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We previously developed an innovative strategy to induce CD8+T lymphocyte-immunity through in vivo engineering of extracellular vesicles (EVs). This approach relies on intramuscular injection of DNA expressing antigens of interest fused at a biologically-inactive HIV-1 Nef protein mutant (Nefmut). Nefmutis very efficiently incorporated into EVs, thus conveying large amounts of fusion proteins into EVs released by transfected cells. This platform proved successful against highly immunogenic tumor-specific antigens. Here, we tested whether antigen-specific CD8+T cell immune responses induced by engineered EVs can counteract the growth of tumors expressing two “self” tumor-associated antigens (TAAs): HOXB7 and Her2/neu. FVB/N mice were injected with DNA vectors expressing Nefmutfused to HOXB7 or Her2/neu, singly and in combination, before subcutaneous implantation of breast carcinoma cells co-expressing HOXB7 and Her2/neu. All mice immunized with the combination vaccine remained tumor-free, whereas groups vaccinated with single Nefmut-fused antigens were only partly protected, with stronger antitumor effects in Her2/neu-immunized mice. Double-vaccinated mice also controlled tumor growth upon a later tumor cell re-challenge. Importantly, co-vaccination also contained tumors in a therapeutic immunization setting. These results showed the efficacy of EV-based vaccination against two TAAs, and represent the first demonstration that HOXB7 may be targeted in multi-antigen immunotherapy strategies.

Background: Eukaryotic cells release vesicles of different sizes under both physiological and pathological conditions. On the basis of the respective biogenesis, extracellular vesicles are classified as apoptotic bodies, microvesicles, and exosomes. Among these, exosomes are considered tools for innovative therapeutic interventions, especially when engineered with effector molecules. The delivery functions of exosomes are favored by a number of typical features. These include their small size (i.e., 50-200 nm), the membrane composition tightly similar to that of producer cells, lack of toxicity, stability in serum as well as other biological fluids, and accession to virtually any organ and tissue including central nervous system. However, a number of unresolved questions still affects the possible use of exosomes in therapy. Among these are the exact identification of both in vitro and ex vivo produced vesicles, their large-scale production and purification, the uploading efficiency of therapeutic macromolecules, and the characterization of their pharmacokinetics. Objective: Here, we discuss two key aspects to be analyzed before considering exosomes as a tool of delivery for the desired therapeutic molecule, i.e., techniques of engineering, and their in vivo biodistribution/ pharmacokinetics. In addition, an innovative approach aimed at overcoming at least part of the obstacles towards a safe and efficient use of exosomes in therapy will be discussed. Conclusion: Several biologic features render exosomes an attractive tool for the delivery of therapeutic molecules. They will surely be a part of innovative therapeutic interventions as soon as few still unmet technical hindrances will be overcome.

Nef is an accessory protein expressed exclusively in primate lentiviruses. It is devoid of enzymatic activities while interacting with several cell proteins as an adaptor/scaffold protein. Intracellular functions of Nef largely account for many pathogenic effects observed in AIDS disease. Nef, despite lacking known secretory pathways, can be detected in plasma of HIV-1-infected patients at the concentration varing from 5 to 10 ng/ml. Remarkably, the levels of Nef in plasma of HIV patients do not correlate with viral load or number of CD4+ T lymphocytes, and persist during antiretroviral therapy. Here, we review literature data describing how Nef can be transmitted from HIV-1- infected cells to bystander ones, and the effects of extracellular Nef in different cell types. Overall, large part of experimental evidences supports the idea that extracellular Nef plays a relevant role in AIDS pathogenesis. Hence, efforts focused on the identification of Nef-inhibiting drugs would be of relevance to establish new therapeutic approaches supporting current antiretroviral therapies.

The identification of still unrevealed mechanisms affecting the anti-HIV CD8T-cell response in HIV-1 infection.

