RESUMEN

BACKGROUND: HIV-1 has well-established mechanisms to disrupt essential pathways in people with HIV, such as inflammation and metabolism. Moreover, diversity of the amino acid sequences in fundamental HIV-1 proteins including Tat and Vif, have been linked to dysregulating these pathways, and subsequently influencing clinical outcomes in people with HIV. However, the relationship between Tat and Vif amino acid sequence variation and specific immune markers and metabolites of the tryptophan-kynurenine (Trp-Kyn) pathway remains unclear. Therefore, this study aimed to investigate the relationship between Tat/Vif amino acid sequence diversity and Trp-Kyn metabolites (quinolinic acid (QUIN), Trp, kynurenic acid (KA), Kyn and Trp/Kyn ratio), as well as specific immune markers (sCD163, suPAR, IL-6, NGAL and hsCRP) in n = 67 South African cART-naïve people with HIV. METHODS: Sanger sequencing was used to determine blood-derived Tat/Vif amino acid sequence diversity. To measure Trp-Kyn metabolites, a LC-MS/MS metabolomics platform was employed using a targeted approach. To measure immune markers, Enzyme-linked immunosorbent assays and the Particle-enhanced turbidimetric assay was used. RESULTS: After adjusting for covariates, sCD163 (p = 0.042) and KA (p = 0.031) were higher in participants with Tat signatures N24 and R57, respectively, and amino acid variation at position 24 (adj R2 = 0.048, ß = -0.416, p = 0.042) and 57 (adj R2 = 0.166, ß = 0.535, p = 0.031) of Tat were associated with sCD163 and KA, respectively. CONCLUSIONS: These preliminary findings suggest that amino acid variation in Tat may have an influence on underlying pathogenic HIV-1 mechanisms and therefore, this line of work merits further investigation.

Asunto(s)

Infecciones por VIH , VIH-1 , Inflamación , Quinurenina , Triptófano , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Humanos , Triptófano/metabolismo , Infecciones por VIH/virología , Infecciones por VIH/genética , Masculino , VIH-1/genética , Adulto , Femenino , Quinurenina/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Persona de Mediana Edad , Biomarcadores/sangre , Receptores de Superficie Celular , Antígenos de Diferenciación Mielomonocítica , Antígenos CD

RESUMEN

AIDS is a serious disease with impaired immune function caused by human immunodeficiency virus (HIV) infection. The treatment of AIDS has always been the focus of global scientific research, and Tat protein is a key regulatory protein in the process of HIV infection. Its high expression is closely related to virus replication, disease progression, etc. The aim of this study is to explore the molecular mechanism of regulating Tat protein expression by using network pharmacology based traditional Chinese medicine for calming the liver and detoxifying. 129 AIDS patients were enrolled in the study and randomly divided into HAART combined with PGJDP treatment and HAART alone treatment groups. The virological response rate, immunological response status (CD4 + T cell level, CD4/CD8) and incidence of abnormal liver function were observed before and 48 weeks after treatment. Using the TCMSP database to obtain the chemical components and targets of the main traditional Chinese medicine components in PGJDP, clinical results indicate that the combination of HAART and PGJDP treatment can improve the virological response rate (P > 0.05); Increase the number of CD4 + T lymphocytes (P > 0.05); Significantly increased CD4/CD8 ratio (P < 0.01); Simultaneously, it significantly reduced the incidence of liver dysfunction (P < 0.01). After screening and analysis, the Chinese herbal medicine for calming liver and detoxifying has the potential to significantly regulate the expression of Tat protein. These Chinese herbal compounds can reduce the expression of Tat protein by affecting key pathways and regulating the expression of related genes, which has potential therapeutic effects on the treatment of AIDS.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida , Terapia Antirretroviral Altamente Activa , Medicamentos Herbarios Chinos , Medicina Tradicional China , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Humanos , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Masculino , Medicamentos Herbarios Chinos/farmacología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Medicina Tradicional China/métodos , Femenino , Adulto , Farmacología en Red , Persona de Mediana Edad , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos

RESUMEN

The human immunodeficiency virus (HIV-1) is highly dependent on a variety of host factors. Beside proteins, host RNA molecules are reported to aid HIV-1 replication and latency maintenance. Here, we implement multiple workflows of native RNA immunoprecipitation and sequencing (nRIPseq) to determine direct host RNA interaction partners of all 18 HIV-1 (poly)proteins. We identify 1,727 HIV-1 protein - human RNA interactions in the Jurkat cell line and 1,558 interactions in SupT1 cells for a subset of proteins, and discover distinct cellular pathways that seem to be used or controlled by HIV-1 on the RNA level: Tat binds mRNAs of proteins involved in the super elongation complex (AFF1-4, Cyclin-T1). Correlation of the interaction scores (based on binding abundancy) allows identifying the highest confidence interactions, for which we perform a small-scale knockdown screen that leads to the identification of three HIV-1 protein binding RNA interactors involved in HIV-1 replication (AFF2, H4C9 and RPLP0).

