RESUMEN
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).
Asunto(s)
Fármacos Anti-VIH , VIH-1 , Productos del Gen vif del Virus de la Inmunodeficiencia Humana , VIH-1/efectos de los fármacos , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Humanos , Relación Estructura-Actividad , Estructura Molecular , Benzamidas/farmacología , Benzamidas/química , Benzamidas/síntesis química , Descubrimiento de Drogas , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Proteolisis/efectos de los fármacos , Simulación de Dinámica MolecularRESUMEN
APOBEC3G (A3G) restricts HIV-1 replication primarily by reducing viral cDNA and inducing G-to-A hypermutations in viral cDNA. HIV-1 encodes virion infectivity factor (Vif) to counteract A3G primarily by excluding A3G viral encapsidation. Even though the Vif-induced exclusion is robust, studies suggest that A3G is still detectable in the virion. The impact of encapsidated A3G in the HIV-1 replication is unclear. Using a highly sensitive next-generation sequencing (NGS)-based G-to-A hypermutation detecting assay, we found that wild-type HIV-1 produced from A3G-expressing T-cells induced higher G-to-A hypermutation frequency in viral cDNA than HIV-1 from non-A3G-expressing T-cells. Interestingly, although the virus produced from A3G-expressing T-cells induced higher hypermutation frequency, there was no significant difference in viral infectivity, revealing a disassociation of cDNA G-to-A hypermutation to viral infectivity. We also measured G-to-A hypermutation in the viral RNA genome. Surprisingly, our data showed that hypermutation frequency in the viral RNA genome was significantly lower than in the integrated DNA, suggesting a mechanism exists to preferentially select intact genomic RNA for viral packing. This study revealed a new insight into the mechanism of HIV-1 counteracting A3G antiviral function and might lay a foundation for new antiviral strategies.
Asunto(s)
Desaminasa APOBEC-3G , ADN Complementario , VIH-1 , Mutación , Humanos , Desaminasa APOBEC-3G/genética , Desaminasa APOBEC-3G/metabolismo , ADN Complementario/genética , ADN Viral/genética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Infecciones por VIH/virología , VIH-1/genética , VIH-1/patogenicidad , Linfocitos T/virología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Replicación Viral/genéticaRESUMEN
One of the major functions of the accessory protein Vif of human immunodeficiency virus type 1 (HIV-1) is to induce the degradation of APOBEC3 (A3) family proteins by recruiting a Cullin5-ElonginB/C-CBFß E3 ubiquitin ligase complex to facilitate viral replication. Therefore, the interactions between Vif and the E3 complex proteins are promising targets for the development of novel anti-HIV-1 drugs. Here, peptides are designed for the Vif-CBFß interaction based on the sequences of Vif mutants with higher affinity for CBFß screened by a yeast surface display platform. We identified two peptides, VMP-63 and VMP-108, that could reduce the infectivity of HIV-1 produced from A3G-positive cells with IC50 values of 49.4 µM and 55.1 µM, respectively. They protected intracellular A3G from Vif-mediated degradation in HEK293T cells, consequently increasing A3G encapsulation into the progeny virions. The peptides could rapidly enter cells after addition to HEK293T cells and competitively inhibit the binding of Vif to CBFß. Homology modeling analysis demonstrated the binding advantages of VMP-63 and VMP-108 with CBFß over their corresponding wild-type peptides. However, only VMP-108 effectively restricted long-term HIV-1 replication and protected A3 functions in non-permissive T lymphocytes. Our findings suggest that competitive Vif-derived peptides targeting the Vif-CBFß interaction are promising for the development of novel therapeutic strategies for acquired immune deficiency syndrome.