Human immunodeficiency virus (HIV) starts to replicate upon virus-cell fusion mediated by the CD4-Envcoreceptor interaction. HIV enters target cells also through endocytosis, a mechanism which rarely leads to HIV replication. Both modes of entry are greatly improved by cell-cell contact. We recently reported that the contact of human primary dendritic cells with HIV-1 infected cells leads to high levels of virus endocytosis and HIV-1 antigen presentation activity in dendritic cells. Here, we provide evidence that the activity of matrix metalloproteinase (MMP)-9 is involved in the mechanism of cell-to-cell HIV-1 endocytosis in DCs. Accordingly, the specific inhibition of MMP-9 led to reduced extents of HIV-1 antigen presentation activity. The identification of cell molecules involved in the cell-to-cell HIV-1 endocytosis would be of significance for better understanding the mechanisms underlying the induction of the anti-HIV adaptive immune response.

AIDS is generally considered to be an immune system disease with a viral etiology. Interestingly, a single HIV-1 product, specifically Nef, recapitulates, at least in part, the pathogenetic effects of the infection. HIV-1 Nef is a 27-kDa regulatory protein lacking any enzymatic activity, but that interacts with several cell proteins, such as an adaptor protein. A surprisingly high number of functions are attributed to Nef, and this review seeks to summarize the most relevant effects of Nef that result from the alteration of both molecular trafficking and intracellular signaling in the host cell. The overall outcome is an increase in viral replication and the impairment of both innate and adaptive immune responses. Unfortunately, although Nef plays a relevant role in AIDS pathogenesis, no Nef-inhibiting drugs are currently available. Thus, finding anti-Nef compounds would contribute to innovative anti-HIV therapeutic approaches.

Human immunodeficiency virus (HIV)-1 Nef is a regulatory protein critically involved in AIDS pathogenesis. We previously demonstrated that extracellular Nef is efficiently internalized by human primary monocyte-derived macrophages (MDMs), thereby activating a number of transcription factors including STATs, MAPKs, IRF-3, and NF-κB. Such an activation state leads to the release of inflammatory factors whose paracrine effects deserve deep consideration. Here, we demonstrate that quiescent CD4 lymphocytes undergo cell activation when cultivated in supernatants from autologous MDMs treated with extracellular wt Nef but not with its counterpart mutated in the 72PxxP75 polyproline domain. Of a pathogenetic relevance, this effect coupled with the sensitization of quiescent CD4 lymphocytes to HIV-1 infection. By microarray assay, we found that the CCL24/eotaxin-2 gene was up-regulated in MDMs treated with wt Nef but not with the 72AxxA75 mutant. In addition, the higher transcription activity correlated with a significant increase of the CCL24/Eotaxin-2 release. Finally, we observed that anti-CCL24/eotaxin-2 antibodies efficiently neutralized the stimulatory effect on CD4 lymphocytes of supernatants from MDMs treated with extracellular Nef. Overall, these data support the idea that CCL24/eotaxin-2 is part of the mechanism of CD4 lymphocyte activation paracrinally induced by Nef.

Hypoxia, a local decrease in oxygen tension, occurring in many pathological processes, modifies macrophage (Mφ) 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‐γ‐activated Mφ. 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 Mφ 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 Mφ lineage‐selective action. Finally, we found that MoMLV‐mediated gene expression in Mφ was also inhibited by picolinic acid, a tryptophan catabolite with hypoxia‐like activity and Mφ‐activating properties, suggesting a pathophysiological role of this molecule in viral resistance and its possible use as an antiviral agent.

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.

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.

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‐1α, that requires de novo protein synthesis but appears independent from the activation of srctyrosine 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.

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 (ΔNGFr, NH2 moiety), and F12Nef (COOH moiety), under the control of the HIV-1 long terminal repeats. In this manner, both the selection marker (ΔNGFr) 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 ΔNGFr/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 ΔNGFr/F12Nef fusion protein represents a novel and powerful tool for an effective anti-HIV-1 gene therapy strategy.

