Natural Target Cells Research Articles

Antiviral effect of cidofovir on parvovirus B19 replication

Parvovirus B19 (B19V) is a human ssDNA virus responsible for a wide range of clinical manifestations, still lacking for a specific antiviral therapy. The identification of compounds active against B19V may add therapeutic options to the treatment of B19V infections, that now entirely relies on symptomatic treatments. In the search for compounds possibly inhibiting B19V replication, a particular focus was raised to cidofovir, an acyclic nucleoside phosphonate broadly active against dsDNA viruses. The present study was aimed at evaluating the effect of cidofovir against B19V in two model systems, the UT7/EpoS1 cell line and erythroid progenitor cells (EPC), generated from peripheral blood mononuclear cells. Experiments were carried out at different multiplicity of infections and cidofovir concentrations (0–500μM) during a course of infection. The effects of cidofovir on B19V replication were assessed by qPCR assays while influence of cidofovir on host cells was measured by cell proliferation and viability assays. Our findings demonstrated that cidofovir has a relevant inhibiting activity on B19V replication within infected UT7/EpoS1, and that the effect on B19V DNA amounts is dose-dependent allowing for the determination of EC50 and EC90 values (7.45–41.27μM, and 84.73–360.7μM, respectively). In EPCs, that constitute a cellular population close to the natural target cells in bone marrow, the inhibitory effect was demonstrated to a lesser extent, however provoking a significant reduction on B19V DNA amounts at 500μM (68.2–92.8%). To test infectivity of virus released from EPCs cultured in the presence of cidofovir, cell culture supernatants were used as inoculum for a further course of infection in UT7/EpoS1 cells, indicating a significant reduction in viral infectivity at 500μM cidofovir. Since the drug did not interfere with the overall cellular DNA synthesis and metabolic activity, the observed effect of cidofovir could be likely related to a specific inhibition of B19V replication.

Tetherin can restrict cell-free and cell-cell transmission of HIV from primary macrophages to T cells.

Bst-2/Tetherin inhibits the release of HIV by tethering newly formed virus particles to the plasma membrane of infected cells. Although the mechanisms of Tetherin-mediated restriction are increasingly well understood, the biological relevance of this restriction in the natural target cells of HIV is unclear. Moreover, whether Tetherin exerts any restriction on the direct cell-cell spread of HIV across intercellular contacts remains controversial. Here we analyse the restriction endogenous Tetherin imposes on HIV transmission from primary human macrophages, one of the main targets of HIV in vivo. We find that the mRNA and protein levels of Tetherin in macrophages are comparable to those in T cells from the same donors, and are highly upregulated by type I interferons. Improved immunocytochemistry protocols enable us to demonstrate that Tetherin localises to the cell surface, the trans-Golgi network, and the macrophage HIV assembly compartments. Tetherin retains budded virions in the assembly compartments, thereby impeding the release and cell-free spread of HIV, but it is not required for the maintenance of these compartments per se. Notably, using a novel assay to quantify cell-cell spread, we show that Tetherin promotes the transfer of virus clusters from macrophages to T cells and thereby restricts the direct transmission of a dual-tropic HIV-1. Kinetic analyses provide support for the notion that this direct macrophage-T cell spread is mediated, at least in part, by so-called virological synapses. Finally, we demonstrate that the viral Vpu protein efficiently downregulates the cell surface and overall levels of Tetherin, and thereby abrogates this HIV restriction in macrophages. Together, our study shows that Tetherin, one of the most potent HIV restriction factors identified to date, can inhibit virus spread from primary macrophages, regardless of the mode of transmission.

Small molecules that inhibit Vif-induced degradation of APOBEC3G.