Asunto(s)
Fármacos Anti-VIH , Subunidad beta del Factor de Unión al Sitio Principal , VIH-1 , Péptidos , Unión Proteica , Productos del Gen vif del Virus de la Inmunodeficiencia Humana , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Humanos , VIH-1/efectos de los fármacos , VIH-1/fisiología , Células HEK293 , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Péptidos/farmacología , Péptidos/metabolismo , Péptidos/química , Fármacos Anti-VIH/farmacología , Replicación Viral/efectos de los fármacos , Diseño de Fármacos , Infecciones por VIH/virología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismoRESUMEN
Virion infectivity factor (Vif), an accessory protein of HIV-1 (human immunodeficiency virus type 1), antagonizes host APOBEC3 protein (apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3) or A3 via proteasomal degradation, facilitating viral replication. HLA (Human leukocyte antigens) alleles, host restriction factors, and error-prone reverse transcription contribute to the global polymorphic dynamics of HIV, impacting effective vaccine design. Our computational analysis of over 50,000 HIV-1 M vif sequences from the Los Alamos National Laboratory (LANL) database (1998-2021) revealed positive selection pressure on the vif gene (nonsynonymous to synonymous ratio, dn/ds=1.58) and an average entropy score of 0.372 in protein level. Interestingly, over the years (1998-2021), a decreasing trend of dn/ds (1.68 to 1.47) and an increasing trend of entropy (0.309 to 0.399) was observed. The predicted mutational frequency against Vif consensus sequence decreased over time (slope = -0.00024, p < 0.0001). Sequence conservation was observed in Vif functional motifs F1, F2, F3, G, BC box, and CBF ß binding region, while variability was observed mainly in N- and C- terminal and Zinc finger region, which were dominantly under immune pressure by host HLA-I-restricted CD8+ T cell. Computational analysis of ∆∆Gstability through protein stability prediction tools suggested that missense mutation may affect Vif stability, especially in the Vif-A3 binding interface. Notably, mutations R17K and Y44F in F1 and G box were predicted to destabilize the Vif-A3 binding interface by altering bond formations with adjacent amino acids. Therefore, our analysis demonstrates Vif adaptation with host physiology by maintaining sequence conservation, especially in A3 interacting functional motifs, highlighting important therapeutic candidate regions of Vif against HIV-1 infections.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Mutación Missense , Polimorfismo Genético , Unión Proteica , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismoRESUMEN
PURPOSE: Despite extensive research, HIV-1 remains a global epidemic with variations in pathogenesis across regions and subtypes. The Viral Infectivity Factor (Vif) protein, which neutralizes the host protein APOBEC3G, has been implicated in differences in clinical outcomes among people living with HIV (PLHIV). Most studies on Vif sequence diversity have focused on subtype B, leaving gaps in understanding Vif variations in HIV-1C regions like South Africa. This study aimed to identify and compare Vif sequence diversity in a cohort of 51 South African PLHIV and other HIV-1C prevalent regions. METHODS: Sanger sequencing was used for Vif analysis in the cohort, and additional sequences were obtained from the Los Alamos database. Molecular modeling and docking techniques were employed to study the influence of subtype-specific variants on Vif-APOBEC3G binding affinity. RESULTS: The findings showed distinct genetic variations between Vif sequences from India and Uganda, while South African sequences had wider distribution and closer relatedness to both. Specific amino acid substitutions in Vif were associated with geographic groups. Molecular modeling and docking analyses consistently identified specific residues (ARGR19, LYS26, TYR30, TYR44, and TRP79) as primary contributors to intermolecular contacts between Vif and APOBEC3G, essential for their interaction. The Indian Vif variant exhibited the highest predicted binding affinity to APOBEC3G among the studied groups. CONCLUSIONS: These results provide insights into Vif sequence diversity in HIV-1C prevalent regions and shed light on differential pathogenesis observed in different geographical areas. The identified Vif amino acid residues warrant further investigation for their diagnostic, prognostic, and therapeutic potential.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , VIH-1/genética , VIH-1/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Pueblo Africano , Desaminasa APOBEC-3G/genéticaRESUMEN
HIV-1 Vif is known to counteract the antiviral activity of human apolipoprotein B mRNA-editing catalytic polypeptide-like (A3), a cytidine deaminase, in various ways. However, the precise mechanism behind this interaction has remained elusive. Within infected cells, Vif forms a complex called VßBCC, comprising CBFß and the components of E3 ubiquitin ligase, Elongin B, Elongin C, and Cullin5. Together with the ubiquitin-conjugating enzyme, VßBCC induces ubiquitination-mediated proteasomal degradation of A3. However, Vif exhibits additional counteractive effects. In this study, we elucidate that VßBCC inhibits deamination by A3G, A3F, and A3B independently of proteasomal degradation. Surprisingly, we discovered that this inhibition for A3G is directly attributed to the interaction between VßBCC and the C-terminal domain of A3G. Previously, it was believed that Vif did not interact with the C-terminal domain. Our findings suggest that inhibiting the interaction between VßBCC and the C-terminal domain, as well as the N-terminal domain known to be targeted for ubiquitination, of A3G may be needed to prevent counteraction by Vif.