Asunto(s)

VIH-1 , Proteoma , Replicación Viral , Humanos , VIH-1/genética , VIH-1/metabolismo , VIH-1/fisiología , Células Jurkat , Proteoma/metabolismo , Replicación Viral/genética , Interacciones Huésped-Patógeno/genética , Unión Proteica , Ciclina T/metabolismo , Ciclina T/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Infecciones por VIH/virología , Infecciones por VIH/metabolismo , Infecciones por VIH/genética , ARN Viral/metabolismo , ARN Viral/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética

RESUMEN

Kaposi sarcoma (KS) is a neoplasm of vascular origin that promotes angiogenesis and the growth of endothelial cells triggered by the Kaposi Sarcoma-associated Herpes Virus (KSHV). When associated with HIV, KSHV becomes more aggressive and rapidly evolves. The HIV-1 TAT protein can be essential in developing AIDS-associated KS by promoting angiogenesis and increasing KSHV replication. Therefore, we evaluated the genetic profile of the first exon of tat gene among groups of people living with HIV (PLHIV) with (case group, n = 36) or without KS, this later with (positive control group, n = 46) and without KSHV infection (negative control group, n = 24); all individuals under antiretroviral therapy. The genetic diversity, the DN/DS ratio, and the genetic entropy of the first exon of tat were higher in the case group, followed by the positive control group, which was higher than the negative control group. The number of tat codons under positive selection was seven in the case group, six in the positive control group, and one in the negative control group. The prevalence of HIV viral loads below the detection limit was equal in the case and positive control groups, which were lower than in the negative control group. The mean CD4+ T cell counts were higher in the negative control group, followed by the positive control group, and followed by the case group. These results emphasize the negative influence of KSHV in antiretroviral treatment, as well as the HIV-specific TAT profile among PLHIV who developed KS.

Asunto(s)

Coinfección , Infecciones por VIH , Herpesvirus Humano 8 , Sarcoma de Kaposi , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Humanos , Sarcoma de Kaposi/virología , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Masculino , Herpesvirus Humano 8/genética , Femenino , Adulto , Persona de Mediana Edad , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Coinfección/virología , Coinfección/tratamiento farmacológico , VIH-1/genética , VIH-1/efectos de los fármacos , Variación Genética , Carga Viral , Antirretrovirales/uso terapéutico , Recuento de Linfocito CD4

RESUMEN

Caffeine is one of the most popular consumed psychostimulants that mitigates several neurodegenerative diseases. Nevertheless, the roles and molecular mechanisms of caffeine in HIV-associated neurocognitive disorders (HAND) remain largely unclear. Transactivator of transcription (Tat) is a major contributor to the neuropathogenesis of HAND in the central nervous system. In the present study, we determined that caffeine (100 µM) treatment significantly ameliorated Tat-induced decreased astrocytic viability, oxidative stress, inflammatory response and excessive glutamate and ATP release, thereby protecting neurons from apoptosis. Subsequently, SIRT3 was demonstrated to display neuroprotective effects against Tat during caffeine treatment. In addition, Tat downregulated SIRT3 expression via activation of EGR1 signaling, which was reversed by caffeine treatment in astrocytes. Overexpression of EGR1 entirely abolished the neuroprotective effects of caffeine against Tat. Furthermore, counteracting Tat or caffeine-induced differential expression of SIRT3 abrogated the neuroprotection of caffeine against Tat-triggered astrocytic dysfunction and neuronal apoptosis. Taken together, our study establishes that caffeine ameliorates astrocytes-mediated Tat neurotoxicity by targeting EGR1/SIRT3 signaling pathway. Our findings highlight the beneficial effects of caffeine on Tat-induced astrocytic dysfunction and neuronal death and propose that caffeine might be a novel therapeutic drug for relief of HAND.

Asunto(s)

Apoptosis , Astrocitos , Cafeína , Proteína 1 de la Respuesta de Crecimiento Precoz , VIH-1 , Neuronas , Transducción de Señal , Sirtuina 3 , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/virología , Astrocitos/patología , Sirtuina 3/genética , Sirtuina 3/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Transducción de Señal/efectos de los fármacos , Cafeína/farmacología , Humanos , Apoptosis/efectos de los fármacos , VIH-1/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Neuronas/virología , Regulación hacia Arriba/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Complejo SIDA Demencia/tratamiento farmacológico , Complejo SIDA Demencia/genética , Complejo SIDA Demencia/metabolismo , Complejo SIDA Demencia/patología , Estimulantes del Sistema Nervioso Central/farmacología , Estimulantes del Sistema Nervioso Central/toxicidad

