immunodeficiency virus type 1 (HIV‐1), cytomegalovirus (CMV), herpes simplex virus 1 (HSV‐1), Ebola virus, vaccinia virus, and influenza virus have been found to incorporate host regulators of complement activation (RCA) into their viral envelopes and, as a result, escape antibody‐dependent complement‐mediated lysis (ADCML). Hepatitis C virus (HCV) is an enveloped virus of the family Flaviviridae and incorporates more than 10 host lipoproteins. Patients chronically infected with HCV develop high‐titer and crossreactive neutralizing antibodies (nAbs), yet fail to clear the virus, raising the possibility that HCV may also use the similar strategy of RCA incorporation to escape ADCML. The current study was therefore undertaken to determine whether HCV virions incorporate biologically functional CD59, a key member of RCA. Our experiments provided several lines of evidence demonstrating that CD59 was associated with the external membrane of HCV particles derived from either Huh7.5.1 cells or plasma samples from HCV‐infected patients. First, HCV particles were captured by CD59‐specific Abs. Second, CD59 was detected in purified HCV particles by immunoblot analysis and in the cell‐free supernatant from HCV‐infected Huh7.5.1 cells, but not from uninfected or adenovirus serotype 5 (Ad5) (a nonenveloped cytolytic virus)‐infected Huh7.5.1 cells by enzyme‐linked immunosorbent assay. Last, abrogation of CD59 function with its blockers increased the sensitivity of HCV virions to ADCML, resulting in a significant reduction of HCV infectivity. Additionally, direct addition of CD59 blockers into plasma samples from HCV‐infected patients increased autologous virolysis. Conclusion: Our study, for the first time, demonstrates that CD59 is incorporated into both cell line‐derived and plasma primary HCV virions at levels that protect against ADCML. This is also the first report to show that direct addition of RCA blockers into plasma from HCV‐infected patients renders endogenous plasma virions sensitive to ADCML. (HEPATOLOGY 2012)

A high-affinity inhibitor of human CD59 enhances complement-mediated virolysis of HIV-1: implications for treatment of HIV-1/AIDS.

Many pathogenic enveloped viruses, including HIV-1, escape complement-mediated virolysis by incorporating host cell regulators of complement activation into their own viral envelope. The presence of complement regulators including CD59 on the external surface of the viral envelope confers resistance to complement-mediated virolysis, which may explain why human pathogenic viruses such as HIV-1 are not neutralized by complement in human fluids, even in the presence of high Ab titers against the viral surface proteins. In this study, we report the development of a recombinant form of the fourth domain of the bacterial toxin intermedilysin (the recombinant domain 4 of intermedilysin [rILYd4]), a 114 aa protein that inhibits human CD59 function with high affinity and specificity. In the presence of rILYd4, HIV-1 virions derived from either cell lines or peripheral blood mononuclear cells of HIV-1–infected patients became highly sensitive to complement-mediated lysis activated by either anti–HIV-1 gp120 Abs or by viral infection-induced Abs present in the plasma of HIV-1–infected individuals. We also demonstrated that rILYd4 together with serum or plasma from HIV-1–infected patients as a source of anti–HIV-1 Abs and complement did not mediate complement-mediated lysis of either erythrocytes or peripheral blood mononuclear cells. These results indicate that rILYd4 may represent a novel therapeutic agent against HIV-1/AIDS

BST-2 expression in human hepatocytes is inducible by all three types of interferons and restricts production of hepatitis C virus.