Asunto(s)
VIH-1 , Productos del Gen vif del Virus de la Inmunodeficiencia Humana , Humanos , Citosina Desaminasa/metabolismo , VIH-1/metabolismo , Unión Proteica , ProteolisisRESUMEN
We have previously reported an HIV-1 mutant designated NL-Y226tac that expresses Vif at an ultra-low level, being replication-defective in high-APOBEC3G cells, such as H9. It carries a synonymous mutation within the splicing SA1 site relative to its parental clone. In order to determine whether a certain mutant(s) emerges during multi-infection cycles, we maintained H9 cells infected with a relatively low or high input of NL-Y226tac for extended time periods. Unexpectedly, we reproducibly identified a g5061a mutation in the SD2b site in the two independent long-term culture experiments that partially increases Vif expression and replication ability. Importantly, the adaptive mutation g5061a was demonstrated to enhance vif mRNA production by activation of the SA1 site mediated through increasing usage of a rarely used SD2b site. In the long-term culture initiated by a high virus input, we additionally found a Y226Fttc mutation at the original Y226tac site in SA1 that fully restores Vif expression and replication ability. As expected, the adaptive mutation Y226Fttc enhances vif mRNA production through increasing the splicing site usage of SA1. Our results here revealed the importance of the SD2b nucleotide sequence in producing vif mRNA involved in the HIV-1 adaptation and of mutual antagonism between Vif and APOBEC3 proteins in HIV-1 adaptation/evolution and survival.
Asunto(s)
VIH-1 , ARN Mensajero/genética , VIH-1/fisiología , Línea Celular , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Replicación Viral/genética , Citidina Desaminasa/genética , MutaciónRESUMEN
HIV-1 must overcome multiple innate antiviral mechanisms to replicate in CD4+ T lymphocytes and macrophages. Previous studies have demonstrated that the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) family of proteins (at least A3D, A3F, A3G, and stable A3H haplotypes) contribute to HIV-1 restriction in CD4+ T lymphocytes. Virus-encoded virion infectivity factor (Vif) counteracts this antiviral activity by degrading A3 enzymes allowing HIV-1 replication in infected cells. In addition to A3 proteins, Vif also targets other cellular proteins in CD4+ T lymphocytes, including PPP2R5 proteins. However, whether Vif primarily degrades only A3 proteins during viral replication is currently unknown. Herein, we describe the development and characterization of A3F-, A3F/A3G-, and A3A-to-A3G-null THP-1 cells. In comparison to Vif-proficient HIV-1, Vif-deficient viruses have substantially reduced infectivity in parental and A3F-null THP-1 cells, and a more modest decrease in infectivity in A3F/A3G-null cells. Remarkably, disruption of A3A-A3G protein expression completely restores the infectivity of Vif-deficient viruses in THP-1 cells. These results indicate that the primary function of Vif during infectious HIV-1 production from THP-1 cells is the targeting and degradation of A3 enzymes. IMPORTANCE HIV-1 Vif neutralizes the HIV-1 restriction activity of A3 proteins. However, it is currently unclear whether Vif has additional essential cellular targets. To address this question, we disrupted A3A to A3G genes in the THP-1 myeloid cell line using CRISPR and compared the infectivity of wild-type HIV-1 and Vif mutants with the selective A3 neutralization activities. Our results demonstrate that the infectivity of Vif-deficient HIV-1 and the other Vif mutants is fully restored by ablating the expression of cellular A3A to A3G proteins. These results indicate that A3 proteins are the only essential target of Vif that is required for fully infectious HIV-1 production from THP-1 cells.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , VIH-1/fisiología , Citidina Desaminasa/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Unión Proteica , Desaminasa APOBEC-3G/metabolismo , Citosina Desaminasa/genética , Citosina Desaminasa/metabolismo , Línea Celular , Células Mieloides/metabolismo , Virión/metabolismo , Desaminasas APOBEC/metabolismoRESUMEN
Great effort has been devoted to discovering the basis of A3G-Vif interaction, the key event of HIV's counteraction mechanism to evade antiviral innate immune response. Here we show reconstitution of the A3G-Vif complex and subsequent A3G ubiquitination in vitro and report the cryo-EM structure of the A3G-Vif complex at 2.8 Å resolution using solubility-enhanced variants of A3G and Vif. We present an atomic model of the A3G-Vif interface, which assembles via known amino acid determinants. This assembly is not achieved by protein-protein interaction alone, but also involves RNA. The cryo-EM structure and in vitro ubiquitination assays identify an adenine/guanine base preference for the interaction and a unique Vif-ribose contact. This establishes the biological significance of an RNA ligand. Further assessment of interactions between A3G, Vif, and RNA ligands show that the A3G-Vif assembly and subsequent ubiquitination can be controlled by amino acid mutations at the interface or by polynucleotide modification, suggesting that a specific chemical moiety would be a promising pharmacophore to inhibit the A3G-Vif interaction.
