Human Immunodeficiency Virus Vif Research Articles

Discovery of L15 as a novel Vif PROTAC degrader with antiviral activity against HIV-1

Viral infectivity factor (Vif) has been recognized as a new therapeutic target for human immunodeficiency virus-1 (HIV-1) infected patients. In our previous work, we have synthesized a novel class of Vif inhibitors with 2-amino-N-(5-hydroxy-2-methoxyphenyl)-6-((4-nitrophenyl)thio)benzamide scaffold, which show obvious activity in HIV-1 infected cells and are also effective against drug-resistant strains. Proteolytic targeting chimera (PROTAC) utilizes the ubiquitin–proteasome system to degrade target proteins, which is well established in the field of cancer, but the antiviral PROTAC molecules are rarely reported. In order to explore the effectiveness of PROTAC in the antiviral area, we designed and synthesized a series of degrader of HIV-1 Vif based on 2-amino-N-(5-hydroxy-2-methoxyphenyl)-6-((4-nitrophenyl)thio)benzamide scaffold. Among them, L15 can degrade Vif protein obviously in a dose-dependent manner and shows certain antivirus activity. Meanwhile, molecular dynamics simulation indicated that the ternary complex formed by L15, Vif, and E3 ligase adopted a reasonable binding mode and maintained a stable interaction. This provided a molecular basis and prerequisite for the selective degradation of the Vif protein by L15. This study reports the HIV-1 Vif PROTAC for the first time and represents the proof-of-concept of PROTACs-based antiviral drug discovery in the field of HIV/ acquired immune deficiency syndrome (AIDS).

Identification of Clinically Relevant HIV Vif Protein Motif Mutations through Machine Learning and Undersampling.

Human Immunodeficiency virus (HIV) and its clinical entity, the Acquired Immunodeficiency Syndrome (AIDS) continue to represent an important health burden worldwide. Although great advances have been made towards determining the way viral genetic diversity affects clinical outcome, genetic association studies have been hindered by the complexity of their interactions with the human host. This study provides an innovative approach for the identification and analysis of epidemiological associations between HIV Viral Infectivity Factor (Vif) protein mutations and four clinical endpoints (Viral load and CD4 T cell numbers at time of both clinical debut and on historical follow-up of patients. Furthermore, this study highlights an alternative approach to the analysis of imbalanced datasets, where patients without specific mutations outnumber those with mutations. Imbalanced datasets are still a challenge hindering the development of classification algorithms through machine learning. This research deals with Decision Trees, Naïve Bayes (NB), Support Vector Machines (SVMs), and Artificial Neural Networks (ANNs). This paper proposes a new methodology considering an undersampling approach to deal with imbalanced datasets and introduces two novel and differing approaches (MAREV-1 and MAREV-2). As theses approaches do not involve human pre-determined and hypothesis-driven combinations of motifs having functional or clinical relevance, they provide a unique opportunity to discover novel complex motif combinations of interest. Moreover, the motif combinations found can be analyzed through traditional statistical approaches avoiding statistical corrections for multiple tests.

Introduction of Mutant APOBEC3G Into CD4+ T-Cells to Resist HIV-1

Introduction: Human immunodeficiency virus 1 (HIV-1) is a potentially lethal retrovirus with high genetic diversity and replication ability. HIV-1 mainly targets human CD4+ T-cells, causing a rapid decline in CD4+ T-cell count after infection. Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit 3G (APOBEC3G or A3G) is a human anti-viral enzyme that blocks early stages of HIV-1 replication by deaminating the cytosine residues to uracil in the viral minus-strand DNA during reverse transcription. Nevertheless, HIV-1 overcomes A3G anti-viral activity by employing Vif accessory protein to degrade A3G. Due to a proline/aspartate point mutation in residue 129 (P129D), an artificial A3G mutant, also known as A3G-P129D, was found to be resistant against diverse Vif variants, including HIV-1 Vif, thereby shedding light on a potential A3G-mediated gene therapy for HIV-1. Methods: We propose that, using CRISPR-Cas9 tools, the P129D mutation will be introduced into A3G expressed by human hematopoietic stem cells. Engineered stem cells will be transplanted into Non-Obese Diabetic Severe Combined Immunodeficient Gamma (NSG) mice, which will later be infected with HIV-1. Following infection, flow cytometry will be used to compare the CD4+ T-cell count in mice that express A3G-P129D versus control mice transplanted with unmodified human CD4+ T-cells. Results: We expect a notably higher CD4+ T-cell count in mice carrying the P129D mutation than in the control group, which would be attributed to the efficient inhibition of HIV-1 replication by the mutant A3G. Discussion: Transforming human A3G to A3G-P129D will prevent Vif-mediated degradation of A3G, allowing A3G to efficiently target HIV-1. Thus, this treatment would ultimately block HIV-1 replication and prevent CD4+ T-cell depletion. Conclusion: Overall, our novel gene therapy approach could open the door to a potential one-time treatment for HIV-1 infected patients.

