Protein Delivery of Cell-Penetrating Zinc-Finger Activators Stimulates Latent HIV-1-Infected Cells.

Despite efforts to develop effective treatments for eradicating HIV-1, a cure has not yet been achieved. Whereas antiretroviral drugs target an actively replicating virus, latent, nonreplicative forms persist during treatment. Pharmacological strategies that reactivate latent HIV-1 and expose cellular reservoirs to antiretroviral therapy and the host immune system have, so far, been unsuccessful, often triggering severe side effects, mainly due to systemic immune activation. Here, we present an alternative approach for stimulating latent HIV-1 expression via direct protein delivery of cell-penetrating zinc-finger activators (ZFAs). Cys2-His2 zinc-fingers, fused to a transcription activation domain, were engineered to recognize the HIV-1 promoter and induce targeted viral transcription. Following conjugation with multiple positively charged nuclear localization signal (NLS) repeats, protein delivery of a single ZFA (3NLS-PBS1-VP64) efficiently internalized HIV-1 latently infected T-lymphocytes and specifically stimulated viral expression. We show that short-term treatment with this ZFA protein induces higher levels of viral reactivation in cell line models of HIV-1 latency than those observed with gene delivery. Our work establishes protein delivery of ZFA as a novel and safe approach toward eradication of HIV-1 reservoirs.

Can asymmetric post-translational modifications regulate the behavior of STAT3 homodimers?

Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous and pleiotropic transcription factor that plays essential roles in normal development, immunity, response to tissue damage and cancer. We have developed a Venus-STAT3 bimolecular fluorescence complementation assay that allows the visualization and study of STAT3 dimerization and protein-protein interactions in living cells. Inactivating mutations on residues susceptible to post-translational modifications (PTMs) (K49R, K140R, K685R, Y705F and S727A) changed significantly the intracellular distribution of unstimulated STAT3 dimers when the dimers were formed by STAT3 molecules that carried different mutations (ie they were "asymmetric"). Some of these asymmetric dimers changed the proliferation rate of HeLa cells. Our results indicate that asymmetric PTMs on STAT3 dimers could constitute a new level of regulation of STAT3 signaling. We put forward these observations as a working hypothesis, since confirming the existence of asymmetric STAT3 homodimers in nature is extremely difficult, and our own experimental setup has technical limitations that we discuss. However, if our hypothesis is confirmed, its conceptual implications go far beyond STAT3, and could advance our understanding and control of signaling pathways.

miRNA profiling of human naive CD4 T cells links miR-34c-5p to cell activation and HIV replication.

Cell activation is a vital step for T-cell memory/effector differentiation as well as for productive HIV infection. To identify novel regulators of this process, we used next-generation sequencing to profile changes in microRNA expression occurring in purified human naive CD4 T cells in response to TCR stimulation and/or HIV infection. Our results demonstrate, for the first time, the transcriptional up-regulation of miR-34c-5p in response to TCR stimulation in naive CD4 T cells. The induction of this miR was further consistently found to be reduced by both HIV-1 and HIV-2 infections. Overexpression of miR-34c-5p led to changes in the expression of several genes involved in TCR signaling and cell activation, confirming its role as a novel regulator of naive CD4 T-cell activation. We additionally show that miR-34c-5p promotes HIV-1 replication, suggesting that its down-regulation during HIV infection may be part of an anti-viral host response.

Distinct roles of N-acetyl and 5-methoxy groups in the antiproliferative and neuroprotective effects of melatonin.

Melatonin (N-acetyl-5-methoxytryptamine) is a highly pleiotropic hormone with antioxidant, antiproliferative, oncolytic and neuroprotective properties. Here, we present evidence that the N-acetyl side chain plays a key role in melatonin's antiproliferative effect in HT22 and sw-1353 cells, but it does so at the expense of antioxidant and neuroprotective properties. Removal of the N-acetyl group enhances the antioxidant and neuroprotective properties of the indole, but it can lead to toxic methamphetamine-like effects in several cell lines. Inhibition of NFkB mimicked melatonin's antiproliferative and antioxidant effects, but not neuroprotection. Our results strongly suggest that neuroprotective and antiproliferative effects of melatonin rely on different parts of the molecule and are likely mediated by different mechanisms. We also predict that melatonin metabolism by target cells could determine whether melatonin inhibits cell proliferation, prevents toxicity or induces cell death (e.g. apoptosis or autophagy). These observations could have important implications for the rational use of melatonin in personalized medicine.