Asunto(s)
VIH-1 , Productos del Gen vif del Virus de la Inmunodeficiencia Humana , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/fisiología , ARN/metabolismo , Antivirales/farmacología , Antivirales/metabolismo , Aminoácidos/metabolismo , Desaminasa APOBEC-3G/química , Citidina Desaminasa/genéticaRESUMEN
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.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Secuencias de Aminoácidos , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Teorema de Bayes , Mutación , Aprendizaje Automático , VIH-1/metabolismoRESUMEN
HSP40/DNAJ family of proteins is the most diverse chaperone family, comprising about 49 isoforms in humans. Several reports have demonstrated the functional role of a few of these isoforms in the pathogenesis of various viruses, including HIV-1. Our earlier study has shown that several isoforms of HSP40 get significantly modulated at the mRNA level during HIV-1 infection in T cells. To explore the biological role of these significantly modulated isoforms, we analyzed their effect on HIV-1 gene expression and virus production using knockdown and overexpression studies. Among these isoforms, DNAJA3, DNAJB1, DNAJB7, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC22, and DNAJC30 seem to positively regulate virus replication, whereas DNAJB3, DNAJB6, DNAJB8, and DNAJC5 negatively regulate virus replication. Further investigation on the infectivity of the progeny virion demonstrated that only DNAJB8 negatively regulates the progeny virion infectivity. It was further identified that DNAJB8 protein is involved in the downregulation of Vif protein, required for the infectivity of HIV-1 virions. DNAJB8 seems to direct Vif protein for autophagic-lysosomal degradation, leading to rescue of the cellular restriction factor APOBEC3G from Vif-mediated proteasomal degradation, resulting in enhanced packaging of APOBEC3G in budding virions and release of less infective progeny virion particles. Finally, our results also indicate that during the early stage of HIV-1 infection, enhanced expression of DNAJB8 promotes the production of less infective progeny virions, but at the later stage or at the peak of infection, reduced expression of DNJAB8 protein allows the HIV-1 to replicate and produce more infective progeny virion particles.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/metabolismo , Proteínas Virales/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Productos del Gen vif/metabolismo , Replicación Viral/fisiología , Virión/metabolismo , Infecciones por VIH/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Desaminasa APOBEC-3G/genética , Desaminasa APOBEC-3G/metabolismo , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Host restriction factor APOBEC3G (A3G) efficiently restricts Vif-deficient HIV-1 by being packaged with progeny virions and causing the G to A mutation during HIV-1 viral DNA synthesis as the progeny virus infects new cells. HIV-1 expresses Vif protein to resist the activity of A3G by mediating A3G degradation. This process requires the self-association of Vif in concert with A3G proteins, protein chaperones, and factors of the ubiquitination machinery, which are potential targets to discover novel anti-HIV drugs. This review will describe compounds that have been reported so far to inhibit viral replication of HIV-1 by protecting A3G from Vif-mediated degradation.