BackgroundHIV-1 Vif is essential for virus replication in natural target cells such as T cells and macrophages. Vif recruits a ubiquitin ligase to degrade restrictive APOBEC3 proteins. APOBEC3G is one of the most potent retroviral restriction factors targeted by Vif and, as such, the Vif-APOBEC3G interaction has emerged as a promising HIV-1 therapeutic target.Methods20,000 small molecules were used in live-cell screens for those that preserve EGFP-APOBEC3G fluorescence and luciferase-APOBEC3G luminescence in the presence of HIV-1 Vif.Results2 compounds with similar core structures preserved APOBEC3G levels in the presence of Vif. 10 μM of compound restored APOBEC3G to levels sufficient for incorporation into vif-proficient virus particles and restriction of virus infectivity. Vif-dependent APOBEC3G polyubiquitination and general proteasomal activity were unaffected at the same concentration.ConclusionsThe small molecules described here preserve APOBEC3G levels and activity in the presence of Vif. These molecules are starting points for further development as antiretrovirals.

Stable Microtubule Subsets Facilitate Early HIV-1 Infection

The density of the host cytoplasm presents pathogens such as HIV-1 with a major impediment to reaching the nucleus. As such, they must exploit host cytoskeletal networks to facilitate their movement.1 Within host cells, microtubule (MT) filaments act as “highways” for the intracellular transport of cargoes mediated by MT-associated motor proteins.2 While it has been established that the MT motor dynein mediates inward movement of HIV-1 to the nucleus,3 studies using MT depolymerizing agents such as nocodazole have produced seemingly conflicting suggestions that MTs are not important for HIV-1 infection. Recent findings relating to the role of specialized stable MT subsets now offer a potential explanation. MTs form polarized filaments; their minus ends are anchored in a perinuclear MT organizing center (MTOC) while their plus ends grow outward to explore or “sense” the intracellular environment and form connections to the cell periphery. As such, in many cell types MTs are highly dynamic, undergoing rapid phases of growth and collapse through the addition and loss of tubulin subunits primarily at the plus end. MT stability is controlled by highly specialized plus-end-binding proteins, called+TIPs. Central to this is the end-binding protein EB1, which recognizes the MT plus end and recruits other+TIPs that control MT dynamics. In response to specific signals, subsets of MTs become stabilized and form long-lived filaments that acquire distinguishing posttranslational tubulin modifications such as detyrosination and acetylation. Although these modifications do not seem to impart stability per se, they are thought to enable selective recognition of stable MT subsets by specific MT motor proteins. As such, these filaments can act as specialized tracks for vesicle trafficking to facilitate processes such as cell polarization. A notable feature of these long-lived MT subsets is their relative insensitivity to MT depolymerizing agents, but their potential contribution to HIV-1 infection has until now gone largely unstudied. We recently found that HIV-1 infection rapidly induces MT stabilization in a range of human cell types,4 including natural target cells such as T cells, microglia, and primary macrophages (Fig. 1). This stabilization appears to occur very soon after viral entry and involves viral targeting of EB1 and the EB1-binding+TIP, Kif4. MT stabilization continues to increase as infection progresses, potentially as more virus enters the cell, triggered by viral proteins released during uncoating or by the activation of signal pathways that regulate MT stability, something we are currently studying. Viral particles also exhibited significant localization to stable MTs and when stable MT formation was blocked using either RNA interference (RNAi) or dominant negative approaches infection was potently suppressed, with viral particles failing to reach the nucleus efficiently. These findings suggest that HIV-1 induces the formation of specialized stable MT subsets early after entry in order to facilitate translocation of viral particles to the nucleus. The importance of these stable MT subsets to early HIV-1 infection offers an explanation for why dynein is important for infection yet MT depolymerizing agents have little effect, and identifies new factors to explore as potential therapeutic targets to treat HIV-1. FIG. 1. HIV-1 induces stable microtubule (MT) formation in primary human macrophages. Primary human macrophages were mock infected (inset image lower left) or infected with pseudotyped HIV-1 and then fixed and stained for tyrosinated (red) and acetylated (green) ...