Reactivation of Latent HIV-1 Expression by Engineered TALE Transcription Factors.

The presence of replication-competent HIV-1 -which resides mainly in resting CD4+ T cells--is a major hurdle to its eradication. While pharmacological approaches have been useful for inducing the expression of this latent population of virus, they have been unable to purge HIV-1 from all its reservoirs. Additionally, many of these strategies have been associated with adverse effects, underscoring the need for alternative approaches capable of reactivating viral expression. Here we show that engineered transcriptional modulators based on customizable transcription activator-like effector (TALE) proteins can induce gene expression from the HIV-1 long terminal repeat promoter, and that combinations of TALE transcription factors can synergistically reactivate latent viral expression in cell line models of HIV-1 latency. We further show that complementing TALE transcription factors with Vorinostat, a histone deacetylase inhibitor, enhances HIV-1 expression in latency models. Collectively, these findings demonstrate that TALE transcription factors are a potentially effective alternative to current pharmacological routes for reactivating latent virus and that combining synthetic transcriptional activators with histone deacetylase inhibitors could lead to the development of improved therapies for latent HIV-1 infection.

Host Factors and HIV-1 Replication: Clinical Evidence and Potential Therapeutic Approaches.

HIV and human defense mechanisms have co-evolved to counteract each other. In the process of infection, HIV takes advantage of cellular machinery and blocks the action of the host restriction factors (RF). A small subset of HIV+ individuals control HIV infection and progression to AIDS in the absence of treatment. These individuals known as long-term non-progressors (LNTPs) exhibit genetic and immunological characteristics that confer upon them an efficient resistance to infection and/or disease progression. The identification of some of these host factors led to the development of therapeutic approaches that attempted to mimic the natural control of HIV infection. Some of these approaches are currently being tested in clinical trials. While there are many genes which carry mutations and polymorphisms associated with non-progression, this review will be specifically focused on HIV host RF including both the main chemokine receptors and chemokines as well as intracellular RF including, APOBEC, TRIM, tetherin, and SAMHD1. The understanding of molecular profiles and mechanisms present in LTNPs should provide new insights to control HIV infection and contribute to the development of novel therapies against AIDS.

HIV-1 Vif protein blocks the cytidine deaminase activity of B-cell specific AID in E. coli by a similar mechanism of action.

HIV-1 Vif protein protects viral replication in non-permissive cells by inducing degradation of APOBEC3G via ubiquitination and proteasomal pathway, although new studies indicate a putative role in Vif's direct inhibition of APOBEC3G. APOBEC3G is member of a homologous family of proteins with cytidine deaminase activity expressed with characteristic tissue specificity, that in humans consist of APOBEC1, APOBEC2, APOBEC3A-H, APOBEC4 and the activation-induced deaminase (AID), a B lymphoid protein necessary for somatic hypermutation, gene conversion and class switch recombination. In this work we show that Vif can counteract AID's activity in E. coli in absence of specific eukaryotic co-factors necessary for AID induced somatic hypermutation, gene conversion and to stimulate class switch recombination in B-cells. We show that AID inhibition is mediated by a direct protein-protein interaction via unique amino acid D118 an homologous mutant responsible for the species-specific restriction of HIV-1 Vif protein existent for APOBEC3G. These results raise the hypothesis that Vif related proteins can act as a broad inhibitor of deaminase activity. Moreover as AID and Vif evolved in different cellular environments, these results may indicate that Vif related proteins might mimic cellular factors that interact with a structural conserved domain of cytidine deaminases during evolution.

HIV-1 Vif can directly inhibit apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G-mediated cytidine deamination by using a single amino acid interaction and without protein degradation.