Asunto(s)
VIH-1 , Productos del Gen vif del Virus de la Inmunodeficiencia Humana , Humanos , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/metabolismo , Línea Celular , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Mutación , Replicación Viral , Desaminasa APOBEC-3G/metabolismoRESUMEN
The APOBEC3 (A3) proteins are host antiviral cellular proteins that hypermutate the viral genome of diverse viral families. In retroviruses, this process requires A3 packaging into viral particles1-4. The lentiviruses encode a protein, Vif, that antagonizes A3 family members by targeting them for degradation. Diversification of A3 allows host escape from Vif whereas adaptations in Vif enable cross-species transmission of primate lentiviruses. How this 'molecular arms race' plays out at the structural level is unknown. Here, we report the cryogenic electron microscopy structure of human APOBEC3G (A3G) bound to HIV-1 Vif, and the hijacked cellular proteins that promote ubiquitin-mediated proteolysis. A small surface explains the molecular arms race, including a cross-species transmission event that led to the birth of HIV-1. Unexpectedly, we find that RNA is a molecular glue for the Vif-A3G interaction, enabling Vif to repress A3G by ubiquitin-dependent and -independent mechanisms. Our results suggest a model in which Vif antagonizes A3G by intercepting it in its most dangerous form for the virus-when bound to RNA and on the pathway to packaging-to prevent viral restriction. By engaging essential surfaces required for restriction, Vif exploits a vulnerability in A3G, suggesting a general mechanism by which RNA binding helps to position key residues necessary for viral antagonism of a host antiviral gene.
Asunto(s)
Desaminasa APOBEC-3G , VIH-1 , Proteolisis , Productos del Gen vif del Virus de la Inmunodeficiencia Humana , Animales , Humanos , Desaminasa APOBEC-3G/antagonistas & inhibidores , Desaminasa APOBEC-3G/química , Desaminasa APOBEC-3G/metabolismo , Desaminasa APOBEC-3G/ultraestructura , VIH-1/metabolismo , VIH-1/patogenicidad , ARN/química , ARN/metabolismo , Ubiquitina/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/ultraestructura , Microscopía por Crioelectrón , Empaquetamiento del Genoma Viral , Primates/virologíaRESUMEN
The seven human APOBEC3 enzymes (APOBEC3A through H, excluding E) are host restriction factors. Most of the APOBEC3 enzymes can restrict HIV-1 replication with different efficiencies. The HIV-1 Vif protein combats APOBEC3-mediated restriction by inducing ubiquitination and degradation in the proteasome. APOBEC3F and APOBEC3G can hetero-oligomerize, which increases their restriction capacity and resistance to Vif. Here we determined if APOBEC3C, APOBEC3F, or APOBEC3G could hetero-oligomerize with APOBEC3H haplotype I. APOBEC3H haplotype I has a short half-life in cells due to ubiquitination and degradation by host proteins, but is also resistant to Vif. We hypothesized that hetero-oligomerization with APOBEC3H haplotype I may result in less Vif-mediated degradation of the interacting APOBEC3 and stabilize APOBEC3H haplotype I, resulting in more efficient HIV-1 restriction. Although we found that all three APOBEC3s could interact with APOBEC3H haplotype I, only APOBEC3F affected APOBEC3H haplotype I by surprisingly accelerating its proteasomal degradation. However, this increased APOBEC3F levels in cells and virions in the absence or presence of Vif and enabled APOBEC3F-mediated restriction of HIV-1 in the presence of Vif. Altogether, the data suggest that APOBEC3 enzymes can co-regulate each other at the protein level and that they cooperate to ensure HIV-1 inactivation rather than evolution.
