RESUMEN
Recent evidence has shown that uncoating and reverse transcription precede nuclear import. These recent breakthroughs have been made possible through the development of innovative biochemical and imaging techniques. This method outlines the biochemical assay used for detecting the presence of the HIV-1 core in the nuclear compartment. In this procedure, human cells are infected with HIV-1NL4-3, with or without the inclusion of PF74, a small molecule that inhibits core entry into the nuclear compartment. Subsequently, cells are separated into cytosolic and nuclear fractions. To assess whether the capsid protein has reached the nuclear compartment, cytosolic and nuclear fractions are subjected to Western blot analysis, utilizing antibodies specific to the HIV-1 capsid protein p24. To validate the true origin of these fractions, Western blot analysis employing antibodies against cytosolic and nuclear markers are also performed. In summary, this assay provides a reliable and efficient means to detect the presence of the HIV-1 capsid protein in the nucleus during infection under various conditions.
Asunto(s)
Cápside , Infecciones por VIH , VIH-1 , Humanos , Western Blotting/métodos , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Proteína p24 del Núcleo del VIH/metabolismo , Proteína p24 del Núcleo del VIH/análisis , Infecciones por VIH/virología , Infecciones por VIH/metabolismo , Fenilalanina/metabolismo , Fenilalanina/análogos & derivadosRESUMEN
Assays to study HIV persistence are crucial to evaluate therapeutic strategies aimed toward an HIV cure. Several assays have been developed to date that rely on the measurement of nucleic acids. In recent years, the advancement of ultrasensitive technologies for the detection of proteins has improved our understanding of the role of translation-competent reservoirs in HIV persistence. In this chapter, we describe the development of an ultrasensitive p24 ELISA that uses planar array technology. This assay allows for the detection of HIV-1 p24 in the low fg/ml range in different biological matrixes, including cell lysates. This assay can be used to investigate the efficacy of latency reversing agents to reactivate HIV or to evaluate the persistence of translation-competent reservoirs in people living with HIV (PWH) in cells or diverse biological fluids.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Proteína p24 del Núcleo del VIH , Infecciones por VIH , VIH-1 , Humanos , Ensayo de Inmunoadsorción Enzimática/métodos , Proteína p24 del Núcleo del VIH/metabolismo , Proteína p24 del Núcleo del VIH/análisis , Infecciones por VIH/virología , Latencia del VirusRESUMEN
The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells' transcriptome and proteome profiles.
Asunto(s)
Productos del Gen tat , VIH-1 , Nanopartículas , ARN Viral , Latencia del Virus , Latencia del Virus/efectos de los fármacos , Latencia del Virus/genética , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , ARN Viral/administración & dosificación , ARN Viral/genética , ARN Viral/metabolismo , Nanopartículas/administración & dosificación , Nanopartículas/química , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Infecciones por VIH/virología , Panobinostat/farmacología , Terapia Antirretroviral Altamente Activa , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Antígenos CD4/genética , Antígenos CD4/metabolismo , VIH-1/efectos de los fármacos , VIH-1/genética , Provirus/efectos de los fármacos , Provirus/genética , Análisis de Expresión Génica de una Sola Célula , Proteína p24 del Núcleo del VIH/genética , Proteína p24 del Núcleo del VIH/metabolismo , ARN Largo no Codificante/metabolismo , Células Cultivadas , Humanos , Ionomicina/farmacologíaRESUMEN
While combination antiretroviral therapy maintains undetectable viremia in people living with HIV (PLWH), a lifelong treatment is necessary to prevent viremic rebound after therapy cessation. This rebound seemed mainly caused by long-lived HIV-1 latently infected cells reverting to a viral productive status. Reversing latency and elimination of these cells by the so-called shock-and-kill strategy is one of the main investigated leads to achieve an HIV-1 cure. Small molecules referred to as latency reversal agents (LRAs) proved to efficiently reactivate latent CD4+ T cells. However, the LRA impact on de novo infection or HIV-1 production in productively infected