The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G), also known as CEM-15, is a host-cell factor involved in innate resistance to retroviral infection. HIV-1 viral infectivity factor (Vif) protein was shown to protect the virus from APOBEC3G-mediated viral cDNA hypermutation. The mechanism proposed for protection of the virus by HIV-1 Vif is mediated by APOBEC3G degradation through ubiquitination and the proteasomal pathway. Here we show that in Escherichia coli the APOBEC3G-induced cytidine deamination is inhibited by expression of Vif without depletion of deaminase. Moreover, inhibition of deaminase-mediated bacterial hypermutation is dependent on a single amino acid substitution D128K that renders APOBEC3G resistant to Vif inhibition. This single amino acid was elegantly proven by other authors to determine species-specific sensitivity. Our results show that in bacteria this single amino acid substitution controls Vif-dependent blocking of APOBEC3G that is dependent on a strong protein interaction. The C-terminal region of Vif is responsible for this strong protein-protein interaction. In conclusion, our experiments suggest a complement to the model of Vif-induced degradation of APOBEC3G by bringing to relevance that deaminase inhibition can also result from a direct interaction with Vif protein.

Functional analysis of Vif protein shows less restriction of human immunodeficiency virus type 2 by APOBEC3G.

Viral infectivity factor (Vif) is one of the human immunodeficiency virus (HIV) accessory proteins and is conserved in the primate lentivirus group. This protein is essential for viral replication in vivo and for productive infection of nonpermissive cells, such as peripheral blood mononuclear cells (PBMC). Vif counteracts an antiretroviral cellular factor in nonpermissive cells named CEM15/APOBEC3G. Although HIV type 1 (HIV-1) Vif protein (Vif1) can be functionally replaced by HIV-2 Vif protein (Vif2), its identity is very small. Most of the functional studies have been carried out with Vif1. Characterization of functional domains of Vif2 may elucidate its function, as well as differences between HIV-1 and HIV-2 infectivity. Our aim was to identify the permissivity of different cell lines for HIV-2 vif-minus viruses. By mutagenesis specific conserved motifs of HIV-2 Vif protein were analyzed, as well as in conserved motifs between Vif1 and Vif2 proteins. Vif2 mutants were examined for their stability, expression, and cellular localization in order to characterize essential domains of Vif2 proteins. Viral replication in various target cells (PBMC and H9, A3.01, U38, and Jurkat cells) and infectivity in single cycle assays in the presence of APOBEC3G were also analyzed. Our results of viral replication show that only PBMC have a nonpermissive phenotype in the absence of Vif2. Moreover, the HIV-1 vif-minus nonpermissive cell line H9 does not show a similar phenotype for vif-negative HIV-2. We also report a limited effect of APOBEC3G in a single-cycle infectivity assay, where only conserved domains between HIV-1 and HIV-2 Vif proteins influence viral infectivity. Taken together, these results allow us to speculate that viral inhibition by APOBEC3G is not the sole and most important determinant of antiviral activity against HIV-2.

Phosphorylation of a novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5 complex that promotes APOBEC3G degradation.

HIV-1 Vif (viral infectivity factor) protein overcomes the antiviral activity of the DNA deaminase APOBEC3G by targeting it for proteasomal degradation. We report here that Vif targets APOBEC3G for degradation by forming an SCF-like E3 ubiquitin ligase containing Cullin 5 and Elongins B and C (Cul5-EloB-EloC) through a novel SOCS (suppressor of cytokine signaling)-box that binds EloC. Vif binding to EloC is negatively regulated by serine phosphorylation in the BC-box motif of the SOCS-box. Vif ubiquitination is promoted by Cul5 in vitro and in vivo, and requires an intact SOCS-box. Thus, autoubiquitination of Vif occurs within the assembled Vif-Cul5 complex, analogous to F-box proteins that are autoubiquitinated within their SCF (Skp1-Cullin-F-box) complex. These findings suggest mechanisms that regulate the assembly and activity of Cul5 E3 complexes through phosphorylation or autoubiquitination of the SOCS-box protein, and identify interactions between Vif and host cell proteins that may be therapeutic targets.

HIV-1 Vif and APOBEC3G: multiple roads to one goal.