Asunto(s)
Seropositividad para VIH , VIH-1 , Humanos , VIH-1/genética , Haplotipos , Citidina Desaminasa , Citoplasma , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Citosina Desaminasa , Desaminasas APOBEC , Aminohidrolasas/genéticaRESUMEN
Human APOBEC3G (A3G) is a virus restriction factor that inhibits HIV-1 replication and triggers lethal hypermutation on viral reverse transcripts. HIV-1 viral infectivity factor (Vif) breaches this host A3G immunity by hijacking a cellular E3 ubiquitin ligase complex to target A3G for ubiquitination and degradation. The molecular mechanism of A3G targeting by Vif-E3 ligase is unknown, limiting the antiviral efforts targeting this host-pathogen interaction crucial for HIV-1 infection. Here, we report the cryo-electron microscopy structures of A3G bound to HIV-1 Vif in complex with T cell transcription cofactor CBF-ß and multiple components of the Cullin-5 RING E3 ubiquitin ligase. The structures reveal unexpected RNA-mediated interactions of Vif with A3G primarily through A3G's noncatalytic domain, while A3G's catalytic domain is poised for ubiquitin transfer. These structures elucidate the molecular mechanism by which HIV-1 Vif hijacks the host ubiquitin ligase to specifically target A3G to establish infection and offer structural information for the rational development of antiretroviral therapeutics.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/metabolismo , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Microscopía por Crioelectrón , Ubiquitina/metabolismo , Unión Proteica , Desaminasa APOBEC-3G/genética , Desaminasa APOBEC-3G/metabolismoRESUMEN
The canonical function of lentiviral Vif proteins is to counteract the mutagenic potential of APOBEC3 antiviral restriction factors. However, recent studies have discovered that Vif proteins from diverse HIV-1 and simian immunodeficiency virus (SIV) isolates degrade cellular B56 phosphoregulators to remodel the host phosphoproteome and induce G2/M cell cycle arrest. Here, we evaluate the conservation of this activity among non-primate lentiviral Vif proteins using fluorescence-based degradation assays and demonstrate that maedi-visna virus (MVV) Vif efficiently degrades all five B56 family members. Testing an extensive panel of single amino acid substitution mutants revealed that MVV Vif recognizes B56 proteins through a conserved network of electrostatic interactions. Furthermore, experiments using genetic and pharmacologic approaches demonstrate that degradation of B56 proteins requires the cellular cofactor cyclophilin A. Lastly, MVV Vif-mediated depletion of B56 proteins induces a potent G2/M cell cycle arrest phenotype. Therefore, remodeling of the cellular phosphoproteome and induction of G2/M cell cycle arrest are ancient and conserved functions of lentiviral Vif proteins, which suggests that they are advantageous for lentiviral pathogenesis.
Asunto(s)
VIH-1 , Virus Visna-Maedi , Animales , Evolución Biológica , Puntos de Control del Ciclo Celular , Productos del Gen vif/genética , Productos del Gen vif/metabolismo , VIH-1/genética , VIH-1/metabolismo , Ovinos , Virus Visna-Maedi/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The mechanisms for the protection of the human body from viral or bacterial agents are extremely diverse. In one such mechanism, an important role belongs to the cytidine deaminase APOBEC3 family, which is the factor of congenital immunity and protects the organism from numerous viral agents. One of the proteins of this family, APOBEC3G, is able to protect against Human Immunodeficiency Virus type 1 in the absence of viral protein Vif. In turn, Vif opposes APOBEC3G action, causing polyubiquity of the protein and degradation in the proteasome. The review describes possible ways to increase the anti-HIV activity of APOBEC3G, giving it resistance to viral protein Vif, as well as potential approaches to the use of modified APOBEC3G in gene therapy for HIV.