Determinants of FIV and HIV Vif sensitivity of feline APOBEC3 restriction factors.

BackgroundFeline immunodeficiency virus (FIV) is a global pathogen of Felidae species and a model system for Human immunodeficiency virus (HIV)-induced AIDS. In felids such as the domestic cat (Felis catus), APOBEC3 (A3) genes encode for single-domain A3Z2s, A3Z3 and double-domain A3Z2Z3 anti-viral cytidine deaminases. The feline A3Z2Z3 is expressed following read-through transcription and alternative splicing, introducing a previously untranslated exon in frame, encoding a domain insertion called linker. Only A3Z3 and A3Z2Z3 inhibit Vif-deficient FIV. Feline A3s also are restriction factors for HIV and Simian immunodeficiency viruses (SIV). Surprisingly, HIV-2/SIV Vifs can counteract feline A3Z2Z3.ResultsTo identify residues in feline A3s that Vifs need for interaction and degradation, chimeric human–feline A3s were tested. Here we describe the molecular direct interaction of feline A3s with Vif proteins from cat FIV and present the first structural A3 model locating these interaction regions. In the Z3 domain we have identified residues involved in binding of FIV Vif, and their mutation blocked Vif-induced A3Z3 degradation. We further identified additional essential residues for FIV Vif interaction in the A3Z2 domain, allowing the generation of FIV Vif resistant A3Z2Z3. Mutated feline A3s also showed resistance to the Vif of a lion-specific FIV, indicating an evolutionary conserved Vif–A3 binding. Comparative modelling of feline A3Z2Z3 suggests that the residues interacting with FIV Vif have, unlike Vif-interacting residues in human A3s, a unique location at the domain interface of Z2 and Z3 and that the linker forms a homeobox-like domain protruding of the Z2Z3 core. HIV-2/SIV Vifs efficiently degrade feline A3Z2Z3 by possible targeting the linker stretch connecting both Z-domains.ConclusionsHere we identified in feline A3s residues important for binding of FIV Vif and a unique protein domain insertion (linker). To understand Vif evolution, a structural model of the feline A3 was developed. Our results show that HIV Vif binds human A3s differently than FIV Vif feline A3s. The linker insertion is suggested to form a homeo-box domain, which is unique to A3s of cats and related species, and not found in human and mouse A3s. Together, these findings indicate a specific and different A3 evolution in cats and human.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0274-9) contains supplementary material, which is available to authorized users.

Characterization of the Interaction of Full-Length HIV-1 Vif Protein with its Key Regulator CBFβ and CRL5 E3 Ubiquitin Ligase Components