The viral infectivity factor, Vif, of human immunodeficiency virus type 1, HIV-1, has long been shown to promote viral replication in vivo and to serve a critical function for productive infection of non-permissive cells, like peripheral blood mononuclear cells (PBMC). Vif functions to counteract an anti-retroviral cellular factor in non-permissive cells named APOBEC3G. The current mechanism proposed for protection of the virus by HIV-1 Vif is to induce APOBEC3G degradation through a ubiquitination-dependent proteasomal pathway. However, a new study published in Retrovirology by Strebel and colleagues suggests that Vif-induced APOBEC3G destruction may not be required for Vif's virus-protective effect. Strebel and co-workers show that Vif and APOBEC3G can stably co-exist, and yet viruses produced under such conditions are fully infectious. This new result highlights the notion that depletion of APOBEC3G is not the sole protective mechanism of Vif and that additional mechanisms exerted by this protein can be envisioned which counteract APOBEC3G and enhance HIV infectivity.

Camelized rabbit-derived VH single-domain intrabodies against Vif strongly neutralize HIV-1 infectivity.

We recently developed a specific single-chain antibody from immunized rabbits to HIV-1 Vif protein that was expressed intracellularly and inhibited reverse transcription and viral replication. The Vif of HIV-1 overcomes the innate antiviral activity of a cytidine deaminase Apobec3G (CEM15) that induces G to A hypermutation in the viral genome, resulting in enhancement of viral replication infectivity. Here, we have developed a minimal scaffold VH fragment with intrabody properties derived from anti-Vif single-chain antibody that was engineered to mimic camelid antibody domains. Non-specific binding of VH by its interface for the light chain variable domain (VL) was prevented through amino acid mutations in framework 2 and 4 (Val37F, G44E, L45R, W47G and W103R). Our results demonstrate that all constructed anti-Vif VH single-domains preserve the antigen-binding activity and specificity in the absence of the parent VL domain. However, only the most highly camelized domains had high levels of intracellular expression. The expression in eukaryotic cells showed that VH single-domains could correctly fold as soluble proteins in the reducing environment. The results demonstrated an excellent correlation between improvements in protein solubility with gradually increasing camelization. Camelized single-domains efficiently bound Vif protein and neutralized its infectivity enhancing function, by reducing late reverse transcripts and proviral integration. The activity of the anti-Vif single-domains was shown to be cell-specific, with inhibitory effects only in cells non-permissive that require Vif for HIV-1 replication. Moreover, cell specificity of anti-Vif intrabodies was correlated with an increase of Apobec3G, which potentiates viral inhibition. The present study strongly suggests that camelization of rabbit VH domains is a potentially useful approach for engineering intrabodies for gene therapy.

Functional neutralization of HIV-1 Vif protein by intracellular immunization inhibits reverse transcription and viral replication.

Human immunodeficiency virus type 1 (HIV-1)-encoded Vif protein is important for viral replication and infectivity. Vif is a cytoplasmic protein that acts during virus assembly by an unknown mechanism, enhancing viral infectivity. The action of Vif in producer cells is essential for the completion of proviral DNA synthesis following virus entry. Therefore, Vif is considered to be an important alternative therapeutic target for inhibition of viral infectivity at the level of viral assembly and reverse transcription. To gain insight into this process, we developed a Vif-specific single-chain antibody and expressed it intracellularly in the cytoplasm. This intrabody efficiently bound Vif protein and neutralized its infectivity-enhancing function. Intrabody-expressing cells were shown to be highly refractory to challenge with different strains of HIV-1 and HIV-1-infected cells. Inhibition of Vif by intrabody expression in the donor cell produced viral particles that do not complete reverse transcription in the recipient cell. The anti-Vif scFv was shown to be specific for Vif protein because its function was observed only in nonpermissive cells (H9, CEM, and U38). Moreover, transduction of peripheral blood mononuclear cells with an HIV-derived retroviral vector expressing Vif intrabody was shown to confer resistance to laboratory-adapted and primary HIV strains. This study provides biochemical evidence for the role of Vif in the HIV-1 lifecycle and validates Vif as a target for the control of HIV-1 infection.