RESUMEN

Antiretroviral treatments have notably extended the lives of individuals with HIV and reduced the occurrence of comorbidities, including ocular manifestations. The involvement of endoplasmic reticulum (ER) stress in HIV-1 pathogenesis raises questions about its correlation with cellular senescence or its role in initiating senescent traits. This study investigated how ER stress and dysregulated autophagy impact cellular senescence triggered by HIV-1 Tat in the MIO-M1 cell line (human Müller glial cells). Cells exposed to HIV-1 Tat exhibited increased vimentin expression combined with markers of ER stress (BiP, p-eIF2α), autophagy (LC3, Beclin-1, p62), and the senescence marker p21 compared to control cells. Western blotting and staining techniques like SA-ß-gal were employed to examine these markers. Additionally, treatments with ER stress inhibitor 4-PBA before HIV-1 Tat exposure led to a decreased expression of ER stress, senescence, and autophagy markers. Conversely, pre-treatment with the autophagy inhibitor 3-MA resulted in reduced autophagy and senescence markers but did not alter ER stress markers compared to control cells. The findings suggest a link between ER stress, dysregulated autophagy, and the initiation of a senescence phenotype in MIO-M1 cells induced by HIV-1 Tat exposure.

Asunto(s)

Autofagia , Senescencia Celular , Estrés del Retículo Endoplásmico , VIH-1 , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Humanos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , VIH-1/fisiología , Línea Celular , Células Ependimogliales/metabolismo , Células Ependimogliales/virología , Infecciones por VIH/virología

RESUMEN

Limited cellular levels of the HIV transcriptional activator Tat are one contributor to proviral latency that might be targeted in HIV cure strategies. We recently demonstrated that lipid nanoparticles containing HIV tat mRNA induce HIV expression in primary CD4 T cells. Here, we sought to further characterize tat mRNA in the context of several benchmark latency reversal agents (LRAs), including inhibitor of apoptosis protein antagonists (IAPi), bromodomain and extra-Terminal motif inhibitors (BETi), and histone deacetylase inhibitors (HDACi). tat mRNA reversed latency across several different cell line models of HIV latency, an effect dependent on the TAR hairpin loop. Synergistic enhancement of tat mRNA activity was observed with IAPi, HDACi, and BETi, albeit to variable degrees. In primary CD4 T cells from durably suppressed people with HIV, tat mRNA profoundly increased the frequencies of elongated, multiply-spliced, and polyadenylated HIV transcripts, while having a lesser impact on TAR transcript frequencies. tat mRNAs alone resulted in variable HIV p24 protein induction across donors. However, tat mRNA in combination with IAPi, BETi, or HDACi markedly enhanced HIV RNA and protein expression without overt cytotoxicity or cellular activation. Notably, combination regimens approached or in some cases exceeded the latency reversal activity of maximal mitogenic T cell stimulation. Higher levels of tat mRNA-driven HIV p24 induction were observed in donors with larger mitogen-inducible HIV reservoirs, and expression increased with prolonged exposure time. Combination LRA strategies employing both small molecule inhibitors and Tat delivered to CD4 T cells are a promising approach to effectively target the HIV reservoir.

Asunto(s)

Infecciones por VIH , VIH-1 , Inhibidores de Histona Desacetilasas , Nanopartículas , Latencia del Virus , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Humanos , Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Linfocitos T CD4-Positivos/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Antígenos VIH/genética , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Latencia del Virus/efectos de los fármacos

RESUMEN

Bacteria-derived outer membrane vesicles (OMVs) can be engineered to incorporate foreign antigens. This study explored the potential of ClearColi™-derived OMVs as a natural adjuvant and a carrier (recombinant OMVs or rOMVs) for development of an innovative therapeutic vaccine candidate harboring HIV-1 Nef and Nef-Tat antigens. Herein, the rOMVs containing CytolysinA (ClyA)-Nef and ClyA-Nef-Tat fusion proteins were isolated from ClearColi™ strain. The presence of Nef and Nef-Tat proteins on their surface (rOMVNef and rOMVNef-Tat) was confirmed by western blotting after proteinase K treatment. Immune responses induced by Nef and Nef-Tat proteins emulsified with Montanide® ISA720 or mixed with OMVs, and also rOMVNef and rOMVNef-Tat were investigated in BALB/c mice. Additionally, the potency of splenocytes exposed to single-cycle replicable (SCR) HIV-1 virions was assessed for the secretion of cytokines in vitro. Our findings showed that the rOMVs as an antigen carrier (rOMVNef and rOMVNef-Tat) induced higher levels of IgG2a, IFN-γ and granzyme B compared to OMVs as an adjuvant (Nef + OMV and Nef-Tat + OMV), and also Montanide® ISA720 (Nef + Montanide and Nef-Tat + Montanide). Moreover, IFN-γ level in splenocytes isolated from mice immunized with rOMVNef-Tat was higher than other regimens after exposure to SCR virions. Generally, ClearColi™-derived rOMVs can serve as potent carriers for developing effective vaccines against HIV-1 infection.