Monocytes/macrophages play a predominant role in the immunologic network by secreting and reacting to a wide range of soluble factors. Human immunodeficiency virus (HIV) infection leads to deep immunologic dysfunctions, also as a consequence of alterations in the pattern of cytokine release. Recent studies on in vivo models demonstrated that the expression of HIV Nef alone mimics many pathogenetic effects of HIV infection. In particular, Nef expression in monocytes/macrophages has been correlated with remarkable modifications in the pattern of secreted soluble factors, suggesting that the interaction of Nef with monocytes/macrophages plays a role in the pathogenesis of acquired immunodeficiency syndrome (AIDS). This study sought to define possible alterations in intracellular signaling induced by Nef in monocytes/macrophages. Results demonstrate that HIV-1 Nef specifically activates both α and β isoforms of the signal transducer and activator of transcription 1 (STAT1). This was observed both by infecting human monocyte-derived macrophages (MDMs) with HIV-1 deletion mutants, and by exploiting the ability of MDMs to internalize soluble, recombinant Nef protein (rNef). STAT1-α activation occurs on phosphorylation of both C-terminal Tyr701 and Ser727 and leads to a strong binding activity. Nef-dependent STAT1 activation is followed by increased expression of both STAT1 and interferon regulatory factor-1, a transcription factor transcriptionally regulated by STAT1 activation. It was also established that Nef-induced STAT1- α/β activation occurs through the secretion of soluble factors. Taken together, the results indicate that HIV-1 Nef could interfere with STAT1-governed intracellular signaling in human monocytes/macrophages.

ABSTRACTHuman immunodeficiency virus type 1F12(HIV-1F12) interferes with the replication of other strains of HIV. Its accessory protein, Nef, is sufficient for this phenotype, where the production and infectivity of HIV are impaired significantly. The analysis of three rare mutations in this Nef protein revealed that these effects could be separated genetically. Moreover, the defect in virus production correlated with the lack of processing of the p55Gagprecursor in the presence of Nef from HIV-1F12. Importantly, the introduction of one of these mutations (E177G) into Nef from HIV-1NL4-3also created a dominant-negative Nef protein. Effects of Nef from HIV-1F12on virus production and Gag processing correlated with its altered subcellular distribution. Moreover, the association with two new cellular proteins with molecular masses of 74 and 75 kDa, which do not interact with other Nef proteins, correlated with the decreased virion infectivity. The identification of a dominant-negative protein for the production and infectivity of HIV suggests that Nef plays an active role at this stage of the viral replicative cycle.

ABSTRACTJurkat T-cell clones, stably expressing the human immunodeficiency virus type 1 (HIV-1) Vpr protein, exhibited an impaired susceptibility to HIV-1 infection. A marked down-modulation of surface CD4 receptors was detected in Vpr-expressing clones with respect to control cells. Likewise, a reduced CD4 expression was also observed in parental Jurkat cells infected with wild-type but not with Vpr-mutant HIV-1. Notably, Vpr-expressing clones were fully susceptible to infection with a vesicular stomatitis virus G protein-pseudotyped HIV-1 virus, indicating that a block at the level of viral entry was responsible for the inhibition of viral replication. The effect exerted by Vpr on HIV replication and CD4 expression suggests that this protein can regulate both the establishment of a productive HIV-1 infection and CD4-mediated T-cell functions.

ABSTRACTF12 human immunodeficiency virus type 1 (HIV-1) nefis a naturally occurring nefmutant cloned from the provirus of a nonproductive, nondefective, and interfering HIV-1 variant (F12-HIV). We have already shown that cells stably transfected with a vector expressing the F12-HIV nefallele do not downregulate CD4 receptors and, more peculiarly, become resistant to the replication of wild type (wt) HIV. In order to investigate the mechanism of action of such an HIV inhibition, the F12-HIVnefgene was expressed in the context of the NL4-3 HIV-1 infectious molecular clone by replacing the wt nefgene (NL4-3/chi). Through this experimental approach we established the following. First, NL4-3/chi and nef-defective (?nef) NL4-3 viral particles behave very similarly in terms of viral entry and HIV protein production during the first replicative cycle. Second, no viral particles were produced from cells infected with NL4-3/chi virions, whatever the multiplicity of infection used. The viral inhibition apparently occurs at level of viral assembling and/or release. Third, this block could not be relieved by in-transexpression of wt nef. Finally, NL4-3/chi reverts to a producer HIV strain when F12-HIV Nef is deprived of its myristoyl residue. Through a CD4 downregulation competition assay, we demonstrated that F12-HIV Nef protein potently inhibits the CD4 downregulation induced by wt Nef. Moreover, we observed a redistribution of CD4 receptors at the cell margin induced by F12-HIV Nef. These observations strongly suggest that F12-HIV Nef maintains the ability to interact with the intracytoplasmic tail of the CD4 receptor molecule. Remarkably, we distinguished the intracytoplasmic tails of Env gp41 and CD4 as, respectively, viral and cellular targets of the F12-HIV Nef-induced viral retention. For the first time, the inhibition of the viral life cycle by means of in-cisexpression of a Nef mutant is here reported. Delineation of the F12-HIV Nef mechanism of action may offer additional approaches to interference with the propagation of HIV infection.

