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Maurizio Federico

Publications and source records attributed to Maurizio Federico.

18 recordsLinked to original sources
PubMed2006

Generation and characterization of a stable cell population releasing fluorescent HIV-1-based Virus Like Particles in an inducible way.

BACKGROUND: The availability of cell lines releasing fluorescent viral particles can significantly support a variety of investigations, including the study of virus-cell interaction and the screening of antiviral compounds. Regarding HIV-1, the recovery of such biologic reagents represents a very hard challenge due to the intrinsic cytotoxicity of many HIV-1 products. We sought to overcome such a limitation by using a cell line releasing HIV-1 particles in an inducible way, and by exploiting the ability of a HIV-1 Nef mutant to be incorporated in virions at quite high levels. RESULTS: Here, we report the isolation and characterization of a HIV-1 packaging cell line, termed 18-4s, able to release valuable amounts of fluorescent HIV-1 based Virus-Like Particles (VLPs) in an inducible way. 18-4s cells were recovered by constitutively expressing the HIV-1 NefG3C mutant fused with the enhanced-green fluorescent protein (NefG3C-GFP) in a previously isolated inducible HIV-1 packaging cell line. The G3C mutation creates a palmitoylation site which results in NefG3C-GFP incorporation into virions greatly exceeding that of the wild type counterpart. Upon induction of 18-4s cells with ponasterone A and sodium butyrate, up to 4 mug/ml of VLPs, which had incorporated about 150 molecules of NefG3C-GFP per viral particle, were released into the culture supernatant. Due to their intrinsic strong fluorescence, the 18-4s VLPs were easily detectable by a novel cytofluorometric-based assay developed here. The treatment of target cells with fluorescent 18-4 VLPs pseudotyped with different glycoprotein receptors resulted in these becoming fluorescent as early as two hours post-challenge. CONCLUSION: We created a stable cell line releasing fluorescent HIV-1 based VLPs upon induction useful for several applications including the study of virus-cell interactions and the screening of antiviral compounds.

Genetic Vectors↗
PubMed2006

In vitro treatment of human monocytes/macrophages with myristoylated recombinant Nef of human immunodeficiency virus type 1 leads to the activation of mitogen-activated protein kinases, IkappaB kinases, and interferon regulatory factor 3 and to the release of beta interferon.

The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-kappaB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the alpha and beta subunits of the IkappaB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.

Enzyme Activation↗
PubMed2006

Hypoxia inhibits Moloney murine leukemia virus expression in activated macrophages.

Hypoxia, a local decrease in oxygen tension, occurring in many pathological processes, modifies macrophage (Mphi) gene expression and function. Here, we provide the first evidence that hypoxia inhibits transgene expression driven by the Moloney murine leukemia virus-long terminal repeats (MoMLV-LTR) in IFN-gamma-activated Mphi. Hypoxia silenced the expression of several MoMLV-LTR-driven genes, including v-myc, enhanced green fluorescence protein, and env, and was effective in different mouse Mphi cell lines and on distinct MoMLV backbone-based viruses. Down-regulation of MoMLV mRNA occurred at the transcriptional level and was associated with decreased retrovirus production, as determined by titration experiments, suggesting that hypoxia may control MoMLV retroviral spread through the suppression of LTR activity. In contrast, genes driven by the CMV or the SV40 promoter were up-regulated or unchanged by hypoxia, indicating a selective inhibitory activity on the MoMLV promoter. It is interesting that hypoxia was ineffective in suppressing MoMLV-LTR-controlled gene expression in T or fibroblast cell lines, suggesting a Mphi lineage-selective action. Finally, we found that MoMLV-mediated gene expression in Mphi was also inhibited by picolinic acid, a tryptophan catabolite with hypoxia-like activity and Mphi-activating properties, suggesting a pathophysiological role of this molecule in viral resistance and its possible use as an antiviral agent.

Animals↗
PubMed2006

HIV-1 Nef protects human-monocyte-derived macrophages from HIV-1-induced apoptosis.

HIV-1 Nef is the regulatory protein expressed earliest and most abundantly in the infection cycle. Its expression has been correlated with a plethora of effects detectable either in producer, target, and bystander cells, as well as in the viral particles. Even if the relationship between Nef expression and apoptosis has been already matter of investigation in infected lymphocytes, whose resistance to HIV infection is however limited to few days, this remains to be investigated in cells that in vivo well resist the HIV cytopathic effect. In such an instance, we were interested in establishing whether Nef influences the apoptotic processes in primary human-monocyte-derived macrophages (MDM). High efficiency HIV-1 infection of MDM allowed us to establish that virus-expressed Nef strongly counteracts the HIV-1-induced apoptosis. The Nef mutant analysis suggested that this effect relies on the interaction with different protein partners and cell compartments. We also observed that the Nef protection to the HIV-1-induced apoptosis correlated with the hyper-phosphorylation and consequent inactivation of the pro-apoptotic Bad protein. On the basis of these results, we propose the Nef anti-apoptotic effect as a relevant part of the mechanism of the in vivo establishment of the HIV macrophage reservoirs.