Human immunodeficiency virus-1 (HIV-1) viral infectivity factor (Vif) is essential for viral replication because of its ability to eliminate the host's antiviral response to HIV-1 that is mediated by the APOBEC3 family of cellular cytidine deaminases. Vif targets these proteins, including APOBEC3G, for polyubiquitination and subsequent proteasome-mediated degradation via the formation of a Cullin5-ElonginB/C-based E3 ubiquitin ligase. Determining how the cellular components of this E3 ligase complex interact with Vif is critical to the intelligent design of new antiviral drugs. However, structural studies of Vif, both alone and in complex with cellular partners, have been hampered by an inability to express soluble full-length Vif protein. Here we demonstrate that a newly identified host regulator of Vif, core-binding factor-beta (CBFβ), interacts directly with Vif, including various isoforms and a truncated form of this regulator. In addition, carboxyl-terminal truncations of Vif lacking the BC-box and cullin box motifs were sufficient for CBFβ interaction. Furthermore, association of Vif with CBFβ, alone or in combination with Elongin B/C (EloB/C), greatly increased the solubility of full-length Vif. Finally, a stable complex containing Vif-CBFβ-EloB/C was purified in large quantity and shown to bind purified Cullin5 (Cul5). This efficient strategy for purifying Vif-Cul5-CBFβ-EloB/C complexes will facilitate future structural and biochemical studies of Vif function and may provide the basis for useful screening approaches for identifying novel anti-HIV drug candidates.

T-cell differentiation factor CBF-β regulates HIV-1 Vif-mediated evasion of host restriction

The human APOBEC3 cytidine deaminases are potent inhibitors of diverse retroviruses, including human immunodeficiency virus-1 (HIV-1). HIV-1 Vif forms an E3 ubiquitin ligase complex with cullin 5 (CUL5), elongin B and elongin C , which promotes the polyubiquitination and degradation of APOBEC3 substrates. Here we demonstrate in human T cells that core binding factor β (CBF-β) is a key regulator of the evasion of HIV-1 from the host defence mediated by APOBEC3. CBF-β, the non-DNA-binding subunit of a heterodimeric transcription factor, regulates the folding and DNA-binding activity of partner RUNX family proteins, which have important roles in the development and differentiation of diverse cell types, including T lymphocytes. In our study, knockdown of endogenous CBF-β blocked Vif-induced APOBEC3G polyubiquitination and degradation. CBF-β was not required for the interaction between Vif and APOBEC3G, yet was essential for the assembly of the Vif-CUL5 E3-ubiquitin-ligase complex. CBF-β proved to be a unique regulator of primate lentiviral Vif and not a general component of the CUL5 E3 ubiquitin ligase. We show that Vif and CBF-β physically interact, and that the amino-terminal region of Vif is required for this interaction. Furthermore, interactions with Vif required regions in CBF-β that are not involved in RUNX protein binding. Considering the importance of the interaction between Vif and CBF-β, disrupting this interaction represents an attractive pharmacological intervention against HIV-1.

On the Solution Conformation and Dynamics of the HIV-1 Viral Infectivity Factor

Human immunodeficiency virus-1 (HIV-1) has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host cell enzymes. HIV-1 Vif [ viral (also called virion) infectivity factor], one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and downregulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of the protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method that is well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation, suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion, indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution, including the APOBEC3G/F binding site and the HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.

APOBEC3H haplotypes and HIV-1 pro-viral vif DNA sequence diversity in early untreated human immunodeficiency virus–1 infection

We examined single nucleotide polymorphisms (SNP) in the APOBEC3 locus on chromosome 22, paired with population sequences of pro-viral human immunodeficiency virus–1 (HIV-1) vif from peripheral blood mononuclear cells, from 96 recently HIV-1–infected treatment-naive adults. We found evidence for the existence of an APOBEC3H linkage disequilibrium (LD) block associated with variation in GA→AA, or APOBEC3F/H signature, sequence changes in pro-viral HIV-1 vif sequence (top 10 significant SNPs with a significant p = 4.8 × 10 −3). We identified a common five position risk haplotype distal to APOBEC3H (A3Hrh). These markers were in high LD (D′ = 1; r 2 = 0.98) to a previously described A3H “RED” haplotype containing a variant (E121) with enhanced susceptibility to HIV-1 Vif. This association was confirmed by a haplotype analysis. Homozygote carriers of the A3Hrh had lower GA->AA (A3F/H) sequence editing upon pro-viral HIV-1 vif sequence ( p = 0.01), and lower HIV-1 RNA levels over time during early, untreated HIV-1 infection, ( p = 0.015 mixed effects model). This effect may be due to enhanced susceptibility of A3H forms to HIV-1 Vif mediated viral suppression of sequence editing activity, slowing viral diversification and escape from immune responses.

Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G

The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif. APOBEC3G is a member of a family of polynucleotide cytosine deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine deaminase or anti-viral activity. Here we report a solution structure of the human APOBEC3G catalytic domain. Five alpha-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five beta-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.

A Zinc-binding Region in Vif Binds Cul5 and Determines Cullin Selection

Human immunodeficiency virus-1 (HIV-1) Vif overcomes the anti-viral activity of APOBEC3G by targeting it for ubiquitination via a Cullin 5-ElonginB-ElonginC (Cul5-EloBC) E3 ligase. Vif associates with Cul5-EloBC through a BC-box motif that binds EloC, but the mechanism by which Vif selectively recruits Cul5 is poorly understood. Here we report that a region of Vif (residues 100-142) upstream of the BC-box binds selectively to Cul5 in the absence of EloC. This region contains a zinc coordination site HX5CX17-18CX3-5H (HCCH), with His/Cys residues at positions 108, 114, 133, and 139 coordinating one zinc ion. The HCCH zinc coordination site, which is conserved among primate lentivirus Vif proteins, does not correspond to any known class of zinc-binding motif. Mutations of His/Cys residues in the HCCH motif impair zinc coordination, Cul5 binding, and APOBEC3G degradation. Mutations of conserved hydrophobic residues (Ile-120, Ala-123, and Leu-124) located between the two Cys residues in the HCCH motif disrupt binding of the zinc-coordinating region to Cul5 and inhibit APOBEC3G degradation. The Vif binding site maps to the first cullin repeat in the N terminus of Cul5. These data suggest that the zinc-binding region in Vif is a novel cullin interaction domain that mediates selective binding to Cul5. We propose that the HCCH zinc-binding motif facilitates Vif-Cul5 binding by playing a structural role in positioning hydrophobic residues for direct contact with Cul5.

Induction of APOBEC3 family proteins, a defensive maneuver underlying interferon-induced anti–HIV-1 activity

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G), a cytidine deaminase, is a recently recognized innate intracellular protein with lethal activity against human immunodeficiency virus (HIV). Packaged into progeny virions, APOBEC3G enzymatic activity leads to HIV DNA degradation. As a counterattack, HIV virion infectivity factor (Vif) targets APOBEC3G for proteasomal proteolysis to exclude it from budding virions. Based on the ability of APOBEC3G to antagonize HIV infection, considerable interest hinges on elucidating its mechanism(s) of regulation. In this study, we provide the first evidence that an innate, endogenous host defense factor has the potential to promote APOBEC3G and rebuke the virus-mediated attempt to control its cellular host. We identify interferon (IFN)-α as a potent inducer of APOBEC3G to override HIV Vif neutralization of APOBEC3 proteins that pose a threat to efficient macrophage HIV replication. Our data provide a new dimension by which IFN-α mediates its antiviral activity and suggest a means to render the host nonpermissive for viral replication.

436. shRNAs Target HIV-1 Vif Gene Coding for SOCS-Box Motif Prevent Virus Escape from RNA Interference-Mediated Inhibition