The Potent Oncogene NPM-ALK Mediates Malignant Transformation of Normal Human CD4+ T Lymphocytes

With this study we have demonstrated that in vitro transduction of normal human CD4(+) T lymphocytes with NPM-ALK results in their malignant transformation. The transformed cells become immortalized and display morphology and immunophenotype characteristic of patient-derived anaplastic large-cell lymphomas. These unique features, which are strictly dependent on NPM-ALK activity and expression, include perpetual cell growth, proliferation, and survival; activation of the key signal transduction pathways STAT3 and mTORC1; and expression of CD30 (the hallmark of anaplastic large-cell lymphoma) and of immunosuppressive cytokine IL-10 and cell-surface protein PD-L1/CD274. Implantation of NPM-ALK-transformed CD4(+) T lymphocytes into immunodeficient mice resulted in formation of tumors indistinguishable from patients' anaplastic large-cell lymphomas. Our findings demonstrate that the key aspects of human carcinogenesis closely recapitulating the features of the native tumors can be faithfully reproduced in vitro when an appropriate oncogene is used to transform its natural target cells; this in turn points to the fundamental role in malignant cell transformation of potent oncogenes expressed in the relevant target cells. Such transformed cells should permit study of the early stages of carcinogenesis, and in particular the initial oncogene-host cell interactions. This experimental design could also be useful for studies of the effects of early therapeutic intervention and likely also the mechanisms of malignant progression.

Poly-proline motif in HIV-2 Vpx is critical for its efficient translation

Human immunodeficiency virus type 2 (HIV-2) carries an accessory protein Vpx that is important for viral replication in natural target cells. In its C-terminal region, there is a highly conserved poly-proline motif (PPM) consisting of seven consecutive prolines, encoded in a poly-pyrimidine tract. We have previously shown that PPM is critical for Vpx expression and viral infectivity. To elucidate the molecular basis underlying this observation, we analysed the expression of Vpx proteins with various PPM mutations by in vivo and in vitro systems. We found that the number and position of consecutive prolines in PPM are important for Vpx expression, and demonstrated that PPM is essential for efficient Vpx translation. Furthermore, mutational analysis to synonymously disrupt the poly-pyrimidine tract suggested that the context of PPM amino acid sequences is required for efficient translation of Vpx. We similarly analysed HIV-1 and HIV-2 Vpr proteins structurally related to HIV-2 Vpx. Expression level of the two Vpr proteins lacking PPM was shown to be much lower relative to that of Vpx, and not meaningfully enhanced by introduction of PPM at the C terminus. Finally, we examined the Vpx of simian immunodeficiency virus from rhesus monkeys (SIVmac), which also has seven consecutive prolines, for PPM-dependent expression. A multi-substitution mutation in the PPM markedly reduced the expression level of SIVmac Vpx. Taken together, it can be concluded that the notable PPM sequence enhances the expression of Vpx proteins from viruses of the HIV-2/SIVmac group at the translational level.

Cell type specificity and structural determinants of IRES activity from the 5′ leaders of different HIV-1 transcripts

Internal ribosome entry site (IRES) RNAs are important regulators of gene expression, but their diverse molecular mechanisms remain partially understood. The HIV-1 gag transcript leader contains an IRES that may be a good model for understanding the function of many other IRESs. We investigated the possibility that this IRES’ function is linked to both the structure of the RNA and its cellular environment. We find that in the context of a bicistronic reporter construct, HIV-1 gag IRES’ activity is cell type-specific, with higher activity in T-cell culture systems that model the natural target cells for HIV-1 infection. This finding underscores how an IRES may be fine tuned to function in certain cells, perhaps owing to cell type-specific protein factors. Using RNA probing and mutagenesis, we demonstrate that the HIV-1 gag IRES does not use pre-folded RNA structure to drive function, a finding that gives insight into how conformationally dynamic IRESs operate. Furthermore, we find that a common exon drives IRES activity in a diverse set of alternatively spliced transcripts. We propose a mechanism in which a structurally plastic RNA element confers the ability to initiate translation internally, and activity from this common element is modulated by 3′ nucleotides added by alternative splicing.