Adult↗
PubMed2005

Selective elimination of HIV-1-infected cells by Env-directed, HIV-1-based virus-like particles.

We recently showed that both replicating and resting cells cultivated with ganciclovir (GCV) were killed when challenged with vesicular stomatitis virus G glycoprotein pseudotyped HIV-1-based virus-like particles (VLPs) carrying the Nef7 (i.e., an HIV-1 Nef mutant incorporating in virions at high levels)/herpes simplex virus-1 thymidine kinase (HSV-TK) fusion product. On this basis, a novel anti-HIV therapeutic approach based on Nef7/TK VLPs expressing X4 or R5 HIV cell receptor complexes has been attempted. We here report that (CD4-CXCR4) and (CD4-CCR5) Nef7-based VLPs efficiently enter cells infected by X4- or R5-tropic HIV-1 strains, respectively. Importantly, the delivery of the VLP-associated Nef7/TK led to cell death upon GCV treatment. Of interest, VLPs were effective also against non-replicating, HIV-1-infected primary human monocyte-derived macrophages. HIV-targeted VLPs represent a promising candidate for the treatment of persistently HIV-1-infected cells that are part of virus reservoirs resistant to HAART therapies.

Adult↗
PubMed2005

HIV-1 Nef regulates the release of superoxide anions from human macrophages.

The NADPH oxidase enzymatic complex participates in the oxidative burst by producing ROS (reactive oxygen species). Altered levels of ROS production may have pathogenetic implications due to the loss of some innate immune functions such as oxidative burst and phagocytosis. Considering that HIV-1 Nef protein plays a primary role in AIDS pathogenesis, by affecting the immune system, we sought to dissect possible effects of Nef on the release of superoxide anions. We show here that the inducible expression of Nef in human phagocytic cells modulates the superoxide release in a biphasic manner. In particular, an early Nef-induced increase of the superoxide release was followed by a dramatic decrease starting from 10 h after the Nef induction. This was observed whatever the presence of cell activators such as GM-CSF (granulocyte/macrophage colony-stimulating factor) or fMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine). Whereas the early increase in superoxide release is probably the result of the already described Nef-dependent activation of PAK-2 (p21-activated kinase 2)-Rac2, we were interested in investigating the mechanisms underlying the late inhibition of superoxide release observed originally. In this regard, we individuated at least three independent requirements for the Nef-induced blockade of superoxide release: (i) the active protein synthesis; (ii) both the membrane localization and the interaction with endocytotic machinery of Nef; and (iii) the release of soluble factor(s). Moreover, we observed that IL-10 (interleukin-10) inhibits superoxide release, whereas its depletion restored NADPH oxidase activity. We propose that the cell membrane-to-lysosome Nef transit leads to the synthesis and release of soluble factor(s) and, among them, IL-10 might significantly contribute to the inhibition of NAPDH oxidase activity.

CD4 Antigens↗
PubMed2005

Cell death induced by the herpes simplex virus-1 thymidine kinase delivered by human immunodeficiency virus-1-based virus-like particles.

HIV-1 Nef incorporates into virions at low levels, likely about 10 molecules per viral particle. Here, we describe a Nef mutant (Nef7) apparently showing more than 100-fold higher efficiency of virion incorporation. Interestingly, Nef7 can act as a cargo molecule for protein delivery into the cells, as its virion incorporation appeared conserved even upon C-terminal fusion with proteins of up to 30 kDa. This was demonstrated first by assessing the intracellular fluorescence of cells challenged with lentivirus-based virus-like particles (VLPs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) and incorporating Nef7 fused with the green fluorescent protein. Furthermore, the biologic activity of products delivered by Nef7-based VLPs was demonstrated by tagging Nef7 with the herpes simplex virus-1 thymidine kinase (HSV-1 TK). In fact, we observed that both cell lines and primary human macrophages challenged with (VSV-G) Nef7/TK VLPs died after 5 to 7 days of treatment with ganciclovir (GCV). In sum, our findings support the notion that Nef7-based VLPs can be considered platforms for original systems of protein delivery. In particular, the here- described Nef7/TK VLPs represent a first applicative example opening the way toward new HSV-1 TK/GCV-based cell suicide therapies circumventing cell gene engineering procedures.