An HUT-78 cell clone (F12) chronically infected by a nonproducer HIV-1 variant (Federico et al., (1989) AIDS Res. Hum. Retroviruses 5, 385-396) is fully resistant to superinfection with HIV-1 or HIV-2. We demonstrate that, in spite of the down-regulation of CD4 receptors, superinfecting-HIV-1 and -HIV-2 cross the F12 plasma membrane (even in the presence of OKT4A monoclonal antibodies) but fail to complete retrotranscription. We utilized a series of polymerase chain reaction primers designed to detect certain steps in the reverse transcription process. Superinfecting-HIV-1 (an African strain) and -HIV-2 are detectable using primers specific for env (for HIV-1), 5' LTR (the R and U5 regions), and vpx (for HIV-2). No amplification is visible when primers amplifying either HIV-2 gag or the "primer binding site" region of 5' LTR of HIV-2 are used. DNA-PCR performed on DNAse-pretreated HIV-1 and HIV-2 stocks failed to show any amplification. This rules out that any extra- or intravirion vital DNA contamination may have interfered with our results. In addition, no DNA amplification was observed in F12 and HUT-78 cells exposed to heat-inactivated HIV-2. Finally, when the nonproducer F12 cells as well as control CEMss cells are transfected with the HIV-1 infectious molecular clone pNL4-3, progeny infectious virus is obtained. These findings indicate that reverse transcription of HIVs superinfecting F12 cells is prevented from completing viral DNA synthesis. A similar block occurs in HIV-1-infected producer cells. When integration of the HIV genome into the F12 genome is achieved via transfection of a molecular clone, the virus life cycle can proceed as in control CEMss cells. Copyright 1993, 1999 Academic Press

Recent findings have shown that the expression of the seven trans-membrane G-protein–coupled CXCR4 (the receptor for the stromal cell-derived factor [SDF]-1 chemokine) is necessary for the entry of T-lymphotropic human immunodeficiency virus (HIV) strains, acting as a coreceptor of the CD4 molecule. In the human system, the role of CXCR4 in HIV infection has been determined through env-mediated cell fusion assays and confirmed by blocking viral entry in CD4+/CXCR4+ cells by SDF-1 pretreatment. We observed that the human megakaryoblastic CD4+ UT-7 cell line fails to express CXCR4 RNA and is fully resistant to HIV entry. Transfection of an expression vector containing the CXCR4 c-DNA rendered UT-7 cells readily infectable by different T-lymphotropic syncytium-inducing HIV-1 and HIV-2 isolates. Interestingly, HIV-1 infection of CXCR4 expressing UT-7 cells (named UT-7/fus) induces the formation of polynucleated cells through a process highly reminiscent of megakaryocytic differentiation and maturation. On the contrary, no morphologic changes were observed in HIV-2–infected UT-7/fus cells. These findings further strengthen the role of CXCR4 as a molecule necessary for the replication of T-lymphotropic HIV-1 and HIV-2 isolates and provide a useful model to study the functional role of CD4 coreceptors in HIV infection.