Asunto(s)

Vacunas contra el SIDA , Adyuvantes Inmunológicos , Infecciones por VIH , VIH-1 , Ratones Endogámicos BALB C , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Animales , Vacunas contra el SIDA/inmunología , Vacunas contra el SIDA/genética , VIH-1/genética , VIH-1/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Ratones , Adyuvantes Inmunológicos/administración & dosificación , Infecciones por VIH/prevención & control , Infecciones por VIH/inmunología , Femenino , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunología , Citocinas/metabolismo , Inmunoglobulina G/sangre , Anticuerpos Anti-VIH/inmunología , Membrana Externa Bacteriana/metabolismo , Desarrollo de Vacunas , Humanos , Portadores de Fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Bazo/inmunología

RESUMEN

The cannabinoid receptor type 1 (CB1R) is a promising therapeutic target for various neurodegenerative diseases, including HIV-1-associated neurocognitive disorder (HAND). However, the therapeutic potential of CB1R by direct activation is limited due to its psychoactive side effects. Therefore, research has focused on indirectly activating the CB1R by utilizing positive allosteric modulators (PAMs). Studies have shown that CB1R PAMs (ZCZ011 and GAT211) are effective in mouse models of Huntington's disease and neuropathic pain, and hence, we assess the therapeutic potential of ZCZ011 in a well-established mouse model of neuroHIV. The current study investigates the effect of chronic ZCZ011 treatment (14 days) on various behavioral paradigms and the endocannabinoid system in HIV-1 Tat transgenic female and male mice. Chronic ZCZ011 treatment (10 mg/kg) did not alter body mass, locomotor activity, or anxiety-like behavior regardless of sex or genotype. However, differential effects were noted in hot plate latency, motor coordination, and recognition memory in female mice only, with ZCZ011 treatment increasing hot plate latency and improving motor coordination and recognition memory. Only minor effects or no alterations were seen in the endocannabinoid system and related lipids except in the cerebellum, where the effect of ZCZ011 was more pronounced in female mice. Moreover, AEA and PEA levels in the cerebellum were positively correlated with improved motor coordination in female mice. In summary, these findings indicate that chronic ZCZ011 treatment has differential effects on antinociception, motor coordination, and memory, based on sex and HIV-1 Tat expression, making CB1R PAMs potential treatment options for HAND without the psychoactive side effects.

Asunto(s)

Endocannabinoides , Ratones Transgénicos , Receptor Cannabinoide CB1 , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Animales , Femenino , Masculino , Endocannabinoides/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB1/genética , Ratones , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Modelos Animales de Enfermedad

RESUMEN

BACKGROUND: HIV-1 produces Tat, a crucial protein for transcription, viral replication, and CNS neurotoxicity. Tat interacts with TAR, enhancing HIV reverse transcription. Subtype C Tat variants (C31S, R57S, Q63E) are associated with reduced transactivation and neurovirulence compared to subtype B. However, their precise impact on Tat-TAR binding is unclear. This study investigates how these substitutions affect Tat-TAR interaction. METHODS: We utilized molecular modelling techniques, including MODELLER, to produce precise three-dimensional structures of HIV-1 Tat protein variants. We utilized Tat subtype B as the reference or wild type, and generated Tat variants to mirror those amino acid variants found in Tat subtype C. Subtype C-specific amino acid substitutions were selected based on their role in the neuropathogenesis of HIV-1. Subsequently, we conducted molecular docking of each Tat protein variant to TAR using HDOCK, followed by molecular dynamic simulations. RESULTS: Molecular docking results indicated that Tat subtype B (TatWt) showed the highest affinity for the TAR element (-262.07), followed by TatC31S (-261.61), TatQ63E (-256.43), TatC31S/R57S/Q63E (-238.92), and TatR57S (-222.24). However, binding free energy analysis showed higher affinities for single variants TatQ63E (-349.2 ± 10.4 kcal/mol) and TatR57S (-290.0 ± 9.6 kcal/mol) compared to TatWt (-247.9 ± 27.7 kcal/mol), while TatC31S and TatC31S/R57SQ/63E showed lower values. Interactions over the protein trajectory were also higher for TatQ63E and TatR57S compared to TatWt, TatC31S, and TatC31S/R57SQ/63E, suggesting that modifying amino acids within the Arginine/Glutamine-rich region notably affects TAR interaction. Single amino acid mutations TatR57S and TatQ63E had a significant impact, while TatC31S had minimal effect. Introducing single amino acid variants from TatWt to a more representative Tat subtype C (TatC31S/R57SQ/63E) resulted in lower predicted binding affinity, consistent with previous findings. CONCLUSIONS: These identified amino acid positions likely contribute significantly to Tat-TAR interaction and the differential pathogenesis and neuropathogenesis observed between subtype B and subtype C. Additional experimental investigations should prioritize exploring the influence of these amino acid signatures on TAR binding to gain a comprehensive understanding of their impact on viral transactivation, potentially identifying them as therapeutic targets.