macrophages remains elusive. Nontoxic doses of bryostatin-1, JQ1, and romidepsin were investigated in human monocyte-derived macrophages (MDMs). Treatment with bryostatin-1 or romidepsin resulted in a downregulation of CD4 and CCR5 receptors, respectively, accompanied by a reduction of R5 tropic virus infection. HIV-1 replication was mainly regulated by receptor modulation for bryostatin-1, while romidepsin effects rely on upregulation of SAMHD1 activity. LRA stimulation of chronically infected cells did not enhance HIV-1 production or gene expression. Surprisingly, bryostatin-1 caused a major decrease in viral production. This effect was not viral strain specific but appears to occur only in myeloid cells. Bryostatin-1 treatment of infected MDMs led to decreased amounts of capsid and matrix mature proteins with little to no modulation of precursors. Our observations revealed that bryostatin-1-treated myeloid and CD4+ T cells respond differently upon HIV-1 infection. Therefore, additional studies are warranted to more fully assess the efficiency of HIV-1 eradicating strategies. IMPORTANCE HIV-1 persists in a cellular latent form despite therapy that quickly propagates infection upon treatment interruption. Reversing latency would contribute to eradicate these cells, closing the gap to a cure. Macrophages are an acknowledged HIV-1 reservoir during therapy and are suspected to harbor latency establishment in vivo. However, the impact of latency reversal agents (LRAs) on HIV-1 infection and viral production in human macrophages is poorly known but nonetheless crucial to probe the safety of this strategy. In this in vitro study, we discovered encouraging antireplicative features of distinct LRAs in human macrophages. We also described a new viral production inhibition mechanism by protein kinase C agonists that is specific to myeloid cells. This study provides new insights into HIV-1 propagation restriction potentials by LRAs in human macrophages and underline the importance of assessing latency reversal strategy on all HIV-1-targeted cells.
Asunto(s)
Fármacos Anti-VIH/farmacología , Brioestatinas/farmacología , VIH-1/efectos de los fármacos , Macrófagos/efectos de los fármacos , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Depsipéptidos/farmacología , Diterpenos/farmacología , Proteína p24 del Núcleo del VIH/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/virología , Receptores CCR5/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Effective function of CD8+ T cells and enhanced innate activation of DCs in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of the combination of 2´3´-c´diAM(PS)2 and Poly I:C as potential adjuvants capable of potentiating DC´s abilities to induce polyfunctional HIV-1 specific CD8+ T-cell responses in vitro and in vivo using a humanized BLT mouse model. Adjuvant combination enhanced TBK-1 phosphorylation and IL-12 and IFN-ß expression on DC and increased their ability to activate polyfunctional HIV-1-specific CD8+ T cells in vitro. Moreover, higher proportions of hBLT mice vaccinated with ADJ-DC exhibited less severe CD4+ T-cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to LN, and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, priming of DC with PolyI:C and STING agonists might be useful for future HIV-1 vaccine studies.
Asunto(s)
Vacunas contra el SIDA , VIH-1 , Vacunas contra el SIDA/metabolismo , Adyuvantes Inmunológicos/farmacología , Animales , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Células Dendríticas , Proteína p24 del Núcleo del VIH/metabolismo , Tejido Linfoide , Ratones , Poli I-C/farmacologíaRESUMEN
Human immunodeficiency virus-1 (HIV-1) persistence in the presence of antiretroviral therapy (ART) has halted the development of curative strategies. Measuring HIV persistence is complex due to the low frequency of cells containing virus in vivo. Most of the commercially available assays to date measure nucleic acid. These assays have the advantage of being highly sensitive and allow for the analysis of sequence diversity, intactness of the HIV genome or evaluation of diverse RNA species. However, these assays are limited in evaluating translational competent viral reservoirs. In here, we developed an ultrasensitive p24 ELISA that uses the Simoa planar array technology that can detect HIV-1 virions and HIV-1 infected cell with limit of detection similar to nucleic acid assays. Furthermore, the assay is optimized to measure very low levels of p24 in different biological fluids without a major loss of sensitivity or reproducibility. Our results demonstrate that the 'homebrew' planar p24 ELISA immunoassay is a broadly applicable new tool to evaluate HIV persistence in diverse biological fluids and cells.