Blotting, Western↗
PubMed2005

T Lymphocytes transduced with a lentiviral vector expressing F12-Vif are protected from HIV-1 infection in an APOBEC3G-independent manner.

The viral infectivity factor (Vif) is an essential component of the HIV-1 infectious cycle. Vif counteracts the action of the cytidine deaminase APOBEC3G (AP3G), which confers nonimmune antiviral defense against HIV-1 to T lymphocytes. Disabling or interfering with the function of Vif could represent an alternative therapeutic approach to AIDS. We have expressed a natural mutant of Vif, F12-Vif, in a VSV-G-pseudotyped lentiviral vector under the Tat-inducible control of the HIV-1 LTR. Conditional expression of F12-Vif prevents replication and spreading of both CXCR4 and CCR5 strains of HIV-1 in human primary T lymphocyte and T cell lines. T cells transduced with F12-Vif release few HIV-1 virions and with reduced infectivity. Several lines of evidence indicate that HIV-1 interference requires the presence of both wild-type and F12-Vif proteins, suggesting a dominant-negative feature of the F12-Vif mutant. Surprisingly, however, the F12-Vif-mediated inhibition does not depend on the reestablishment of the AP3G function.

APOBEC-3G Deaminase↗
PubMed2004

HIV-1 Nef enhances both membrane expression and virion incorporation of Env products. A model for the Nef-dependent increase of HIV-1 infectivity.

The expression of human immunodeficiency virus Nef increases the viral infectivity through mechanisms still not fully elucidated. Here we report that wild-type (wt) human immunodeficiency virus, type 1 (HIV-1), particles were neutralized by higher concentrations of either anti-Env glycoprotein (gp) 41 antibodies or recombinant soluble human CD4 compared with Deltanef HIV-1. This appeared to be the result of a Nef-induced increase of virion incorporation of both gp41 (transmembrane (TM)) and surface gp120 Env products likely originating from enhanced steady-state levels of cell membrane-associated Env products. This, in turn, seemed to be the consequence of a reduced retention of the Env precursor. Most interesting, we found that both the Nef-directed increase of Env membrane expression and the Nef-induced enhancement of HIV-1 infectivity relied on the presence of the intracytoplasmic domain of TM, supporting the hypothesis of a functional correlation between these effects. Mutagenesis studies allowed us to establish that the two leucine residues at the TM C terminus, which are part of a sorting motif involved in the control of Env membrane expression, and the 181-210-residue Nef C-terminal region were critically involved in the Nef/Env functional interaction. In conclusion, we propose that Nef increases the infectivity of HIV-1 at least in part by enhancing the amounts of Env products incorporated into virus particles.

Gene Expression Regulation, Viral↗
PubMed2004

Inhibition of HIV-1 replication in monocyte-derived macrophages by Mycobacterium tuberculosis.

Controversial results have been obtained in studies of the effect of Mycobacterium tuberculosis on human immunodeficiency virus type 1 (HIV-1) replication in cells of the macrophage lineage. In the present study, monocyte-derived macrophages (MDMs), previously incubated for 2 days with heat-inactivated M. tuberculosis, were infected with HIV-1. M. tuberculosis consistently inhibited viral replication, and a similar result also was observed in the presence of supernatants from M. tuberculosis-stimulated MDMs, which indicates that this effect was mediated by soluble factors. Although CCR5-binding chemokines were induced by M. tuberculosis stimulation, the results of neutralization experiments indicated that it is unlikely that they were responsible for viral suppression. Inhibition occurred mainly after viral entry (demonstrated by use of a vesicular stomatitis virus G-pseudotyped HIV-1 and by analysis of HIV-1 early and late reverse-transcription products). Therefore, M. tuberculosis-induced factors may inhibit in vitro HIV-1 replication in macrophages by affecting an early postentry step in the HIV-1 cycle.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Targeting the Nef induced increase of HIV infectivity.

Nef is a regulatory protein expressed exclusively by HIV-1/2 and SIV. It is critical for the optimal viral infectivity and for the destruction of the host immune system. This renders Nef a rather attractive therapeutic target. The most affordable point(s) of attack for effective anti-Nef therapeutic strategies can be individuated on the basis of a detailed knowledge of the mechanisms underlying the Nef induced enhancement of infectivity. However, the emerging picture still appears rather complex and not always coherent, so that additional, intensive endeavours in basic research are required for an effective exploitation of the Nef induced increase of infectivity as a therapeutic target.