RNA interference (RNAi) is powerful cellular mechanism in which double-stranded small interfering RNAs (siRNAs) induce sequence-specific degradation of homologous RNA molecules. Recent studies have shown that human immunodeficiency virus (HIV) replication can be effectively inhibited by intracellularly expressed siRNA or shRNA for short term. Since RNAi is highly sequence specific and HIV mutates rapidly, long-term inhibition of HIV replication is still a challenge for RNAi mediated virus gene therapy. Several groups report that HIV can escape RNAi inhibition as early as 21 days post challenge by target site point or deletion mutations. In this study, we developed a novel strategy to target the HIV Vif protein SOCS-box motif coding region using stably expressed shRNAs. HIV-1 Vif (viral infectivity factor) protein overcomes the antiviral activity of the host cellular DNA deaminase APOBEC3G by acting through Cul5-ElonginB-ElonginC E3 ubiquitin ligase. Sequence alignment of the Vif protein with other well-studied E3 ubiquitin complex binding proteins shows Vif contains the highly conserved SOCS-box motif that is the important binding domain to Cul5 complex. Mutations in this motif impeded the protein's ability to bind to this complex. For this reason, we designed two shRNAs targeting the highly conserved Vif SOCS-box motif-coding regions. Wild type virus will be directly inhibited by shRNA mediated RNAi. Although the virus may mutate and escape from RNAi via mutations in the target sites, the mutated SOCS-box motif will lose its ability to direct APOBEC3G to the proteasome, thereby allowing its incorporation into the virion, which will lead to defective virus. Our preliminary data shows the two stably expressed shRNAs can effectively inhibit HIV replication in the short term and reduce virus infectivity over long-term culture. There are no virus mutations with the shRNA target sites that we could detect up to one month after the initial challenge with HIV-1 IIIB. However, extended incubation shows one of the two target sites acquired a single point mutation after two months in culture. This point mutation is considerably delayed in comparison comparing with other reported escape mutations. The amino acid encoded by this mutation resulted in a very conservative change from Leu to Ile. The function of Vif with this amino acid changed mutation needs to be further studied. Nevertheless, we have yet to detect a mutation at the other conserved site in Vif. Thus, our results provide a novel strategy in targeting a highly conserved motif in an essential viral protein coding region which directly or indirectly can prevent virus escape from RNAi inhibition.

APOBEC3G is a single-stranded DNA cytidine deaminase and functions independently of HIV reverse transcriptase.

In the absence of the viral vif gene, human immunodeficiency virus (HIV) may be restricted by the APOBEC3G gene on chromosome 22. The role of the HIV Vif protein is to exclude host cell APOBEC3G from the budding virion. As APOBEC3G shows sequence homology to cytidine deaminases, it is presumed that in the absence of Vif, cytidine residues in the cDNA are deaminated yielding uracil. It is not known if additional proteins mediate APOBEC3G function or if deamination occurs in concert with reverse transcription. This report describes an in vitro assay showing that Baculovirus derived APOBEC3G alone extensively deaminates cDNA independently of reverse transcriptase. It reproduces the dinucleotide context typical of G --> A hypermutants derived from a Delta(vif) virus. By using an RNaseH- form of reverse transcriptase, it was shown that the cDNA has to be free of its RNA template to allow deamination. APOBEC3G deamination of dC or dCTP was not detected. In short, APOBEC3G is a single-stranded DNA cytidine deaminase capable of restricting retroviral replication.

DNA Deamination Mediates Innate Immunity to Retroviral Infection

CEM15/APOBEC3G is a cellular protein required for resistance to infection by virion infectivity factor (Vif)-deficient human immunodeficiency virus (HIV). Here, using a murine leukemia virus (MLV)-based system, we provide evidence that CEM15/APOBEC3G is a DNA deaminase that is incorporated into virions during viral production and subsequently triggers massive deamination of deoxycytidine to deoxyuridine within the retroviral minus (first)-strand cDNA, thus providing a probable trigger for viral destruction. Furthermore, HIV Vif can protect MLV from this CEM15/APOBEC3G-dependent restriction. These findings imply that targeted DNA deamination is a major strategy of innate immunity to retroviruses and likely also contributes to the sequence variation observed in many viruses (including HIV).

Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein.

Viruses have developed diverse non-immune strategies to counteract host-mediated mechanisms that confer resistance to infection. The Vif (virion infectivity factor) proteins are encoded by primate immunodeficiency viruses, most notably human immunodeficiency virus-1 (HIV-1). These proteins are potent regulators of virus infection and replication and are consequently essential for pathogenic infections in vivo. HIV-1 Vif seems to be required during the late stages of virus production for the suppression of an innate antiviral phenotype that resides in human T lymphocytes. Thus, in the absence of Vif, expression of this phenotype renders progeny virions non-infectious. Here, we describe a unique cellular gene, CEM15, whose transient or stable expression in cells that do not normally express CEM15 recreates this phenotype, but whose antiviral action is overcome by the presence of Vif. Because the Vif:CEM15 regulatory circuit is critical for HIV-1 replication, perturbing the circuit may be a promising target for future HIV/AIDS therapies.