Asunto(s)

Sustitución de Aminoácidos , VIH-1 , Simulación de Dinámica Molecular , Unión Proteica , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , VIH-1/genética , VIH-1/clasificación , VIH-1/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Duplicado del Terminal Largo de VIH/genética , Aminoácidos/genética , Aminoácidos/metabolismo , Modelos Moleculares

RESUMEN

BACKGROUND: Heterologous combinations in vaccine design are an effective approach to promote T cell activity and antiviral effects. The goal of this study was to compare the homologous and heterologous regimens targeting the Nef-Tat fusion antigen to develop a human immunodeficiency virus-1 (HIV-1) therapeutic vaccine candidate. METHODS: At first, the DNA and protein constructs harboring HIV-1 Nef and the first exon of Tat as linked form (pcDNA-nef-tat and Nef-Tat protein) were prepared in large scale and high purity. The generation of the Nef-Tat protein was performed in the E. coli expression system using an IPTG inducer. Then, we evaluated and compared immune responses of homologous DNA prime/ DNA boost, homologous protein prime/ protein boost, and heterologous DNA prime/protein boost regimens in BALB/c mice. Finally, the ability of mice splenocytes to secret cytokines after exposure to single-cycle replicable (SCR) HIV-1 was compared between immunized and control groups in vitro. RESULTS: The nef-tat gene was successfully subcloned in eukaryotic pcDNA3.1 (-) and prokaryotic pET-24a (+) expression vectors. The recombinant Nef-Tat protein was generated in the E. coli Rosetta strain under optimized conditions as a clear band of ~ 35 kDa detected on SDS-PAGE. Moreover, transfection of pcDNA-nef-tat into HEK-293T cells was successfully performed using Lipofectamine 2000, as confirmed by western blotting. The immunization studies showed that heterologous DNA prime/protein boost regimen could significantly elicit the highest levels of Ig- G2a, IFN-γ, and Granzyme B in mice as compared to homologous DNA/DNA and protein/protein regimens. Moreover, the secretion of IFN-γ was higher in DNA/protein regimens than in DNA/DNA and protein/protein regimens after exposure of mice splenocytes to SCR HIV-1 in vitro. CONCLUSION: The chimeric HIV-1 Nef-Tat antigen was highly immunogenic, especially when applied in a heterologous prime/ boost regimen. This regimen could direct immune response toward cellular immunity (Th1 and CTL activity) and increase IFN-γ secretion after virus exposure.

Asunto(s)

Vacunas contra el SIDA , VIH-1 , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión , Vacunas de ADN , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Animales , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Vacunas contra el SIDA/inmunología , Vacunas contra el SIDA/genética , Vacunas de ADN/inmunología , Vacunas de ADN/genética , VIH-1/inmunología , VIH-1/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Ratones , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Humanos , Femenino , Linfocitos T/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Inmunización Secundaria , Citocinas/metabolismo , Escherichia coli/genética , Escherichia coli/inmunología

RESUMEN

Antigen presenting cells (APCs)-derived exosomes are nano-vesicles that can induce antigen-specific T cell responses, and possess therapeutic effects in clinical settings. Moreover, dendritic cells (DCs)-based vaccines have been developed to combat human immunodeficiency virus-1 (HIV-1) infection in preclinical and clinical trials. We investigated the immunostimulatory effects (B- and T-cells activities) of DCs- and exosomes-based vaccine constructs harboring HIV-1 Nefmut-Tat fusion protein as an antigen candidate and heat shock protein 70 (Hsp70) as an adjuvant in mice. The modified DCs and engineered exosomes harboring Nefmut-Tat protein or Hsp70 were prepared using lentiviral vectors compared to electroporation, characterized and evaluated by in vitro and in vivo immunological tests. Our data indicated that the engineered exosomes induced high levels of total IgG, IgG2a, IFN-γ, TNF-α and Granzyme B. Moreover, co-injection of exosomes harboring Hsp70 could significantly increase the secretion of antibodies, cytokines and Granzyme B. The highest levels of IFN-γ and TNF-α were observed in exosomes harboring Nefmut-Tat combined with exosomes harboring Hsp70 (Exo-Nefmut-Tat + Exo-Hsp70) regimen after single-cycle replicable (SCR) HIV-1 exposure. Generally, Exo-Nefmut-Tat + Exo-Hsp70 regimen can be considered as a promising safe vaccine candidate due to high T-cells (Th1 and CTL) activity and its maintenance against SCR HIV-1 exposure.

Asunto(s)

Vacunas contra el SIDA , Células Dendríticas , Exosomas , VIH-1 , Proteínas HSP70 de Choque Térmico , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Exosomas/inmunología , Exosomas/metabolismo , Células Dendríticas/inmunología , Animales , VIH-1/inmunología , VIH-1/genética , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/genética , Vacunas contra el SIDA/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Ratones , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Humanos , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Citocinas/metabolismo

RESUMEN

HIV-1 infection remains a public health problem with no cure. Although antiretroviral therapy (ART) is effective for suppressing HIV-1 replication, it requires lifelong drug administration due to a stable reservoir of latent proviruses and may cause serious side effects and drive the emergence of drug-resistant HIV-1 variants. Gene therapy represents an alternative approach to overcome the limitations of conventional treatments against HIV-1 infection. In this study, we constructed and investigated the antiviral effects of an HIV-1 Tat-dependent conditionally replicating adenovirus, which selectively replicates and expresses the diphtheria toxin A chain (Tat-CRAds-DTA) in HIV-1-infected cells both in vitro and in vivo. We found that Tat-CRAds-DTA could specifically induce cell death and inhibit virus replication in HIV-1-infected cells mediated by adenovirus proliferation and DTA expression. A low titer of progeny Tat-CRAds-DTA was also detected in HIV-1-infected cells. In addition, Tat-CRAds-DTA showed no apparent cytotoxicity to HIV-1-negative cells and demonstrated significant therapeutic efficacy against HIV-1 infection in a humanized mouse model. The findings in this study highlight the potential of Tat-CRAds-DTA as a new gene therapy for the treatment of HIV-1 infection.