Asunto(s)
VIH-1 , Productos del Gen vif del Virus de la Inmunodeficiencia Humana , Desaminasa APOBEC-3G/genética , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Terapia Genética , VIH-1/metabolismo , Humanos , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3s) are host restriction factors that inhibit viral replication. Viral infectivity factor (Vif), a human immunodeficiency virus type 1 (HIV-1) accessory protein, mediates the degradation of APOBEC3s by forming the Vif-E3 complex, in which core-binding factor beta (CBFß) is an essential molecular chaperone. Here, we screened nonfunctional Vif mutants with high affinity for CBFß to inhibit HIV-1 in a dominant negative manner. We applied the yeast surface display technology to express Vif random mutant libraries, and mutants showing high CBFß affinity were screened using flow cytometry. Most of the screened Vif mutants containing random mutations of different frequencies were able to rescue APOBEC3G (A3G). In the subsequent screening, three of the mutants restricted HIV-1, recovered G-to-A hypermutation, and rescued APOBEC3s. Among them, Vif-6M showed a cross-protection effect toward APOBEC3C, APOBEC3F, and African green monkey A3G. Stable expression of Vif-6M in T lymphocytes inhibited the viral replication in newly HIV-1-infected cells and the chronically infected cell line H9/HXB2. Furthermore, the expression of Vif-6M provided a survival advantage to T lymphocytes infected with HIV-1. These results suggest that dominant negative Vif mutants acting on the Vif-CBFß target potently restrict HIV-1. IMPORTANCE Antiviral therapy cannot eliminate HIV and exhibits disadvantages such as drug resistance and toxicity. Therefore, novel strategies for inhibiting viral replication in patients with HIV are urgently needed. APOBEC3s in host cells are able to inhibit viral replication but are antagonized by HIV-1 Vif-mediated degradation. Therefore, we screened nonfunctional Vif mutants with high affinity for CBFß to compete with the wild-type Vif (wtVif) as a potential strategy to assist with HIV-1 treatment. Most screened mutants rescued the expression of A3G in the presence of wtVif, especially Vif-6M, which could protect various APOBEC3s and improve the incorporation of A3G into HIV-1 particles. Transduction of Vif-6M into T lymphocytes inhibited the replication of the newly infected virus and the chronically infected virus. These data suggest that Vif mutants targeting the Vif-CBFß interaction may be promising in the development of a new AIDS therapeutic strategy.
Asunto(s)
Subunidad beta del Factor de Unión al Sitio Principal , Infecciones por VIH , VIH-1 , Productos del Gen vif del Virus de la Inmunodeficiencia Humana , Desaminasas APOBEC/genética , Desaminasas APOBEC/metabolismo , Animales , Línea Celular , Chlorocebus aethiops , Subunidad beta del Factor de Unión al Sitio Principal/genética , VIH-1/genética , VIH-1/fisiología , Interacciones Huésped-Patógeno , Humanos , Linfocitos T/virología , Replicación Viral , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
APOBEC3G (A3G) is a member of cytosine deaminase family with a variety of innate immune functions. It displays activities against retrovirus and retrotransposon by inhibition of virus infectivity factor (Vif)-deficient HIV-1 replication. The interaction between A3G N-terminal domain and Vif directs the cellular Cullin 5 E3-ubiquitin ligase complex to ubiquitinate A3G, and leads to A3G proteasomal degradation, which is a potential target for anti-HIV drug. Currently, there are very few reports about stable small molecules targeting the interaction between A3G and Vif. In this study, we screened two series of small molecules containing carbamyl sulfamide bond or disulfide bond as bridges of two different aromatic rings. Five asymmetrical disulfides were successfully identified against interaction between A3G and Vif with the IC 50 values close to or smaller than 1â µM, especially, not through covalently binding with A3G or Vif. They restore the A3G expression in the presence of Vif by inhibiting Vif-induced A3G ubiquitination and degradation. This study opens a way to the discovery of new anti-HIV drugs.
Asunto(s)
Infecciones por VIH , Productos del Gen vif del Virus de la Inmunodeficiencia Humana , Desaminasa APOBEC-3G , Línea Celular , Citidina Desaminasa/química , Citidina Desaminasa/metabolismo , Disulfuros , Infecciones por VIH/tratamiento farmacológico , Humanos , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
OBJECTIVES: In children, achieving an acceptable degree of accuracy from dietary or physical activity (PA) assessments remains a challenge. Children tend to overestimate their time spent in daily PA and underestimate their dietary intake of fat and sugar. Because parents play a key role in family lifestyle decisions, including children's food choices and PA levels, it is important to investigate the responses of parents regarding their children's lifestyle habits. We aimed to develop a Self-Administered Parental Assessment of Children's Lifestyle (SAPLACL) questionnaire and test its feasibility and reliability in 191 parents (29 fathers and 162 mothers). RESULTS: For each part of the questionnaire, the rate of missing or improper responses ranged from 0 to 24%. The highest proportion of problems in understanding was reported for the dietary intake dimension, especially for snacking in front of the TV. Some difficulty was also found regarding the question on leisure PA. Test-retest agreement was observed in 54.7-100% of the respondents. Overall, the kappa coefficients were favorable. Thus, the parent self-report questionnaire is a valid and accurate tool for analyzing children's lifestyle habits in France.