Bone marrow stromal cell antigen 2 (BST-2, also known as tetherin, CD317, or HM1.24) has recently been identified as a host restriction factor against diverse families of enveloped viruses. However, the effects of BST-2 on the life cycle of hepatitis C virus (HCV), an enveloped RNA virus, remain unclear and controversial. Here we demonstrated that human hepatocytes including Huh7.5.1 cells, primary human hepatocytes (PHHs), and HepG2 cells constitutively expressed low to moderate levels of endogenous BST-2 on the cell surface, which could be robustly up-regulated by all three types of interferons (IFNs) such as IFN-α, IFN-γ, and IFN-λ. IFN-α and IFN-γ showed a synergistic effect in induction of BST-2 expression on human hepatocytes. Over-expression of BST-2 by BST-2-expressing vector transfection or up-regulation of BST-2 by IFN stimulation markedly suppressed HCV production, whereas shRNA-mediated depletion of endogenous BST-2 significantly enhanced HCV production in infected Huh7.5.1 cells. IFN-mediated anti-HCV activity was partially but significantly diminished by shRNA-mediated knockdown of BST-2 expression, indicating that BST- 2 upregulation is directly involved in IFN-mediated inhibition of HCV production. We also found that both BST-2 and HCV core co-localized with intracellular lipid droplets (LDs), suggesting that BST-2-HCV interaction may take place around LDs as LDs constitute an important intracellular organelle for HCV assembly and replication. Taken together, our data suggest that BST-2 is a host restriction factor against HCV, and induction of BST-2 in hepatocytes could be one of the mechanisms by which current HCV standard therapy (IFN-α plus ribavirin) achieves a sustained virological response (SVR).

Direct effects of HIV-1 Tat on excitability and survival of primary dorsal root ganglion neurons: possible contribution to HIV-1-associated pain.

The vast majority of people living with human immunodeficiency virus type 1 (HIV-1) have pain syndrome, which has a significant impact on their quality of life. The underlying causes of HIV-1-associated pain are not likely attributable to direct viral infection of the nervous system due to the lack of evidence of neuronal infection by HIV-1. However, HIV-1 proteins are possibly involved as they have been implicated in neuronal damage and death. The current study assesses the direct effects of HIV-1 Tat, one of potent neurotoxic viral proteins released from HIV-1-infected cells, on the excitability and survival of rat primary dorsal root ganglion (DRG) neurons. We demonstrated that HIV-1 Tat triggered rapid and sustained enhancement of the excitability of small-diameter rat primary DRG neurons, which was accompanied by marked reductions in the rheobase and resting membrane potential (RMP), and an increase in the resistance at threshold (RTh). Such Tat-induced DRG hyperexcitability may be a consequence of the inhibition of cyclin-dependent kinase 5 (Cdk5) activity. Tat rapidly inhibited Cdk5 kinase activity and mRNA production, and roscovitine, a well-known Cdk5 inhibitor, induced a very similar pattern of DRG hyperexcitability. Indeed, pre-application of Tat prevented roscovitine from having additional effects on the RMP and action potentials (APs) of DRGs. However, Tat-mediated actions on the rheobase and RTh were accelerated by roscovitine. These results suggest that Tat-mediated changes in DRG excitability are partly facilitated by Cdk5 inhibition. In addition, Cdk5 is most abundant in DRG neurons and participates in the regulation of pain signaling. We also demonstrated that HIV-1 Tat markedly induced apoptosis of primary DRG neurons after exposure for longer than 48 h. Together, this work indicates that HIV-1 proteins are capable of producing pain signaling through direct actions on excitability and survival of sensory neurons.

Association of Neisseria gonorrhoeae OpaCEA with Dendritic Cells Suppresses Their Ability to Elicit an HIV-1-Specific T Cell Memory Response

Infection with Neisseria gonorrhoeae (N. gonorrhoeae) can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa) proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on CD4+ T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1)-specific cytotoxic T-lymphocyte (CTL) responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific OpaCEA, but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, OpaCEA binding to CEACAM1 reduced the DCs’ capacity to stimulate an allogeneic T cell proliferative response. Moreover, OpaCEA-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with OpaCEA-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain why infection of N. gonorrhoeae fails to trigger an effective specific immune response or develop immune memory, and may affect the potent synergy between gonorrhea and HIV-1 infection.