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Proteína p24 del Núcleo del VIH/metabolismo , Infecciones por VIH/diagnóstico , Infecciones por VIH/virología , VIH-1/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Proteína p24 del Núcleo del VIH/inmunología , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Human immunodeficiency virus (HIV) infection inflicts significant economic and social burdens on many countries worldwide. Given the substantial morbidity and mortality from HIV infection, there is an urgent need for accurate and early detection of the virus. In this study, immunofluorescence and visual techniques are described that detect the HIV-1 p24 antigen, which relied on selective recognition of Ag+/Ag nanoparticles (Ag NPs) and Cu2+/Cu+ using cadmium telluride quantum dots (CdTe QDs). After the sandwich immunoreactions were accomplished, the alkaline phosphatase (ALP) hydrolyzed L-ascorbic acid 2-phosphate (AAP) to form ascorbic acid (AA) that further reduces Ag+ and Cu2+ to Ag NPs and Cu+, respectively. This method was highly sensitive and selective and could detect as low as 1 pg/mL of p24 antigen by naked eyes and had a good linearity in the concentration range 1-100 pg/mL. When using Ag+ and Cu2+ as media, the limit of detection (LOD) of the new method was 0.3 pg/mL and 0.2 pg/mL, respectively. Compared with clinical electrochemiluminescence immunoassay (ECLIA) results and clinical data, this method demonstrated good consistency for the quantification of HIV-1 p24 antigen in 34 clinical serum samples. In addition, this method could accurately distinguish HIV from other viruses and infections such as hepatitis B virus, systemic lupus erythematosus, hepatitis C virus, Epstein-Barr virus, cytomegalovirus, lipemia, and hemolysis. Therefore, our dual-mode analysis method may provide additional solutions to identify clinical HIV infection. An immunofluorescence and visualization dual-mode strategy for the detection of p24 antigen was constructed based on immune recognition reaction and a phenomenon that cadmium telluride quantum dots (CdTe QDs) can selectively recognize Ag+/Ag nanoparticles (Ag NPs) and Cu2+/Cu+.
Asunto(s)
Compuestos de Cadmio/química , Proteína p24 del Núcleo del VIH/metabolismo , VIH-1/metabolismo , Inmunoensayo/métodos , Telurio/química , Fluorescencia , HumanosRESUMEN
HIV-1 capsid is comprised of over a hundred p24 protein molecules, arranged as either pentamers or hexamers. Three p24 mutants with amino acid substitutions in capsid N-terminal domain protein were examined: G60W (α3-4 loop), M68T (helix 4), and P90T (α4-5 loop), which exhibited no viability for biological activity. One common structural feature of the three p24 N-domain mutants, examined by NMR, was the long-range effect of more ß-structures at the ß2-strand in the N-terminal region compared with the wild-type. In addition, the presence of fewer helical structures was observed in M68T and P90T, beyond the broad area from helix 1 to the C-terminal part of helix 4. This suggests that both N-terminal beta structures and helices play important roles in the formation of p24 hexamers and pentamers. Next, compared with P90T, we examined cis-conformation or trans-conformation of wild-type adopted by isomerization at G89-P90. Since P90T mutant adopts only a trans-conformation, comparison of chemical shifts and signal intensities between each spectra revealed that the major peaks (about 85%) in the spectrum of wild-type correspond to trans-conformation. Furthermore, it was indicated that the region in cis-conformation (minor; 15%) was more stabilized than that observed in trans-conformation, based on the analyses of heteronuclear Overhauser effect as well as the order-parameter. Therefore, it was concluded that the cis-conformation is more favorable than the trans-conformation for the interaction between the p24 N-terminal domain and cyclophilin-A. This is because HIV-1 with a P90T protein, which adopts only a trans-conformation, is associated with non-viability of biological activity.