Animals↗
PubMed2003

Human immunodeficiency virus type 1 (HIV-1) Nef activates STAT3 in primary human monocyte/macrophages through the release of soluble factors: involvement of Nef domains interacting with the cell endocytotic machinery.

Increasing evidence indicates that the expression of the human immunodeficiency virus-1 (HIV-1) Nef protein significantly influences the activation state of the host cell. Here we report that Nef specifically activates STAT3 in primary human monocyte-derived macrophages (MDM). This was demonstrated by both single-cycle infection experiments driven by Vesicular Stomatitis virus glycoprotein (VSV-G) pseudotyped HIV-1 and treatment with exogenous recombinant Nef. The analysis of the effects of Nef mutants revealed that domains of the C-terminal flexible loop interacting with the cell endocytotic machinery are involved in the STAT3 activation. In particular, our data suggest that the Nef-dependent STAT3 activation relies on the targeting of Nef to the late endosome/lysosome compartment. In addition, we found that Nef activates STAT3 through a mechanism mediated by the release of soluble factor(s), including MIP-1alpha, that requires de novo protein synthesis but appears independent from the activation of src tyrosine kinases. The results presented here support the idea that the first intervention of Nef in the intracellular signaling of monocyte-macrophages could generate, by means of the release of soluble factor(s), a secondary wave of activation that could be of a potential pathogenetic significance.

Acute-Phase Proteins↗
PubMed2003

HIV-1 Nef induces the release of inflammatory factors from human monocyte/macrophages: involvement of Nef endocytotic signals and NF-kappa B activation.

It has been recently reported that the endogenous expression of HIV-1 Nef in human monocyte/macrophages induces the release of chemokines and other as yet unidentified soluble factors leading to multiple effects of pathogenic significance, such as the recruitment and activation of quiescent lymphocytes. However, the description of underlying molecular mechanisms remained elusive. We recently demonstrated that human monocyte-derived macrophages (MDM) efficiently internalize soluble rNef, thereby inducing effects largely resembling those observed in cells endogenously expressing Nef. By exploiting the rNef/MDM model, we sought to gain more insights on the molecular mechanisms underlying the response of MDM to Nef. Array analysis for the detection of transcripts from a large number of monokines, chemokines, cytokines, and receptors thereof showed that MDM promptly responded to rNef treatment by increasing the transcription of genes for several inflammatory factors. Analysis of supernatants revealed that rNef treatment induced the release of macrophage inflammatory proteins 1alpha and 1beta, IL-1beta, IL-6, and TNF-alpha. Conversely, rNefs mutated in domains critical for the interaction with the endocytotic machinery (i.e., EE155-156QQ, and DD174-175AA) were ineffective. Interestingly, we found that the Nef-dependent release of inflammatory factors correlated with the activation of the NF-kappaB transcription factor, mainly in its p50/p50 homodimeric form, and in a de novo protein synthesis-independent manner. Our data add new hints supporting the idea that the presence of Nef is per se heavily detrimental for monocyte/macrophages and relative cross-talking cell types.

Adult↗
PubMed2003

Vav exchange factor counteracts the HIV-1 Nef-mediated decrease of plasma membrane GM1 and NF-AT activity in T cells.

Several findings support the importance of GM1-enriched lipid microdomains of plasma membrane and of Vav, an essential regulator of actin cytoskeletal rearrangement, in the regulation of T cell activation. Moreover, a functional link among lipid microdomains, Vav and the HIV product Nef has been described. These observations suggest that Nef can modify plasma membrane GM1, affecting the behavior of HIV-infected cells towards antigen recognition and Vav towards counteracting such an effect. We observed that Nef expression, either following viral infection or ectopic expression, significantly decreased the level of plasma membrane GM1 in unstimulated T cells. This down-regulation was associated with the inhibition of NF-AT activation, but not with NF-kappaB activation induced by TCR engagement. Dissecting the signaling pathway that regulates NF-AT activation, we found that Nef inhibited exclusively the Ca(2+)/calcineurin cascade, whereas the JNK cascade and AP-1 transcriptional activity were not affected. Our evidence that Vav overexpression counteracted both the Nef-induced decrease of GM1 expression and the inhibition of NF-AT activity, suggests a novel mechanism by which Nef may interfere with TCR-mediated activation through the modulation of intracellular trafficking and clustering of GM1-enriched microdomains at the cell surface.

Calcium Signaling↗
PubMed2002

Inducible expression of the deltaNGFr/F12Nef fusion protein as a new tool for anti-human immunodeficiency virus type 1 gene therapy.