Primary Human Macrophages Serve as Vehicles for Vaccinia Virus Replication and Dissemination

ABSTRACT Human monocytic and professional antigen-presenting cells have been reported only to exhibit abortive infections with vaccinia virus (VACV). We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, but not human AB serum-derived cells, were permissive to VACV replication. The titers of infectious virions in both cell-free supernatants and cellular lysates of infected M1 and M2 markedly increased in a time-dependent manner. The majority of virions produced in permissive MDMs were extracellular enveloped virions (EEV), a secreted form of VACV associated with long-range virus dissemination, and were mainly found in the culture supernatant. Infected MDMs formed VACV factories, actin tails, virion-associated branching structures, and cell linkages, indicating that MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. VACV replication was sensitive to inhibitors against the Akt and Erk1/2 pathways that can be activated by VACV infection and M-CSF stimulation. Classical activation of MDMs by lipopolysaccharide (LPS) plus gamma interferon (IFN-γ) stimulation caused no effect on VACV replication, while alternative activation of MDMs by interleukin-10 (IL-10) or LPS-plus-IL-1β treatment significantly decreased VACV production. The IL-10-mediated suppression of VACV replication was largely due to Stat3 activation, as a Stat3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data demonstrate that primary human macrophages are permissive to VACV replication. After infection, these cells produce EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread. IMPORTANCE Our results provide critical information to the burgeoning fields of cancer-killing (oncolytic) virus therapy with vaccinia virus (VACV). One type of macrophage (M2) is considered a common presence in tumors and is associated with poor prognosis. Our results demonstrate a preference for VACV replication in M2 macrophages and could assist in designing treatments and engineering poxviruses with special considerations for their effect on M2 macrophage-containing tumors. Additionally, this work highlights the importance of macrophages in the field of vaccine development using poxviruses as vectors. The understanding of the dynamics of poxvirus-infected foci is central in understanding the effectiveness of the immune response to poxvirus-mediated vaccine vectors. Monocytic cells have been found to be an important part of VACV skin lesions in mice in controlling the infection as well as mediating virus transport out of infected foci.

Provirus Activation Plus CD59 Blockage Triggers Antibody-Dependent Complement-Mediated Lysis of Latently HIV-1–Infected Cells

Latently HIV-1–infected cells are recognized as the last barrier toward viral eradication and cure. To purge these cells, we combined a provirus stimulant with a blocker of human CD59, a key member of the regulators of complement activation, to trigger Ab-dependent complement-mediated lysis. Provirus stimulants including prostratin and histone deacetylase inhibitors such as romidepsin and suberoylanilide hydroxamic acid activated proviruses in the latently HIV-1–infected T cell line ACH-2 as virion production and viral protein expression on the cell surface were induced. Romidepsin was the most attractive provirus stimulant as it effectively activated proviruses at nanomolar concentrations that can be achieved clinically. Antiretroviral drugs including two protease inhibitors (atazanavir and darunavir) and an RT inhibitor (emtricitabine) did not affect the activity of provirus stimulants in the activation of proviruses. However, saquinavir (a protease inhibitor) markedly suppressed virus production, although it did not affect the percentage of cells expressing viral Env on the cell surface. Provirus-activated ACH-2 cells expressed HIV-1 Env that colocalized with CD59 in lipid rafts on the cell surface, facilitating direct interaction between them. Blockage of CD59 rendered provirus-activated ACH-2 cells and primary human CD4+ T cells that were latently infected with HIV-1 sensitive to Ab-dependent complement-mediated lysis by anti–HIV-1 polyclonal Abs or plasma from HIV-1–infected patients. Therefore, a combination of provirus stimulants with regulators of complement activation blockers represents a novel approach to eliminate HIV-1.

Primary Human Leukocyte Subsets Differentially Express Vaccinia Virus Receptors Enriched in Lipid Rafts

ABSTRACT Poxviruses, including vaccinia virus (VV) and canarypox virus (ALVAC), do not indiscriminately infect all cell types of the primary human leukocytes (PHLs) that they encounter but instead demonstrate an extremely strong bias toward infection of monocytes and monocyte lineage cells. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias of cell surface protein-dependent binding to monocytes, B cells, and activated T cells to a similar degree and to neutrophils to a much lesser extent. Resting T cells and resting NK cells exhibited only trace amounts of VV binding. Activated T cells, however, became permissive to VV binding, infection, and replication, while activated NK cells still resisted VV binding. VV binding strongly colocalized with lipid rafts on the surfaces of all VV binding-susceptible PHL subsets, even when lipid rafts were relocated to cell uropods upon cell polarization. Immunosera raised against detergent-resistant membranes (DRMs) from monocytes or activated T cells, but not resting T cells, effectively cross-blocked VV binding to and infection of PHL subsets. CD29 and CD98, two lipid raft-associated membrane proteins that had been found to be important for VV entry into HeLa cells, had no effect on VV binding to and infection of primary activated T cells. Our data indicate that PHL subsets express VV protein receptors enriched in lipid rafts and that receptors are cross-presented on all susceptible PHLs.