APOBEC3G Impairs the Multimerization of the HIV-1 Vif Protein in Living Cells

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in nonpermissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G [A3G]) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55(Gag). Recently, we showed that oligomerization of Vif into high-molecular-mass complexes induces Vif folding and influences its binding to high-affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy (FLIM)- and fluorescence resonance energy transfer (FRET)-based assays to investigate Vif-Vif interactions in living cells. By using two N-terminally tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain ((161)PPLP(164)) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate in Vif oligomerization. When coexpressed together with Pr55(Gag), Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.

Human rElafin Inhibits HIV-1 Replication in Its Natural Target Cells

Trappin-2/elafin is a novel innate immune factor that belongs to the serine protease inhibitor family and has known antibacterial, antifungal, and antiviral properties. In this study, we further investigated the anti-HIV activity of elafin using different cellular models and both X4– and R5–HIV-1 laboratory strains. We compared the antiviral activity of human recombinant elafin (rElafin) with three well-known antiretroviral drugs, AZT, tenofovir, and enfuvirtide. We have found that when the virus is pre-incubated with rElafin prior to the infection of the cells, HIV-1 replication is significantly inhibited. In target T cells and human peripheral blood mononuclear cells, maximal inhibition was achieved using submicromolar concentrations, and rElafin was found to be as potent as enfuvirtide, showing its potential for therapeutic application. We also show data on the mechanism of the antiviral activity of rElafin. We have demonstrated that rElafin neither binds to CD4, CXCR4, or CCR5 host cell receptors, nor to the viral glycoproteins gp120 and gp41. Furthermore, in our cell-to-cell fusion assays, in contrast to enfuvirtide, rElafin failed to block cell fusion. Altogether our results indicate that rElafin interferes with HIV replication at the early steps of its cycle but with a different mechanism of action than enfuvirtide. This study provides the first experimental evidence that elafin inhibits HIV replication in its natural target cells; therefore, elafin might have potential for its development as a new anti-HIV drug or microbicide.

Normal development and function but impaired memory phenotype of CD8+ T cells in transgenic mice expressing HIV-1 Nef in its natural target cells

We studied the impact of HIV Nef on CD8+ T cells in a mouse model of AIDS, the CD4C/HIVNef transgenic (Tg) mice. We found that negative and positive thymic selections of CD8+ T cells proceeded normally in Nef Tg mice bred respectively with HY or OT-1 TCR Tg mice. Tg peripheral CD8+ T cells showed an activated phenotype and enhanced cell division in vivo and proliferated efficiently when stimulated in vitro with antigenic peptide. When challenged with LCMVArmstrong, Nef Tg mice developed a strong acute CD8+ T cell response and cleared the virus as efficiently as wild-type mice. However, maintenance of LCMV-specific CD8+ memory T cells was impaired in Nef Tg mice, a defect partially rescued by adoptive transfer of non-Tg naïve CD4+ T cells. Thus, despite severe abnormalities of their precursors, the double-positive CD4+CD8+ thymocytes, Tg CD8+ T cells have conserved important functions.

Classification of human natural killer cells based on migration behavior and cytotoxic response

AbstractDespite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of the basic functional heterogeneity in individual NK cell behavior. Using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, in the present study, we were able to quantify how the cytotoxic response varied between individual NK cells. Strikingly, approximately half of the NK cells did not kill any target cells at all, whereas a minority of NK cells was responsible for a majority of the target cell deaths. These dynamic cytotoxicity data allowed categorization of NK cells into 5 distinct classes. A small but particularly active subclass of NK cells killed several target cells in a consecutive fashion. These “serial killers” delivered their lytic hits faster and induced faster target cell death than other NK cells. Fast, necrotic target cell death was correlated with the amount of perforin released by the NK cells. Our data are consistent with a model in which a small fraction of NK cells drives tumor elimination and inflammation.