Asunto(s)

Adenoviridae , Toxina Diftérica , Terapia Genética , Vectores Genéticos , Infecciones por VIH , VIH-1 , Replicación Viral , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Humanos , VIH-1/genética , Toxina Diftérica/genética , Animales , Adenoviridae/genética , Infecciones por VIH/terapia , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Ratones , Terapia Genética/métodos , Vectores Genéticos/genética , Modelos Animales de Enfermedad , Línea Celular , Células HEK293 , Expresión Génica , Fragmentos de Péptidos

RESUMEN

The human immunodeficiency virus (HIV) integrates into the host genome forming latent cellular reservoirs that are an obstacle for cure or remission strategies. Viral transcription is the first step in the control of latency and depends upon the hijacking of the host cell RNA polymerase II (Pol II) machinery by the 5' HIV LTR. Consequently, "block and lock" or "shock and kill" strategies for an HIV cure depend upon a full understanding of HIV transcriptional control. The HIV trans-activating protein, Tat, controls HIV latency as part of a positive feed-forward loop that strongly activates HIV transcription. The recognition of the TATA box and adjacent sequences of HIV essential for Tat trans-activation (TASHET) of the core promoter by host cell pre-initiation complexes of HIV (PICH) has been shown to be necessary for Tat trans-activation, yet the protein composition of PICH has remained obscure. Here, DNA-affinity chromatography was employed to identify the mitotic deacetylase complex (MiDAC) as selectively recognizing TASHET. Using biophysical techniques, we show that the MiDAC subunit DNTTIP1 binds directly to TASHET, in part via its CTGC DNA motifs. Using co-immunoprecipitation assays, we show that DNTTIP1 interacts with MiDAC subunits MIDEAS and HDAC1/2. The Tat-interacting protein, NAT10, is also present in HIV-bound MiDAC. Gene silencing revealed a functional role for DNTTIP1, MIDEAS, and NAT10 in HIV expression in cellulo. Furthermore, point mutations in TASHET that prevent DNTTIP1 binding block the reactivation of HIV by latency reversing agents (LRA) that act via the P-TEFb/7SK axis. Our data reveal a key role for MiDAC subunits DNTTIP1, MIDEAS, as well as NAT10, in Tat-activated HIV transcription and latency. DNTTIP1, MIDEAS and NAT10 emerge as cell cycle-regulated host cell transcription factors that can control activated HIV gene expression, and as new drug targets for HIV cure strategies.

Asunto(s)

Regulación Viral de la Expresión Génica , Infecciones por VIH , VIH-1 , Regiones Promotoras Genéticas , Latencia del Virus , Humanos , VIH-1/genética , VIH-1/fisiología , Infecciones por VIH/virología , Infecciones por VIH/metabolismo , Infecciones por VIH/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Transcripción Viral

RESUMEN

Fragile X syndrome (FXS) arises from the loss of fragile X messenger ribonucleoprotein (FMRP) needed for normal neuronal excitability and circuit functions. Recent work revealed that FMRP contributes to mossy fiber long-term potentiation by adjusting the Kv4 A-type current availability through interactions with a Cav3-Kv4 ion channel complex, yet the mechanism has not yet been defined. In this study using wild-type and Fmr1 knock-out (KO) tsA-201 cells and cerebellar sections from male Fmr1 KO mice, we show that FMRP associates with all subunits of the Cav3.1-Kv4.3-KChIP3 complex and is critical to enabling calcium-dependent shifts in Kv4.3 inactivation to modulate the A-type current. Specifically, upon depolarization Cav3 calcium influx activates dual-specific phosphatase 1/6 (DUSP1/6) to deactivate ERK1/2 (ERK) and lower phosphorylation of Kv4.3, a signaling pathway that does not function in Fmr1 KO cells. In Fmr1 KO mouse tissue slices, cerebellar granule cells exhibit a hyperexcitable response to membrane depolarizations. Either incubating Fmr1 KO cells or in vivo administration of a tat-conjugated FMRP N-terminus fragment (FMRP-N-tat) rescued Cav3-Kv4 function and granule cell excitability, with a decrease in the level of DUSP6. Together these data reveal a Cav3-activated DUSP signaling pathway critical to the function of a FMRP-Cav3-Kv4 complex that is misregulated in Fmr1 KO conditions. Moreover, FMRP-N-tat restores function of this complex to rescue calcium-dependent control of neuronal excitability as a potential therapeutic approach to alleviating the symptoms of FXS.