An activated Th17-prone T cell subset involved in chronic graft-versus-host disease sensitive to pharmacological inhibition

Chronic graft-versus-host disease (cGvHD) remains a major complication of allogeneic stem cell transplantation requiring novel therapies. CD146 and CCR5 are expressed by activated T cells and associated with increased T cell migration capacity and Th17 polarization. We performed a multiparametric flow cytometry analysis in a cohort of 40 HSCT patients together with a cGvHD murine model to understand the role of CD146-expressing subsets. We observed an increased frequency of CD146+ CD4 T cells in the 20 patients with active cGvHD with enhanced RORγt expression. This Th17-prone subset was enriched for cells coexpressing CD146 and CCR5 that harbor mixed Th1/Th17 features and were more frequent in cGvHD patients. Utilizing a murine cGvHD model with bronchiolitis obliterans (BO), we observed that donor T cells from CD146-deficient mice versus those from WT mice caused significantly reduced pulmonary cGvHD. Reduced cGvHD was not the result of failed germinal center B cell or T follicular helper cell generation. Instead, CD146-deficient T cells had significantly lower pulmonary macrophage infiltration and T cell CCR5, IL-17, and IFN-γ coexpression, suggesting defective pulmonary end-organ effector mechanisms. We, thus, evaluated the effect of TMP778, a small-molecule RORγt activity inhibitor. TMP778 markedly alleviated cGvHD in murine models similarly to agents targeting the Th17 pathway, such as STAT3 inhibitor or IL-17-blocking antibody. Our data suggest CD146-expressing T cells as a cGvHD biomarker and suggest that targeting the Th17 pathway may represent a promising therapy for cGvHD.

HIV-1 Coinfection Profoundly Alters Intrahepatic Chemokine but Not Inflammatory Cytokine Profiles in HCV-Infected Subjects

The pathogenesis of accelerated liver damage in subjects coinfected with hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) remains largely unknown. Recent studies suggest that ongoing chronic liver inflammation is responsible for the liver injury in HCV-infected patients. We aimed to determine whether HIV-1 coinfection altered intrahepatic inflammatory profiles in HCV infection, thereby hastening liver damage. We used a real-time RT-PCR-based array to comparatively analyze intrahepatic inflammation gene profiles in liver biopsy specimens from HCV-infected (n = 16), HCV/HIV-1-coinfected (n = 8) and uninfected (n = 8) individuals. We then used human hepatocytes to study the molecular mechanisms underlying alternations of the inflammatory profiles. Compared with uninfected individuals, HCV infection and HCV/HIV-1 coinfection markedly altered expression of 59.5% and 50.0% of 84 inflammation-related genes tested, respectively. Among these genes affected, HCV infection up-regulated the expression of 24 genes and down-regulated the expression of 26 genes, whereas HCV/HIV-1 coinfection up-regulated the expression of 21 genes and down-regulated the expression of 21 genes. Compared with HCV infection, HCV/HIV-1 coinfection did not dramatically affect intrahepatic gene expression profiles of cytokines and their receptors, but profoundly altered expression of several chemokine genes including up-regulation of the CXCR3-associated chemokines. Human hepatocytes produced these chemokines in response to virus-related microbial translocation, viral protein stimulation, and antiviral immune responses. Conclusions HIV-1 coinfection profoundly alters intrahepatic chemokine but not cytokine profiles in HCV-infected subjects. The altered chemokines may orchestrate the tissue-specific and cell-selective trafficking of immune cells and autoimmunity to accelerate liver disease in HCV/HIV-1 coinfection.