Asunto(s)
Sustitución de Aminoácidos , Proteína p24 del Núcleo del VIH/química , VIH-1/química , Modelos Moleculares , Mutación Missense , Proteína p24 del Núcleo del VIH/genética , Proteína p24 del Núcleo del VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , Conformación Proteica en Hélice alfa , Dominios ProteicosRESUMEN
Although HIV infection inhibits interferon responses in its target cells in vitro, interferon signatures can be detected in vivo soon after sexual transmission, mainly attributed to plasmacytoid dendritic cells (pDCs). In this study, we examined the physiological contributions of pDCs to early HIV acquisition using coculture models of pDCs with myeloid DCs, macrophages and the resting central, transitional and effector memory CD4 T cell subsets. pDCs impacted infection in a cell-specific manner. In myeloid cells, HIV infection was decreased via antiviral effects, cell maturation and downregulation of CCR5 expression. In contrast, in resting memory CD4 T cells, pDCs induced a subset-specific increase in intracellular HIV p24 protein expression without any activation or increase in CCR5 expression, as measured by flow cytometry. This increase was due to reactivation rather than enhanced viral spread, as blocking HIV entry via CCR5 did not alter the increased intracellular p24 expression. Furthermore, the load and proportion of cells expressing HIV DNA were restricted in the presence of pDCs while reverse transcriptase and p24 ELISA assays showed no increase in particle associated reverse transcriptase or extracellular p24 production. In addition, pDCs also markedly induced the expression of CD69 on infected CD4 T cells and other markers of CD4 T cell tissue retention. These phenotypic changes showed marked parallels with resident memory CD4 T cells isolated from anogenital tissue using enzymatic digestion. Production of IFNα by pDCs was the main driving factor for all these results. Thus, pDCs may reduce HIV spread during initial mucosal acquisition by inhibiting replication in myeloid cells while reactivating latent virus in resting memory CD4 T cells and retaining them for immune clearance.
Asunto(s)
Células Dendríticas/virología , Infecciones por VIH/virología , VIH/inmunología , Interferón-alfa/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Células Dendríticas/inmunología , Citometría de Flujo , VIH/genética , VIH/fisiología , Proteína p24 del Núcleo del VIH/genética , Proteína p24 del Núcleo del VIH/metabolismo , Infecciones por VIH/inmunología , Humanos , Células Mieloides/inmunología , Células Mieloides/virología , FenotipoRESUMEN
Defining tissue and plasma-specific prophylactic drug concentrations is central to pre-exposure prophylaxis product development for sexual transmission of HIV-1. Pharmacokinetic (PK) data from study RMP-02/MTN-006 comparing single dose oral tenofovir disoproxil fumarate with single and multiple dose rectal tenofovir (TFV) gel administration in HIV-1 seronegative adults was used to construct a multicompartment plasma-rectal tissue population PK model for TFV and tenofovir-diphosphate (TFVdp) in plasma and rectal tissue. PK data were collected in five matrices: TFV (plasma, rectal tissue homogenate), TFVdp (peripheral blood mononuclear cells, rectal mononuclear cells (MMCs), rectal tissue homogenate). A viral growth compartment and a delayed effect compartment for p24 antigen expression measured from an ex vivo explant assay described HIV-1 infection and replication. Using a linear PK/pharmacodynamic model, MMC TFVdp levels over 9,000 fmol/million cells in the explant assay provided apparent viral replication suppression down to 1%. Parameters were estimated using NONMEM version 7.4.
Asunto(s)
Fármacos Anti-VIH/farmacocinética , Infecciones por VIH/prevención & control , Leucocitos Mononucleares/efectos de los fármacos , Tenofovir/farmacocinética , Replicación Viral/efectos de los fármacos , Administración Oral , Administración Rectal , Adulto , Anciano , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacología , Disponibilidad Biológica , Desarrollo de Medicamentos/métodos , Femenino , Geles/farmacología , Geles/uso terapéutico , Proteína p24 del Núcleo del VIH/efectos de los fármacos , Proteína p24 del Núcleo del VIH/metabolismo , Seronegatividad para VIH/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Modelos Teóricos , Profilaxis Pre-Exposición/métodos , Recto/citología , Recto/efectos de los fármacos , Tenofovir/administración & dosificación , Tenofovir/farmacologíaRESUMEN