Asunto(s)

Calcio , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Ratones Noqueados , Neuronas , Animales , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Ratones , Masculino , Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/fisiopatología , Neuronas/metabolismo , Calcio/metabolismo , Ratones Endogámicos C57BL , Canales de Potasio Shal/metabolismo , Canales de Potasio Shal/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo

RESUMEN

Antiretroviral therapy-naive people living with HIV possess less fat than people without HIV. Previously, we found that HIV-1 transactivator of transcription (TAT) decreases fat in ob/ob mice. The TAT38 (a.a. 20-57) is important in the inhibition of adipogenesis and contains three functional domains: Cys-ZF domain (a.a. 20-35 TACTNCYCAKCCFQVC), core-domain (a.a. 36-46, FITKALGISYG), and protein transduction domain (PTD)(a.a. 47-57, RAKRRQRRR). Interestingly, the TAT38 region interacts with the Cyclin T1 of the P-TEFb complex, of which expression increases during adipogenesis. The X-ray crystallographic structure of the complex showed that the Cys-ZF and the core domain bind to the Cyclin T1 via hydrophobic interactions. To prepare TAT38 mimics with structural and functional similarities to TAT38, we replaced the core domain with a hydrophobic aliphatic amino acid (from carbon numbers 5 to 8). The TAT38 mimics with 6-hexanoic amino acid (TAT38 Ahx (C6)) and 7-heptanoic amino acid (TAT38 Ahp (C7)) inhibited adipogenesis of 3T3-L1 potently, reduced cellular triglyceride content, and decreased body weight of diet-induced obese (DIO) mice by 10.4-11 % in two weeks. The TAT38 and the TAT38 mimics potently repressed the adipogenic transcription factors genes, C/EBPα, PPARγ, and SREBP1. Also, they inhibit the phosphorylation of PPARγ. The TAT peptides may be promising candidates for development into a drug against obesity or diabetes.

Asunto(s)

Adipogénesis , PPAR gamma , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Animales , PPAR gamma/metabolismo , Adipogénesis/efectos de los fármacos , Ratones , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/genética , Células 3T3-L1 , Humanos , Regulación de la Expresión Génica , Ratones Obesos , Masculino , Ciclina T/metabolismo , Obesidad/metabolismo , Adipocitos/metabolismo , Ratones Endogámicos C57BL , Proteínas Potenciadoras de Unión a CCAAT

RESUMEN

Viruses provide vital insights into gene expression control. Viral transactivators, with other viral and cellular proteins, regulate expression of self, other viruses, and host genes with profound effects on infected cells, underlying inflammation, control of immune responses, and pathogenesis. The multifunctional Tat proteins of lentiviruses (HIV-1, HIV-2, and SIV) transactivate gene expression by recruiting host proteins and binding to transacting responsive regions (TARs) in viral and host RNAs. SARS-CoV-2 nucleocapsid participates in early viral transcription, recruits similar cellular proteins, and shares intracellular, surface, and extracellular distribution with Tat. SARS-CoV-2 nucleocapsid interacting with the replication-transcription complex might, therefore, transactivate viral and cellular RNAs in the transcription and reactivation of self and other viruses, acute and chronic pathogenesis, immune evasion, and viral evolution. Here, we show, by using primary and secondary structural comparisons, that the leaders of SARS-CoV-2 and other coronaviruses contain TAR-like sequences in stem-loops 2 and 3. The coronaviral nucleocapsid C-terminal domains harbor a region of similarity to TAR-binding regions of lentiviral Tat proteins, and coronaviral nonstructural protein 12 has a cysteine-rich metal binding, dimerization domain, as do lentiviral Tat proteins. Although SARS-CoV-1 nucleocapsid transactivated gene expression in a replicon-based study, further experimental evidence for coronaviral transactivation and its possible implications is warranted.

Asunto(s)

COVID-19 , VIH-1 , Humanos , VIH-1/fisiología , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Activación Transcripcional , Duplicado del Terminal Largo de VIH , COVID-19/genética , Productos del Gen tat/genética , Lentivirus/genética , Expresión Génica , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , ARN Viral/metabolismo

RESUMEN

Opioid overdose deaths have dramatically increased by 781% from 1999 to 2021. In the setting of HIV, opioid drug abuse exacerbates neurotoxic effects of HIV in the brain, as opioids enhance viral replication, promote neuronal dysfunction and injury, and dysregulate an already compromised inflammatory response. Despite the rise in fentanyl abuse and the close association between opioid abuse and HIV infection, the interactive comorbidity between fentanyl abuse and HIV has yet to be examined in vivo. The HIV-1 Tat-transgenic mouse model was used to understand the interactive effects between fentanyl and HIV. Tat is an essential protein produced during HIV that drives the transcription of new virions and exerts neurotoxic effects within the brain. The Tat-transgenic mouse model uses a glial fibrillary acidic protein (GFAP)-driven tetracycline promoter which limits Tat production to the brain and this model is well used for examining mechanisms related to neuroHIV. After 7 days of fentanyl exposure, brains were harvested. Tight junction proteins, the vascular cell adhesion molecule, and platelet-derived growth factor receptor-ß were measured to examine the integrity of the blood brain barrier. The immune response was assessed using a mouse-specific multiplex chemokine assay. For the first time in vivo, we demonstrate that fentanyl by itself can severely disrupt the blood-brain barrier and dysregulate the immune response. In addition, we reveal associations between inflammatory markers and tight junction proteins at the blood-brain barrier.