Expression of the human immunodeficiency virus type 1 (HIV-1) Nef triple mutant F12Nef strongly inhibits HIV-1 replication. We exploited such a unique feature in a novel anti-HIV-1 gene therapy design by constructing an HIV-1 Tat-defective lentivirus vector expressing the product of fusion between the low-affinity human nerve growth factor receptor truncated in its intracytoplasmic domain (deltaNGFr, NH(2) moiety), and F12Nef (COOH moiety), under the control of the HIV-1 long terminal repeats. In this manner, both the selection marker (deltaNGFr) and the anti-HIV-1 effector are comprised in the same fusion protein, the expression of which is targetable by HIV-1 infection. Such a vector was proved to transduce human cells efficiently and, on HIV-1 infection, it expressed high levels of the fusion protein. In addition, strong antiviral activity of the deltaNGFr/F12Nef-expressing vector was demonstrated in cell lines as well as in primary cell cultures challenged with T- or M-tropic HIV-1 isolates. Thus, the HIV-1-targetable expression of the deltaNGFr/F12Nef fusion protein represents a novel and powerful tool for an effective anti-HIV-1 gene therapy strategy.

CD4-Positive T-Lymphocytes↗
PubMed2001

Genetic and functional analysis of the human immunodeficiency virus (HIV) type 1-inhibiting F12-HIVnef allele.

The primary human immunodeficiency virus type 1 (HIV-1) Nef mutant F12-HIVNef is characterized by three rare amino acid substitutions, G(140)E, V(153)L and E(177)G. It was reported previously that the expression of F12-HIVNef in the context of the highly productive NL4-3 HIV-1 strain blocks virus replication at the level of virus assembly and/or release by a mechanism depending on the presence of the CD4 intracytoplasmic tail. Here, it is reported that NL4-3 HIV-1 strains expressing F12-HIVnef alleles that were back-mutated in each amino acid substitution readily replicated in CD4(+) cells. Attempting to correlate possible functional alterations with antiviral effects, both F12-HIVNef and its back mutants were tested in terms of well-characterized markers of Nef expression. Both F12-HIVNef and its G(177)E back mutant did not down-regulate CD4 as the consequence of a greatly reduced rate of CD4 internalization. On the other hand, F12-HIVNef as well as the E(140)G and L(153)V back mutants failed to activate the p62 Nef-associated kinase (p62NAK). Thus, only F12-HIVNef was defective in both accelerated rates of CD4 internalization and p62NAK activation, whereas at least one Nef function was restored in all of the back mutants. Infection of cells expressing Nef-resistant CD4 molecules with HIV-1 strains encoding F12-HIVNef back mutants showed that both the lack of accelerated CD4 endocytosis and an, as yet, still unidentified function are required for the F12-HIVNef inhibitory phenotype. These results provide a detailed functional analysis of the F12-HIVnef allele and support the idea that both CD4 accelerated internalization and p62NAK activation are part of the essential steps in the virus replication cycle.

Alleles↗
PubMed2000

T-tropic human immunodeficiency virus (HIV) type 1 Nef protein enters human monocyte-macrophages and induces resistance to HIV replication: a possible mechanism of HIV T-tropic emergence in AIDS.

Increasing interest has been devoted to the role that monocyte-macrophages play in the pathogenesis of AIDS. The hypothesis of an involvement in AIDS pathogenesis of human/simian immunodeficiency virus (HIV/SIV) Nef also is currently under evaluation by many investigators. The original basis of this hypothesis came from evidence that monkeys infected with a nef-deleted SIV strain failed to develop simian AIDS. Here, we show that treatment of human monocyte-derived macrophages (MDM) with recombinant HIV-1 Nef protein (rNef) induces a strong inhibition of the replication of either macrophage (M-) or dual-tropic HIV-1 strains. Through cytofluorimetric analyses, we detected internalization of FITC-conjugated rNef in MDM as early as 6 h after treatment. Confocal microscope observations demonstrated that the intracellular distribution of internalized rNef was identical to that of endogenously produced Nef. Down-regulation of the CD4 HIV receptor detected upon rNef treatment of MDM suggested that the rNef-induced HIV inhibition occurred at the virus entry step. This deduction was strengthened by the observation that CD4-independent infection was totally insensitive to rNef treatment. The specificity of all observed effects was demonstrated by immunodepletion of rNef. Finally, we showed that the resistance to HIV replication induced by rNef treatment in MDM favours the spread of T-tropic over M-tropic HIV strains in doubly infected CD4(+) lymphocyte-MDM co-cultures. We propose that extracellular Nef contributes to AIDS pathogenesis by inducing resistance to M-tropic HIV replication in MDM, thereby facilitating the switching from M- to T-tropic HIV prevalence that correlates frequently with AIDS progression.

Acquired Immunodeficiency Syndrome↗