The emphasis of the present study is to evaluate a natural product and the potential microbicide activity using a dual chamber infection method. Malva sylvestris extracts and fractions were screened for anti-HIV activity by measuring the virus-antibody neutralization. Plant extracts with strong antiviral activity working in nanomolar or picomolar range can be used to enhance the activity of synthetic compounds and work as anti-HIV agents. The aqueous fraction (AF) of M. sylvestris demonstrated antiviral activity in a model with epithelial and blood cell lines. The AF showed an effective antiviral potential on the TZM-bl cells with reduction scores higher than 60% of infectivity. Quantification of p24 in the supernatant of the co-culture model demonstrated a reduction in the number of viral particles after AF treatment (p < 0.05). Cytokines were quantified and all signaling inflammatory markers; IL1-alpha, IL-beta, IL-6, IL-8 and GM-CSF (p < 0.05) were modulated by positive control and AF treatments. In particular, IL-6 had lower levels of expression in Malva groups when compared to the Zidovudine positive control group. Natural occurring derivatives of M. sylvestris demonstrated to work inhibiting reverse transcriptase enzyme action. M. sylvestris contains highly potential anti-HIV-1 BaL components and may be considered a potential source for new formulations in the development of topical microbicides.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Malva/química , Animales , Fármacos Anti-VIH/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fraccionamiento Químico , Citocinas/metabolismo , Proteína p24 del Núcleo del VIH/metabolismo , Humanos , Ratones , Extractos Vegetales/farmacologíaRESUMEN
Virus-like particles (VLPs) can be used as nano-carriers and antigen-display systems in vaccine development and therapeutic applications. Conjugation of peptides or whole proteins to VLPs can be achieved using different methods such as the SpyTag/SpyCatcher system. Here we investigate the conjugation of tandem Hepatitis B core (tHBcAg) VLPs and the model antigen GFP in vivo in Nicotiana benthamiana. We show that tHBcAg VLPs could be successfully conjugated with GFP in the cytosol and ER without altering VLP formation or GFP fluorescence. Conjugation in the cytosol was more efficient when SpyCatcher was displayed on tHBcAg VLPs instead of being fused to GFP. This effect was even more obvious in the ER, showing that it is optimal to display SpyCatcher on the tHBcAg VLPs and SpyTag on the binding partner. To test transferability of the GFP results to other antigens, we successfully conjugated tHBcAg VLPs to the HIV capsid protein P24 in the cytosol. This work presents an efficient strategy which can lead to time and cost saving post-translational, covalent conjugation of recombinant proteins in plants.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/metabolismo , Proteínas Recombinantes , Vacunas de Partículas Similares a Virus/metabolismo , Proteínas Fluorescentes Verdes , Proteína p24 del Núcleo del VIH/metabolismo , Péptidos , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Ingeniería de Proteínas , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Here, a fully integrated multicolor immunosensor was developed for sensitive and reliable semiquantitative analysis of HIV-1 p24, which integrates the multistep reactions of horseradish peroxidase (HRP)-linked immunoassay and gold nanorod (AuNR)-based multicolor assay into a single microfluidic chip. The HRP-linked immunoassay functions by moving magnetic beads bound to a capture antibody through different aqueous phases containing immunoassay reagents. HRP-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) is used to mediate AuNRs etching for producing various color changes. Multiple etching processes can be activated by simple mixing of the reagents from the reagent storage reservoir. The fully integrated strategy with sample-in answer-out capability is initiated by simple chip manipulation and finally concluded by converting recognition of antigen-antibody into a vivid color variation for direct visualization and semiquantitative analysis. By bare eye observation, our integrated multicolor immunosensor allows sensitive and reliable semiquantitative analysis of HIV-1 p24 within 1 h. The microfluidic chip device demonstrated here simplifies the operation significantly and thus allows broader application of a multicolor immunosensor for point of care (POC) testing in low-resource settings.
Asunto(s)
Técnicas Biosensibles/métodos , Proteína p24 del Núcleo del VIH/metabolismo , VIH-1/patogenicidad , Inmunoensayo/métodos , Microfluídica/métodos , HumanosRESUMEN
Despite combined antiretroviral therapy (cART), HIV infection in the CNS persists with reported increases in activation of macrophages (MΦ), microglia, and surrounding astrocytes/neurons, conferring HIV-induced inflammation. Chronic inflammation results in HIV-associated neurocognitive disorders (HAND) with reported occurrence of up to half of individuals with HIV infection. The existing HAND mouse model used by laboratories including ours, and the effect of novel agents on its pathology present with labor-intensive and time-consuming limitations since brain sections and immunohistochemistry assays have to be performed and analyzed. A novel flow cytometry-based system to objectively quantify phenotypic effects of HIV using a SCID mouse HAND model was developed which demonstrated that the HIV-infected mice had significant increases in astrogliosis, loss of neuronal dendritic marker, activation of murine microglia, and human macrophage explants compared to uninfected control mice. HIV p24 could also be quantified in the brains of the infected mice. Correlation of these impairments with HIV-induced brain inflammation and previous behavioral abnormalities studies in mice suggests that this model can be used as a fast and relevant throughput methodology to quantify preclinical testing of novel treatments for HAND.
Asunto(s)
Encéfalo/metabolismo , Disfunción Cognitiva/genética , Modelos Animales de Enfermedad , Gliosis/genética , Infecciones por VIH/genética , VIH-1/genética , Animales , Astrocitos/metabolismo , Astrocitos/virología , Biomarcadores/metabolismo , Encéfalo/virología , Disfunción Cognitiva/complicaciones , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/virología , Expresión Génica , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/complicaciones , Gliosis/metabolismo , Gliosis/virología , Proteína p24 del Núcleo del VIH/genética , Proteína p24 del Núcleo del VIH/metabolismo , Infecciones por VIH/complicaciones , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/metabolismo , VIH-1/patogenicidad , Humanos , Inflamación , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/metabolismo , Macrófagos/metabolismo , Macrófagos/virología , Masculino , Ratones , Ratones SCID , Microglía/metabolismo , Microglía/virología , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/metabolismo , Neuronas/virología , FenotipoRESUMEN
Quantifying the inducible HIV reservoir provides an estimate of the frequency of quiescent HIV-infected cells in humans as well as in animal models, and can help ascertain the efficacy of latency reversing agents (LRAs). The quantitative viral outgrowth assay (QVOA) is used to measure inducible, replication competent HIV and generate estimations of reservoir size. However, traditional QVOA is time and labor intensive and requires large amounts of lymphocytes. Given the importance of reproducible and accurate assessment of both reservoir size and LRA activity in cure strategies, efforts to streamline the QVOA are of high priority. We developed a modified QVOA, the Digital ELISA Viral Outgrowth or DEVO assay, with ultra-sensitive p24 readout, capable of femtogram detection of HIV p24 protein in contrast to the picogram limitations of traditional ELISA. For each DEVO assay, 8-12 × 106 resting CD4 + T cells from aviremic, ART-treated HIV + participants are plated in limiting dilution and maximally stimulated with PHA, IL-2 and uninfected allogeneic irradiated PBMC. CD8-depleted PHA blasts from an uninfected donor or HIV-permissive cells (e.g., Molt4/CCR5) are added to the cultures and virus allowed to amplify for 8-12 days. HIV p24 from culture supernatant is measured at day 8 by Simoa (single molecule array, ultra-sensitive p24 assay) confirmed at day 12, and infectious units per million CD4 + T cells (IUPM) are calculated using the maximum likelihood method. In all DEVO assays performed, HIV p24 was detected in the supernatant of cultures as early as 8 days post stimulation. Importantly, DEVO IUPM values at day 8 were comparable or higher than traditional QVOA IUPM values obtained at day 15. Interestingly, DEVO IUPM values were similar with or without the addition of allogeneic CD8-depleted target PHA blasts or HIV permissive cells traditionally used to expand virus. The DEVO assay uses fewer resting CD4 + T cells and provides an assessment of reservoir size in less time than standard QVOA. This assay offers a new platform to quantify replication competent HIV during limited cell availability. Other potential applications include evaluating LRA activity, and measuring clearance of infected cells during latency clearance assays.
Asunto(s)
Proteína p24 del Núcleo del VIH/metabolismo , Infecciones por VIH/diagnóstico , Infecciones por VIH/virología , VIH-1/fisiología , Carga Viral , Replicación Viral , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Ensayo de Inmunoadsorción Enzimática , Infecciones por VIH/inmunología , Humanos , Sensibilidad y EspecificidadRESUMEN
Our recent publication illustrates the critical role of phenylalanine-mediated aromatic-aromatic interactions in determining the assembly of peptidic ß-sheets. However, the effect of phenylalanine number on regulating the assembly efficacy of peptidic ß-sheets remains poorly understood. We herein evaluate the assembly efficacy of ß-sheets of a series of oligopeptides which contain 0, 1, 2, or 3 phenylalanine in their molecular backbones. In our assembly system, two phenylalanine (2F) is the minimum number for driving the assembly of ß-sheets of oligopeptides. Oligopeptides with three phenylalanine (3F) show significantly increased assembly efficacy of ß-sheets compared to that with 2F. These results suggest a positive correlation between the phenylalanine number and assembly efficacy of ß-sheets. By improving the assembly efficacy of ß-sheets, we further develop a highly sensitive HIV analytical system in which the specific binding of ß-sheets with Congo Red induces enhanced fluorescence. For HIV p24 detection, the 3F-based analytical system (0.61 pg/mL) shows a significantly lower limit of detection (LOD) than the 2F-based analytical system (2.44 pg/mL), both of which are more sensitive than commercial ELISA (5 pg/mL) used in the clinic. This work not only illustrates the effect of phenylalanine number on regulating the assembly efficacy of ß-sheets but also provides a guideline for the construction of a highly sensitive analytical system of disease diagnosis.
Asunto(s)
Proteína p24 del Núcleo del VIH/sangre , VIH/química , Conformación Proteica en Lámina beta/efectos de los fármacos , Sangre/virología , Rojo Congo/química , Rojo Congo/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Proteína p24 del Núcleo del VIH/química , Proteína p24 del Núcleo del VIH/metabolismo , Humanos , Límite de Detección , Fenilalanina/química , Unión ProteicaRESUMEN
HIV-1 is a global health crisis that has infected more than 37 million people. Latent reservoirs throughout the body are a major hurdle when it comes to eradicating the virus. In our previous study, we found that exosomes, a type of extracellular vesicle (EV), from uninfected cells activate the transcription of HIV-1 in latent infected cells, regardless of combination antiretroviral therapy (cART). In this study, we investigated the specific mechanism behind the EV activation of latent HIV-1. We found that phosphorylated c-Src is present in EVs of various cell lines and has the ability to activate downstream proteins such as EGFR, initiating a signal cascade. EGFR is then able to activate the PI3K/AKT/mTOR pathway, resulting in the activation of STAT3 and SRC-1, culminating in the reversal of HIV-1 latency. This was verified by examining levels of HIV-1 TAR, genomic RNA and HIV-1 Gag p24 protein in cell lines and primary cells. We found that EVs containing c-Src rescued HIV-1 despite the presence of inhibitors, validating the importance of EV-associated c-Src in latent HIV-1 activation. Lastly, we discovered an increased recruitment of p300 and NF-κB in the nucleus of EV-treated infected cells. Collectively, our data suggest that EV-associated c-Src is able to activate latent HIV-1 via the PI3K/AKT/mTOR pathway and SRC-1/p300-driven chromatin remodeling. These findings could aid in designing new strategies to prevent the reactivation of latent HIV-1 in patients under cART.
Asunto(s)
Exosomas/metabolismo , VIH-1/crecimiento & desarrollo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Activación Viral/fisiología , Latencia del Virus/fisiología , Línea Celular Tumoral , Proteína p300 Asociada a E1A/metabolismo , Receptores ErbB/metabolismo , Vesículas Extracelulares/metabolismo , Proteína p24 del Núcleo del VIH/metabolismo , Infecciones por VIH , Humanos , Células Jurkat , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Unión al ARN/metabolismo , Factor de Transcripción STAT3/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transcripción Genética/genética , Activación Transcripcional/genética , Células U937RESUMEN
Fostemsavir/temsavir is an investigational HIV-1 entry inhibitor currently in late-stage clinical trials. Although it holds promise to be a first-in-class Env-targeted entry inhibitor for the clinic, issues with bioavailability relegate its use to salvage therapies only. As such, the development of a small molecule HIV-1 entry inhibitor that can be used in standard combination antiretroviral therapy (cART) remains a longstanding goal for the field. We previously demonstrated the ability of extending the chemotypes available to this class of inhibitor as the first step towards this overarching goal. In addition to poor solubility, metabolic stability is a crucial determinant of bioavailability. Therefore, in this short communication, we assess the metabolic stabilities of five of our novel chemotype entry inhibitors. We found that changing the piperazine core region of temsavir alters the stability of the compound in human liver microsome assays. Moreover, we identified an entry inhibitor with more than twice the metabolic stability of temsavir and demonstrated that the orientation of the core replacement is critical for this increase. This work further demonstrates the feasibility of our long-term goal-to design an entry inhibitor with improved drug-like qualities-and warrants expanded studies to achieve this.
Asunto(s)
Fármacos Anti-VIH/farmacología , Inhibidores de Fusión de VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Organofosfatos/química , Piperazinas/química , Triazoles/metabolismo , Compuestos de Azabiciclo/síntesis química , Compuestos de Azabiciclo/química , Compuestos de Azabiciclo/farmacología , Azetidinas/síntesis química , Azetidinas/química , Azetidinas/farmacología , Cromatografía Liquida , Células HEK293 , Proteína p24 del Núcleo del VIH/química , Proteína p24 del Núcleo del VIH/metabolismo , Inhibidores de Fusión de VIH/metabolismo , Infecciones por VIH/virología , Humanos , Microsomas Hepáticos/virología , Organofosfatos/farmacología , Piperazinas/farmacología , Unión Proteica , Pirrolidinas/síntesis química , Pirrolidinas/química , Pirrolidinas/farmacología , Espectrometría de Masas en Tándem , Triazoles/síntesis química , Triazoles/farmacología , Triazoles/toxicidadRESUMEN
BACKGROUND: Different approaches have been investigated to develop a preventive or therapeutic vaccine, although none of them has been fully practical. Therapeutic vaccines against HIV-1 have been studied with the aim of eliminating the virus from reservoir cells with or without HAART (Highly Active Antiretroviral Therapy). Fusion proteins with the most immunogenic features among conserved regions can facilitate this achievement in such a variable virus. To achieve the most immunogenic and also conserved regions, bioinformatics tools are widely used to predict antigens' features before applying them. OBJECTIVE: This study aimed at the in vitro evaluation of p24 -Nef fusion protein based on the previous in silico design to achieve a potential therapeutic subunit vaccine against HIV-1. METHODS: The truncated form of p24-Nef using AAY flexible linker and the full protein were expressed and evaluated in the prokaryotic system and confirmed by western blotting. We also used pcDNA3.1 to transfect Lenti-X 293T cells. Moreover, lentiviral vectors were applied to produce recombinant virions harboring the genes of interest and cell transduction. RESULTS: Both fusion proteins in a truncated and a full form were expressed and confirmed by Anti Nef polyclonal antibody in western blotting. Recombinant virions were generated and transduced Lenti-X 293T cells confirming by immunofluorescence microscope and p24 ELISA assay kit. Transduced cells were analyzed by SDS-PAGE and western blotting, which resulted in approved protein expression. CONCLUSION: Fusion protein of p24 and Nef is well expressed in eukaryotic cell lines according to its pre-evaluated features by bioinformatics tools.
Asunto(s)
Proteína p24 del Núcleo del VIH/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Células HEK293 , Proteína p24 del Núcleo del VIH/genética , VIH-1/inmunología , Humanos , Lentivirus/genética , Transducción Genética , Vacunas Virales , Virión , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The tripartite-motif protein, TRIM5α, is an innate immune sensor that potently restricts retrovirus infection by binding to human immunodeficiency virus capsids. Higher-ordered oligomerization of this protein forms hexagonally patterned structures that wrap around the viral capsid, despite an anomalously low affinity for the capsid protein (CA). Several studies suggest TRIM5α oligomerizes into a lattice with a symmetry and spacing that matches the underlying capsid, to compensate for the weak affinity, yet little is known about how these lattices form. Using a combination of computational simulations and electron cryo-tomography imaging, we reveal the dynamical mechanisms by which these lattices self-assemble. Constrained diffusion allows the lattice to reorganize, whereas defects form on highly curved capsid surfaces to alleviate strain and lattice symmetry mismatches. Statistical analysis localizes the TRIM5α binding interface at or near the CypA binding loop of CA. These simulations elucidate the molecular-scale mechanisms of viral capsid cellular compartmentalization by TRIM5α.