ABSTRACTWe previously demonstrated that expression of the nonproducer F12-human immunodeficiency virus type 1 (HIV-1) variant induces a block in the replication of superinfecting HIV that does not depend on the down-regulation of CD4 HIV receptors. In order to individuate the gene(s) involved in F12-HIV-induced interference, vectors expressing each of the nine F12-HIV proteins were transfected in HIV-susceptible HeLa CD4 cells. Pools of cell clones stably producing each viral protein were infected with HIV-1, and virus release was measured in terms of reverse transcriptase activity in supernatants. We hereby demonstrate that HeLa CD4 cells expressing the F12-HIV gag, vif, ornefgene were resistant, to different degrees, to infection with T-cell-line-adapted HIV-1 strains. Conversely, expression of either the tat, rev, or vpuF12-HIV gene increased the rate of HIV release, and no apparent effects on HIV replication were observed in cells expressing either the F12-HIVvpr, pol, or envgene. No variation of CD4 exposure was detected in any of the uninfected HeLa CD4 pools. These data indicate that F12-HIV homologous viral interference is the consequence of the synergistic anti-HIV effects of Gag, Vif, and Nef proteins. Retrovirus vectors expressing F12-HIV vifornefallowed us to further establish that the expression of each mutated protein (i) inhibits the replication of clinical HIV-1 isolates as well, (ii) impairs the infectivity of the virus released by cells chronically infected with HIV-1, and (iii) limitedly to F12-HIV Vif protein, induces HIV resistance in both vif-permissive and vif-nonpermissive cells. The levels of action of F12-HIV vifand nefanti-HIV effects were also determined. We observed that HIV virions emerging from the first viral cycle on F12-HIV vif-expressing cells, although released in unaltered amounts, had a strongly reduced ability to initiate the retrotranscription process when they reinfected parental HeLa CD4 cells. Differently, we observed that expression of F12-HIV Nef protein affects the HIV life cycle at the level of viral assembling and/or release. For the first time, an inhibitory effect on the HIV life cycle in both acutely and chronically infected cells induced by mutated Vif and Nef HIV-1 proteins is described. These genes could thus be proposed as new useful reagents for anti-HIV gene therapy.

HIV-1 belongs to the Lentiviridae subfamily of the Retroviridae virus family. Lentiviruses (from Latin lenti, slow, for the long incubation times of the diseases) are enveloped viruses carrying two copies of single-strand positive (i.e., codifying) RNA and are considered the etiologic agents of acquired immunodeficiency syndromes from a broad range of animal species. Most likely as the consequence of the adaptation of the viruses to the respective natural hosts, lentiviruses evolved a set of genes coding for non structural proteins. These, defined as regulatory or accessory genes, are six in the case of HIV-1, and termed, starting from the 5' end of the genome, vif, vpr, tat, rev, vpu and nef. Even if in in vitro systems the virus survives the loss of function of each of these genes, except tat and rev, the fact that the far most part of in vivo HIV-1 isolates expresses the whole set of functional regulatory proteins strongly suggests that they play functions critical for the in vivo propagation. Ho wever, while in the case of vif, tat and rev the mechanisms underlying their pivotal functions have been revealed in great detail, the roles of vpr, vpu and nef in the viral replication appear at the present more intriguing. In this respect, the case of nef is paradigmatic. This protein, evolved exclusively in SIV and HIV lentiviruses, has been found interacting with a surprisingly high number of cell proteins and also with Gag and Env HIV-1 products. This led the investigators to envision different models that, while at least in part reflecting the ability of HIV to replicate in different cell types and in different cell activation states, appear however not always consistent. Both the uniqueness and the complexity of the Nef mode of action suggested to pay particular attention to such a fascinating and still partly mysterious viral protein in this issue. Anti-HIV compounds part of the high active anti-retroviral therapy (HAART), at the present the most effective firewall against the viral spread, recognize exclusively HIV structural proteins as targets, in particular the integrase, the reverse transcriptase, and the transmembrane envelope subunit. Clearly, due to the well established limits of efficacy of HAART, expanding the drug targets towards the HIV regulatory proteins and / or the relative cell protein partners appears mandatory. In this issue, each HIV-1 regulatory protein is described in detail in terms of structure, function and strategies of pharmacological attack already proven and / or of a potential exploiting. Of course, the reader will be easily aware that the drug research developed much more efficiently for those proteins whose mechanism of action has been more accurately revealed, i.e. Tat and Rev. In the case of Vif, the recent outstanding outbreaks on its mechanism of action will certainly allow the design of innovative drugs inte rfering with its functions in a short time. On the other hand, the recovery of effective anti-HIV compounds targeting the remainder regulatory proteins will be facilitated by hopeful advancements in the knowledge of the respective effects on the viral replication. In conclusion, several lines of evidences support the idea that the HIV-1 regulatory proteins represent the heel of Achilles of the virus. In a near future, effective drugs against one or more HIV regulatory proteins could contribute in developing strategies of HIV eradication still not affordable by HAART currently in use.