Antiherpetic potential of 6-bromoindirubin-3′-acetoxime (BIO-acetoxime) in human oral epithelial cells

Glycogen synthase kinase 3 (GSK-3) functions in the regulation of glycogen metabolism, in the cell cycle, and in immune responses and is targeted by some viruses to favor the viral life cycle. Inhibition of GSK-3 by 6-bromoindirubin-3'-acetoxime (BIO-acetoxime), a synthetic derivative of a compound from the Mediterranean mollusk Hexaplex trunculus, protects cells from varicella infection. In this study, we examined the effects of BIO-acetoxime against herpes simplex virus-1 (HSV-1) infection in human oral epithelial cells, which represent a natural target cell type. The results revealed that BIO-acetoxime relieves HSV-1-induced cytopathic effects and apoptosis. We also found that BIO-acetoxime reduced viral yields and the expression of different classes of viral proteins. Furthermore, addition of BIO-acetoxime before, simultaneously with or after HSV-1 infection significantly reduced viral yields. Collectively, BIO-acetoxime may suppress viral gene expression and protect oral epithelial cells from HSV-1 infection. These results suggest the possible involvement of GSK-3 in HSV-1 infection.

Prophylactic, therapeutic and neutralizing effects of zinc oxide tetrapod structures against herpes simplex virus type-2 infection

The attachment of Herpes simplex virus type-2 (HSV-2) to a target cell requires ionic interactions between negatively charged cell surface co-receptor heparan sulfate (HS) and positively charged residues on viral envelop glycoproteins, gB and gC. Effective blocking of this first step of HSV-2 pathogenesis demonstrates significant prophylactic effects against the viral disease; any in vitro therapeutic effects of blocking this interaction, however, are not clear. Here, we provide new evidence that zinc oxide tetrapod micro-nanostructures synthesized by flame transport approach significantly block HSV-2 entry into target cells and, in addition, demonstrate the potential to stop the spread of the virus among already infected cells. The zinc oxide tetrapods (ZnOTs) also exhibit the ability to neutralize HSV-2 virions. Natural target cells such as human vaginal epithelial and HeLa cells showed highly reduced infectivity when infected with HSV-2 virions that were pre-incubated with the ZnOTs. The mechanism behind the ability of ZnOTs to prevent, neutralize or reduce HSV-2 infection relies on their ability to bind the HSV-2 virions. We used fluorescently labeled ZnOTs and GFP-expressing HSV-2 virions to demonstrate the binding of the ZnOTs with HSV-2. We also show that the binding and hence, the antiviral effects of ZnOTs can be enhanced by illuminating the ZnOTs with UV light. Our results provide new insights into the anti-HSV-2 effects of ZnOT and rationalize their development as a HSV-2 trapping agent for the prevention and/or treatment of infection. The observed results also demonstrate that blocking HSV-2 attachment can have prophylactic as well as therapeutic applications.

Determination of the Changes in Human Leukocytes Inflammatory Genes Expression after Infection with Human Papillomavirus

Keratinocytes are the natural target cells for infection by humanpapillomaviruses (HPVs), most of which cause benign epithelial hyperplasias (warts). HPVs, cause lesions that can progress to carcinomas. Inflammatory genes such as those coding for cytokines, chemokines and other genes are induced in response to a viral infection. HPV effect on host cellular responses is critically important to explore the molecular mechanisms of viral-host interaction. The aims of this study were to detect changes in the mRNA expression in a panel of 84 inflammatory genes in the leukocyte of three HPV infected patients using real time-PCR array technology. Nine inflammatory genes showed a significant gene up regulation ranging between 16.897 mean fold change for Interleukin-1 alpha and 353.804mean fold change for Interferon-alpha2 gene. The up regulation of mRNA of nine geneswith high significance (P > 0.05) might suggest that the host-viral interaction is a cellular response against viral effect. In conclusion,this study revealed several alterations of many Leukocyte`s inflammatory gene expressions as a response against HPV. The common up regulated genes after HPV infections stimulate the activity of different immunological functional pathways represented by cytokine-cytokine receptor interaction, NOD-like receptor signaling pathway, and Toll-like receptor signaling pathway.

Ultrasonic standing waves for dynamic micro-array cytometry

We describe a novel platform for dynamic micro-array cytometry (DMAC), i.e., parallel screening of individual cell-cell interactions based on ultrasonic standing wave aggregation and positioning of cells in a multi-well microplate. Upon ultrasound actuation, clusters containing one or a few cells are quickly formed and retained in a precise location synchronously in each of the 100 wells on the microplate. By combining the acoustic cell handling tool with high-resolution fluorescence microscopy, detailed time-lapse monitoring of individual cell-cell interactions in a highly parallel manner is possible. Of particular interest in our group is to study the long-term interaction between natural killer (NK) cells and different target cells at the level of single cells. In this talk we demonstrate both theoretically and experimentally how to design a microchip capable of trapping and positioning individual cells by ultrasound in a highly parallel manner, and with a spatial accuracy of the order of a cell diameter. We quantify the cell cluster motility with and without retained ultrasound exposure during 17 h, and we report on the viability of cells when exposed to continuous ultrasound for up to three days. Finally, we quantify the heterogeneity of NK cells’ cytotoxicity against cancer cells.

Proteomic characterization of a novel structural protein ORF5a of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) emerged in the late 1980s in both Europe and North America is an arterivirus causing great economic losses in the pig industry worldwide. Recently, a novel structural protein, ORF5a, encoded by an alternative ORF of a subgenomic mRNA encoding the major envelope glycoprotein, GP5, has been discovered in all arteriviruses, suggesting its important role in arterivirology. The present study attempted to investigate compensatory changes of cellular gene expression in natural target cells regulated by the ORF5a. We thus established sublines of PAM cells to stably express the PRRSV ORF5a protein and assessed alterations in cellular protein productions of ORF5a-expressing PAM (PAM-ORF5a) cells at different time courses by the use of proteomic analysis. A total of 36 protein spots were initially found to be differentially expressed in PAM-ORF5a cells compared with normal PAM cells by high-resolution two-dimensional gel electrophoresis (2DE). Of these spots, 16 protein spots with statistically significant alteration, including 13 up-regulated and 3 down-regulated protein spots, were picked out for subsequent protein identification by peptide mass fingerprinting after matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS). The altered cellular proteins identified in this study were grouped into the functions associated with a variety of cellular processes such as cell growth, cytoskeleton networks and cell communication, metabolism, protein biosynthesis, RNA processing, and transportation. The proteomics data will provide valuable information for better understanding the specific cellular response to the novel ORF5a protein during PRRSV replication.

The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication

The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented.

HIV-1 Nef mobilizes lipid rafts in macrophages through a pathway that competes with ABCA1-dependent cholesterol efflux

HIV infection, through the actions of viral accessory protein Nef, impairs activity of cholesterol transporter ABCA1, inhibiting cholesterol efflux from macrophages and elevating the risk of atherosclerosis. Nef also induces lipid raft formation. In this study, we demonstrate that these activities are tightly linked and affect macrophage function and HIV replication. Nef stimulated lipid raft formation in macrophage cell line RAW 264.7, and lipid rafts were also mobilized in HIV-1-infected human monocyte-derived macrophages. Nef-mediated transfer of cholesterol to lipid rafts competed with the ABCA1-dependent pathway of cholesterol efflux, and pharmacological inhibition of ABCA1 functionality or suppression of ABCA1 expression by RNAi increased Nef-dependent delivery of cholesterol to lipid rafts. Nef reduced cell-surface accessibility of ABCA1 and induced ABCA1 catabolism via the lysosomal pathway. Despite increasing the abundance of lipid rafts, expression of Nef impaired phagocytic functions of macrophages. The infectivity of the virus produced in natural target cells of HIV-1 negatively correlated with the level of ABCA1. These findings demonstrate that Nef-dependent inhibition of ABCA1 is an essential component of the viral replication strategy and underscore the role of ABCA1 as an innate anti-HIV factor.

MRNA-Selective Translation Induced by FSH in Primary Sertoli Cells

FSH is a key hormonal regulator of Sertoli cell secretory activity, required to optimize sperm production. To fulfil its biological function, FSH binds a G protein-coupled receptor, the FSH-R. The FSH-R-transduced signaling network ultimately leads to the transcription or down-regulation of numerous genes. In addition, recent evidence has suggested that FSH might also regulate protein translation. However, this point has never been demonstrated conclusively yet. Here we have addressed this issue in primary rat Sertoli cells endogenously expressing physiological levels of FSH-R. We observed that, within 90 min of stimulation, FSH not only enhanced overall protein synthesis in a mammalian target of rapamycin-dependent manner but also increased the recruitment of mRNA to polysomes. m(7)GTP pull-down experiments revealed the functional recruitment of mammalian target of rapamycin and p70 S6 kinase to the 5'cap, further supported by the enhanced phosphorylation of one of p70 S6 kinase targets, the eukaryotic initiation factor 4B. Importantly, the scaffolding eukaryotic initiation factor 4G was also recruited, whereas eukaryotic initiation factor 4E-binding protein, the eukaryotic initiation factor 4E generic inhibitor, appeared to play a minor role in translational regulations induced by FSH, in contrast to what is generally observed in response to anabolic factors. This particular regulation of the translational machinery by FSH stimulation might support mRNA-selective translation, as shown here by quantitative RT-PCR amplification of the c-fos and vascular endothelial growth factor mRNA but not of all FSH target mRNA, in polysomal fractions. These findings add a new level of complexity to FSH biological roles in its natural target cells, which has been underappreciated so far.

In vitro-activated tumor-bearing host T cells and the effectiveness of tumor vaccine immunotherapy

BACKGROUND AND OBJECTIVEVaccination during periods of lymphopenia may facilitate immune responses to weak self-antigens and enhance antitumor immunity. The objective of this study was to determine the effectiveness of tumor vaccine immunotherapy combined with immune reconstruction using tumor-bearing host immune cells in lymphopenia, and to investigate the role of tumor-bearing host T cells activated in vitro during immunotherapy.DESIGN AND SETTINGAnimal study conducted in the First Affiliated Hospital of Xi’an Jiaotong University from January 2009 to January 2010.PATIENTS AND METHODSLymphopenia was induced by cyclophosphamide. A reconstituted immune system with different syngeneic lymphocytes was employed, including lymphocytes from naïve rats (unsensitized group), tumor-bearing rats (tumor-bearing group), and tumor-bearing rats activated in vitro (activated group). All rats were immunized with granulocyte-macrophage colony-stimulating factor (GM-CSF)-modified NuTu-19 ovarian cancer (GM-CSF/NuTu-19) cells. Tumor vaccine-draining lymph nodes (TVDLNs) were harvested, and then stimulated to induce effector T cells (TE). TE were then adoptively transferred to rats bearing a 3-day pre-established abdominal tumor (NuTu-19), and the survival rate was calculated.RESULTSCompared with the unsensitized group, the levels of interleukin-2 (IL-2) were significantly lower in the tumor-bearing group, whereas that of IL-4 were significantly higher (P<.05). The number of CD4+ T cells secreting interferon-γ and the specific cytotoxicity of CD8+ cytotoxic T lymphocytes were significantly lower (P<.05). The survival was significantly higher in the activated group compared with the other groups.CONCLUSIONSLymphocytes from tumor-bearing rats activated in vitro can effectively reverse the immunosuppressive effects of tumor-bearing hosts.