Asunto(s)

Barrera Hematoencefálica , Fentanilo , VIH-1 , Ratones Transgénicos , Enfermedades Neuroinflamatorias , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/virología , Ratones , Fentanilo/farmacología , VIH-1/efectos de los fármacos , VIH-1/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Enfermedades Neuroinflamatorias/genética , Enfermedades Neuroinflamatorias/patología , Enfermedades Neuroinflamatorias/virología , Infecciones por VIH/virología , Infecciones por VIH/genética , Infecciones por VIH/patología , Infecciones por VIH/tratamiento farmacológico , Modelos Animales de Enfermedad , Analgésicos Opioides/farmacología , Analgésicos Opioides/efectos adversos , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Proteínas de Uniones Estrechas/genética , Humanos , Encéfalo/efectos de los fármacos , Encéfalo/virología , Encéfalo/metabolismo , Encéfalo/patología , Trastornos Relacionados con Opioides/genética , Trastornos Relacionados con Opioides/patología , Trastornos Relacionados con Opioides/metabolismo

RESUMEN

Despite the success of combination antiretroviral therapy, people living with human immunodeficiency virus (HIV) still have an increased risk of Epstein-Barr virus (EBV)-associated B cell malignancies. In the HIV setting, B cell physiology is altered by coexistence with HIV-infected cells and the chronic action of secreted viral proteins, for example, HIV-1 Tat that, once released, efficiently penetrates noninfected cells. We modeled the chronic action of HIV-1 Tat on B cells by ectopically expressing Tat or TatC22G mutant in two lymphoblastoid B cell lines. The RNA-sequencing analysis revealed that Tat deregulated the expression of hundreds of genes in B cells, including the downregulation of a subset of major histocompatibility complex (MHC) class II-related genes. Tat-induced downregulation of HLA-DRB1 and HLA-DRB5 genes led to a decrease in HLA-DR surface expression; this effect was reproduced by coculturing B cells with Tat-expressing T cells. Chronic Tat presence decreased the NF-ᴋB pathway activity in B cells; this downregulated NF-ᴋB-dependent transcriptional targets, including MHC class II genes. Notably, HLA-DRB1 and surface HLA-DR expression was also decreased in B cells from people with HIV. Tat-induced HLA-DR downregulation in B cells impaired EBV-specific CD4+ T cell response, which contributed to the escape from immune surveillance and could eventually promote B cell lymphomagenesis in people with HIV.

Asunto(s)

Linfocitos B , Infecciones por Virus de Epstein-Barr , Infecciones por VIH , Linfoma , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Humanos , Regulación hacia Abajo , Herpesvirus Humano 4/genética , Infecciones por VIH/genética , VIH-1/genética , Cadenas HLA-DRB1 , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética

RESUMEN

During the antiretroviral era, individuals living with HIV continue to experience milder forms of HIV-associated neurocognitive disorder (HAND). Viral proteins, including Tat, play a pivotal role in the observed alterations within the central nervous system (CNS), with mitochondrial dysfunction emerging as a prominent hallmark. As a result, our objective was to examine the expression of genes associated with mitophagy and mitochondrial biogenesis in the brain exposed to the HIV-1 Tat protein. We achieved this by performing bilateral stereotaxic injections of 100 ng of HIV-1 Tat into the hippocampus of Sprague-Dawley rats, followed by immunoneuromagnetic cell isolation. Subsequently, we assessed the gene expression of Ppargc1a, Pink1, and Sirt1-3 in neurons using RT-qPCR. Additionally, to understand the role of Tert in telomeric dysfunction, we quantified the activity and expression of Tert. Our results revealed that only Ppargc1a, Pink1, and mitochondrial Sirt3 were downregulated in response to the presence of HIV-1 Tat in hippocampal neurons. Interestingly, we observed a reduction in the activity of Tert in the experimental group, while mRNA levels remained relatively stable. These findings support the compelling evidence of dysregulation in both mitophagy and mitochondrial biogenesis in neurons exposed to HIV-1 Tat, which in turn induces telomeric dysfunction.

Asunto(s)

Infecciones por VIH , VIH-1 , Trastornos Neurocognitivos , Sirtuina 3 , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Animales , Ratas , Productos del Gen tat/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Trastornos Neurocognitivos/metabolismo , Trastornos Neurocognitivos/virología , Neuronas/metabolismo , Biogénesis de Organelos , Proteínas Quinasas/metabolismo , Ratas Sprague-Dawley , Sirtuina 3/genética , Sirtuina 3/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma