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1.
Nat Microbiol ; 4(11): 1840-1850, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31611641

RESUMO

The initial steps of HIV replication in host cells prime the virus for passage through the nuclear pore and drive the establishment of a productive and irreparable infection1,2. The timely release of the viral genome from the capsid-referred to as uncoating-is emerging as a critical parameter for nuclear import, but the triggers and mechanisms that orchestrate these steps are unknown. Here, we identify ß-karyopherin Transportin-1 (TRN-1) as a cellular co-factor of HIV-1 infection, which binds to incoming capsids, triggers their uncoating and promotes viral nuclear import. Depletion of TRN-1, which we characterized by mass spectrometry, significantly reduced the early steps of HIV-1 infection in target cells, including primary CD4+ T cells. TRN-1 bound directly to capsid nanotubes and induced dramatic structural damage, indicating that TRN-1 is necessary and sufficient for uncoating in vitro. Glycine 89 on the capsid protein, which is positioned within a nuclear localization signal in the cyclophilin A-binding loop, is critical for engaging the hydrophobic pocket of TRN-1 at position W730. In addition, TRN-1 promotes the efficient nuclear import of both viral DNA and capsid protein. Our study suggests that TRN-1 mediates the timely release of the HIV-1 genome from the capsid protein shell and efficient viral nuclear import.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , beta Carioferinas/química , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Linfócitos T CD4-Positivos/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Deleção de Genes , Células HEK293 , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/metabolismo , Células HeLa , Humanos , Espectrometria de Massas , Modelos Moleculares , Sinais de Localização Nuclear , Ligação Proteica , Conformação Proteica , RNA Viral/metabolismo , Desenvelopamento do Vírus , beta Carioferinas/genética
2.
Eur J Med Chem ; 183: 111699, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31561045

RESUMO

The recent burst of explorations on heat shock protein 90 (HSP90) in virus research supports its emergence as a promising target to overcome the drawbacks of current antiviral therapeutic regimen. In continuation of our efforts towards the discovery of novel anti-retroviral molecules, we designed, synthesized fifteen novels 2-isoxazol-3-yl-acetamide based compounds (2a-o) followed by analysis of their anti-HIV activity and cytotoxicity studies. 2a-b, 2e, 2j, and 2l-m were found to be active with inhibitory potentials >80% at their highest non-cytotoxic concentration (HNC). Further characterization of anti-HIV activity of these molecules suggests that 2l has ∼3.5 fold better therapeutic index than AUY922, the second generation HSP90 inhibitor. The anti-HIV activity of 2l is a cell type, virus isolate and viral load independent phenomena. Interestingly, 2l does not significantly modulate viral enzymes like Reverse Transcriptase (RT), Integrase (IN) and Protease (PR) as compared to their known inhibitors in a cell free in vitro assay system at its HNC. Further, 2l mediated inhibition of HSP90 attenuates HIV-1 LTR driven gene expression. Taken together, structural rationale, modeling studies and characterization of biological activities suggest that this novel scaffold can attenuate HIV-1 replication significantly within the host and thus opens a new horizon to develop novel anti-HIV therapeutic candidates.


Assuntos
Acetamidas/farmacologia , Androstenóis/farmacologia , Fármacos Anti-HIV/farmacologia , Descoberta de Drogas , HIV-1/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Acetamidas/síntese química , Acetamidas/química , Androstenóis/síntese química , Androstenóis/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Relação Dose-Resposta a Droga , HIV-1/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
3.
Molecules ; 24(18)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31489889

RESUMO

HIV protease inhibitors against the viral protease are often hampered by drug resistance mutations in protease and in the viral substrate Gag. To overcome this drug resistance and inhibit viral maturation, targeting Gag alongside protease rather than targeting protease alone may be more efficient. In order to successfully inhibit Gag, understanding of its drug resistance mutations and the elicited structural changes on protease binding needs to be investigated. While mutations on Gag have already been mapped to protease inhibitor resistance, there remain many mutations, particularly the non-cleavage mutations, that are not characterized. Through structural studies to unravel how Gag mutations contributes to protease drug resistance synergistically, it is thus possible to glean insights to design novel Gag inhibitors. In this review, we discuss the structural role of both novel and previously reported Gag mutations in PI resistance, and how new Gag inhibitors can be designed.


Assuntos
Farmacorresistência Viral , HIV-1/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Desenho de Drogas , Protease de HIV/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Relação Estrutura-Atividade
4.
ACS Appl Mater Interfaces ; 11(38): 34586-34594, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31483592

RESUMO

The human immunodeficiency virus 1 (HIV-1) capsid serves as a binding platform for proteins and small molecules from the host cell that regulate various steps in the virus life cycle. However, there are currently no quantitative methods that use assembled capsid lattices to measure host-pathogen interaction dynamics. Here we developed a single-molecule fluorescence biosensor using self-assembled capsid tubes as biorecognition elements and imaged capsid binders using total internal reflection fluorescence microscopy in a microfluidic setup. The method is highly sensitive in its ability to observe and quantify binding, to obtain dissociation constants, and to extract kinetics with an extended application of using more complex analytes that can accelerate characterization of novel capsid binders.


Assuntos
Técnicas Biossensoriais , Capsídeo , HIV-1 , Dispositivos Lab-On-A-Chip , Capsídeo/química , Capsídeo/metabolismo , HIV-1/química , HIV-1/metabolismo , Humanos , Microscopia de Fluorescência
5.
Eur J Endocrinol ; 181(4): 451-459, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31430720

RESUMO

Objectives: Adipose tissue (AT) density measurement may provide information about AT quality among people living with HIV. We assessed AT density and evaluated relationships between AT density and immunometabolic biomarker concentrations in men with HIV. Design: Cross-sectional analysis of men enrolled in the Multicenter AIDS Cohort Study. Methods: Abdominal visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) density (Hounsfield units, HU; less negative = more dense) were quantified from computed tomography (CT) scans. Multivariate linear regression models described relationships between abdominal AT density and circulating biomarker concentrations. Results: HIV+ men had denser SAT (-95 vs -98 HU HIV-, P < 0.001), whereas VAT density was equivalent by HIV serostatus men (382 HIV-, 462 HIV+). Historical thymidine analog nucleoside reverse transcriptase inhibitor (tNRTI) use was associated with denser SAT but not VAT. In adjusted models, a 1 s.d. greater SAT or VAT density was associated with higher levels of adiponectin, leptin, HOMA-IR and triglyceride:HDL cholesterol ratio and lower hs-CRP concentrations in HIV- men. Conversely, in HIV+ men, each s.d. greater SAT density was not associated with metabolic parameter improvements and was significantly (P < 0.05) associated with higher systemic inflammation. Trends toward higher inflammatory biomarker concentrations per 1 s.d. greater VAT density were also observed among HIV+ men. Conclusions: Among men living with HIV, greater SAT density was associated with greater systemic inflammation independent of SAT area. AT density measurement provides additional insight into AT density beyond measurement of AT quantity alone, and may have implications for metabolic disease risk.


Assuntos
Adiposidade/fisiologia , Soropositividade para HIV/sangue , Soropositividade para HIV/diagnóstico , HIV-1/metabolismo , Gordura Subcutânea/metabolismo , Biomarcadores/sangue , Estudos de Coortes , Estudos Transversais , Infecções por HIV/sangue , Infecções por HIV/diagnóstico , Soropositividade para HIV/imunologia , HIV-1/imunologia , Humanos , Inflamação/sangue , Inflamação/diagnóstico , Inflamação/virologia , Masculino , Pessoa de Meia-Idade
6.
Nucleic Acids Res ; 47(14): 7676-7689, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31424549

RESUMO

The potent antiretroviral protein APOBEC3G (A3G) specifically targets and deaminates deoxycytidine nucleotides, generating deoxyuridine, in single stranded DNA (ssDNA) intermediates produced during HIV replication. A non-catalytic domain in A3G binds strongly to RNA, an interaction crucial for recruitment of A3G to the virion; yet, A3G displays no deamination activity for cytidines in viral RNA. Here, we report NMR and molecular dynamics (MD) simulation analysis for interactions between A3Gctd and multiple substrate or non-substrate DNA and RNA, in combination with deamination assays. NMR ssDNA-binding experiments revealed that the interaction with residues in helix1 and loop1 (T201-L220) distinguishes the binding mode of substrate ssDNA from non-substrate. Using 2'-deoxy-2'-fluorine substituted cytidines, we show that a 2'-endo sugar conformation of the target deoxycytidine is favored for substrate binding and deamination. Trajectories of the MD simulation indicate that a ribose 2'-hydroxyl group destabilizes the π-π stacking of the target cytosine and H257, resulting in dislocation of the target cytosine base from the catalytic position. Interestingly, APOBEC3A, which can deaminate ribocytidines, retains the ribocytidine in the catalytic position throughout the MD simulation. Our results indicate that A3Gctd catalytic selectivity against RNA is dictated by both the sugar conformation and 2'-hydroxyl group.


Assuntos
Desaminase APOBEC-3G/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , RNA/metabolismo , Desaminase APOBEC-3G/química , Desaminase APOBEC-3G/genética , Biocatálise , Citidina/química , Citidina/metabolismo , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Desaminação , HIV-1/genética , HIV-1/metabolismo , Humanos , Ligação Proteica , RNA/química , RNA/genética , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Especificidade por Substrato , Vírion/genética , Vírion/metabolismo
8.
Oxid Med Cell Longev ; 2019: 3403206, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31217837

RESUMO

Objective: To evaluate the role of caveolin-1 (Cav-1) in HIV-1 Tat-induced dysfunction of tight junction and amyloid ß-peptide- (Aß-) transferring proteins. Methods: A Cav-1 shRNA interference target sequence was cloned into the lentiviral vector pHBLV-U6-Scramble-ZsGreen-Puro and verified by double enzyme digestion and DNA sequencing. Human cerebral microvascular endothelium (HBEC-5i) cells were transduced with viral particles made in 293T cells by transfection with lentiviral packaging plasmids. HBEC-5i cells transduced with Cav-1 shRNA or Ctr shRNA were exposed to HIV-1 Tat for 24 h, and the protein and mRNA levels of the tight junction protein occludin, Aß-transferring protein, receptor for advanced glycation end products (RAGE), low-density lipoprotein receptor-related protein- (LRP-) 1, and RhoA were evaluated with Western blot and real-time reverse transcription polymerase chain reaction (qRT-PCR) assays, respectively. Results: After sequencing, an RNA interference recombinant lentivirus expressing a vector targeting Cav-1 was successfully established. The recombined lentiviral particles were made by using 293T cells to package the recombined lentiviral vector. A stable monoclonal cell line with strong GFP expression was acquired with a Cav-1 knockdown rate of 85.7%. The occludin protein and mRNA levels in the Ctr shRNA group were decreased with HIV-1 Tat exposure but were upregulated in the Cav-1 shRNA group. The HIV-1 Tat-induced alterations of RAGE and LRP-1 protein and mRNA levels in the Ctr shRNA group were attenuated in the Cav-1 shRNA group. The RhoA protein levels in the Ctr shRNA group were upregulated by HIV-1 Tat exposure but were downregulated in the Cav-1 shRNA group. Conclusion: These results show that HIV-1 Tat-induced downregulation of occludin and LRP-1 and upregulation of RAGE and RhoA may result in the accumulation of Aß in the brain. Silencing the Cav-1 gene with shRNA plays a key role in the protection against HIV-1 Tat-induced dysfunction of the blood-brain barrier and Aß accumulation.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Caveolina 1/genética , HIV-1/metabolismo , Junções Íntimas/metabolismo , Humanos , Transfecção
9.
PLoS Comput Biol ; 15(6): e1007150, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194731

RESUMO

A coarse-grain computational method integrates biophysical and structural data to generate models of HIV-1 genomic RNA, nucleocapsid and integrase condensed into a mature ribonucleoprotein complex. Several hypotheses for the initial structure of the genomic RNA and oligomeric state of integrase are tested. In these models, integrase interaction captures features of the relative distribution of gRNA in the immature virion and increases the size of the RNP globule, and exclusion of nucleocapsid from regions with RNA secondary structure drives an asymmetric placement of the dimerized 5'UTR at the surface of the RNP globule.


Assuntos
HIV-1 , RNA Viral , Ribonucleoproteínas , Proteínas Virais , Biologia Computacional , HIV-1/química , HIV-1/metabolismo , Simulação de Dinâmica Molecular , RNA Viral/química , RNA Viral/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Vírion , Montagem de Vírus
10.
Nucleic Acids Res ; 47(13): 7105-7117, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31199872

RESUMO

The HIV-1 Rev response element (RRE) RNA element mediates the nuclear export of intron containing viral RNAs by forming an oligomeric complex with the viral protein Rev. Stem IIB and nearby stem II three-way junction nucleate oligomerization through cooperative binding of two Rev molecules. Conformational flexibility at this RRE region has been shown to be important for Rev binding. However, the nature of the flexibility has remained elusive. Here, using NMR relaxation dispersion, including a new strategy for directly observing transient conformational states in large RNAs, we find that stem IIB alone or when part of the larger RREII three-way junction robustly exists in dynamic equilibrium with non-native excited state (ES) conformations that have a combined population of ∼20%. The ESs disrupt the Rev-binding site by changing local secondary structure, and their stabilization via point substitution mutations decreases the binding affinity to the Rev arginine-rich motif (ARM) by 15- to 80-fold. The ensemble clarifies the conformational flexibility observed in stem IIB, reveals long-range conformational coupling between stem IIB and the three-way junction that may play roles in cooperative Rev binding, and also identifies non-native RRE conformational states as new targets for the development of anti-HIV therapeutics.


Assuntos
Genes env , HIV-1/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Magnésio/metabolismo , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo
11.
Mol Cell ; 75(1): 172-183.e9, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31178355

RESUMO

Ribosomal frameshifting during the translation of RNA is implicated in human disease and viral infection. While previous work has uncovered many details about single RNA frameshifting kinetics in vitro, little is known about how single RNA frameshift in living systems. To confront this problem, we have developed technology to quantify live-cell single RNA translation dynamics in frameshifted open reading frames. Applying this technology to RNA encoding the HIV-1 frameshift sequence reveals a small subset (∼8%) of the translating pool robustly frameshift. Frameshifting RNA are translated at similar rates as non-frameshifting RNA (∼3 aa/s) and can continuously frameshift for more than four rounds of translation. Fits to a bursty model of frameshifting constrain frameshifting kinetic rates and demonstrate how ribosomal traffic jams contribute to the persistence of the frameshifting state. These data provide insight into retroviral frameshifting and could lead to alternative strategies to perturb the process in living cells.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , HIV-1/genética , Fases de Leitura Aberta , Osteoblastos/metabolismo , RNA Viral/genética , Imagem Individual de Molécula/métodos , Pareamento de Bases , Linhagem Celular Tumoral , HIV-1/metabolismo , Humanos , Modelos Genéticos , Conformação de Ácido Nucleico , Sondas de Oligonucleotídeos/síntese química , Sondas de Oligonucleotídeos/genética , Sondas de Oligonucleotídeos/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Osteoblastos/virologia , RNA Viral/química , RNA Viral/metabolismo , Coloração e Rotulagem/métodos
12.
BMC Womens Health ; 19(1): 63, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068152

RESUMO

BACKGROUND: A high endogenous progesterone luteal state in the menstrual cycle has been independently associated with Human Immunodeficiency Virus (HIV) incidence in epidemiological studies. Hormonal contraception particularly high dose Depot Medroxyprogesterone Acetate (DMPA) is also thought to increase the risk of HIV acquisition. Inconsistent reports of this association have led us to hypothesize that unsuppressed endogenous progesterone level in women who reported hormonal contraception (HC) use may be an explanation for increased vulnerability to HIV. METHODS: This pilot study was a secondary cross-sectional analysis of data and laboratory testing of stored specimens collected from women who participated in the SAMRC HIV prevention MDP 301 trial during 2005-2009 in South Africa. Serum progesterone levels were measured in 39 women at the point of first positive HIV diagnosis during study follow-up and 36 women who remained HIV uninfected at the 52-week study exit visit. RESULTS: Overall, the median (IQR) progesterone level in 49 women using hormonal contraception was 0.39 ng/ml (IQR 0.13-0.45) and 48 (97.9%) women had a progesterone level < 3.0 ng/ml suggestive of adequate progesterone suppression for contraceptive efficacy. After excluding the one woman with a progesterone level of > 3.0 ng/ml, the median progesterone level in women using DMPA remained marginally higher at 0.42 ng/ml (IQR 0.27-0.45) than women using Norethisterone Enanthate (NET-EN) (0.31 ng/ml; IQR 0.13-0.41, p = 0.061). For women using hormonal contraception, the median progesterone level did not differ between women with recent HIV infection or women who remained HIV negative (0.39 vs 0.38 ng/ml, p = 0.959). Similarly, the median progesterone level in women using DMPA or NET-EN did not differ by HIV status (0.43 vs 0.41 ng/ml, p = 0.905; 0.24 vs 0.31 ng/ml, p = 0.889). CONCLUSION: Among women using hormonal contraception, DMPA or NET-EN we did not observe a significant difference in progesterone levels between women with recently acquired HIV infection and women who remained HIV negative. Our findings suggest that endogenous progesterone levels remain suppressed in the presence of hormonal contraception and are not likely to be associated with HIV acquisition.


Assuntos
Anticoncepcionais Femininos/efeitos adversos , Infecções por HIV/etiologia , Soropositividade para HIV/sangue , Acetato de Medroxiprogesterona/efeitos adversos , Progesterona/sangue , Adulto , Anticoncepção/estatística & dados numéricos , Estudos Transversais , Feminino , Infecções por HIV/prevenção & controle , HIV-1/metabolismo , Humanos , Incidência , Projetos Piloto , África do Sul , Adulto Jovem
13.
Soft Matter ; 15(22): 4525-4540, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31099376

RESUMO

The complex-type glycan shields of eukaryotic cells have a core layer of mannose residues buried under tiers of sugars that end with sialic acid (SA) residues. We investigate if the self-latching of mannose residues, earlier reported in pure monolayer studies, also manifests in the setting of a complex-type glycan shield. Would distal SA residues impede access to the mannose core? The interactions of mannobiose-, SA-, and lactose-coated probes with the complex-type VSV-G glycan shield on an HIV pseudovirus were studied with force-spectroscopy and gold-nanoparticle solutions. In force spectroscopy, the sugar probes can be forced to sample the depths of the glycan shield, whereas with sugar-coated nanoparticles, only interactions permitted by freely-diffusive contact occur. Deep-indentation mechanics was performed to verify the inferred structure of the engineered virus and to isolate the glycan shield layer for subsequent interaction studies. The adhesion between the sugar-probes and complex-type glycan shield was deconvoluted by comparing against the cross- and self- adhesions between the sugars in pure monolayers. Results from complementing systems were consistent with mannobiose-coated probes latching to the mannose core in the glycan shield, unhindered by the SA and distal sugars, with a short-range 'brittle' release of adhesion resulting in tightly coated viruses. SA-Coated probes, however, adhere to the terminal SA layer of a glycan shield with long-range and mechanically 'tough' adhesions resulting in large-scale virus aggregation. Lactose-coated probes exhibit ill-defined adherence to sialic residues. The selection and positioning of sugars within a glycan shield can influence how carbohydrate surfaces of different composition adhere.


Assuntos
HIV-1/química , Manose/química , Glicoproteínas de Membrana/química , Ácido N-Acetilneuramínico/química , Proteínas do Envelope Viral/química , Células HEK293 , HIV-1/genética , HIV-1/metabolismo , Humanos , Manose/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Nanopartículas Metálicas/química , Ácido N-Acetilneuramínico/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
14.
Mol Biol (Mosk) ; 53(2): 240-255, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31099774

RESUMO

Currently, more than 37 million individuals worldwide are infected with the human immunodeficiency virus (HIV). Antiretroviral therapy may control the viral infection but is incapable of eradicating it. It is important to understand how cells respond to HIV-1 infection and what cellular factors are involved in this process to develop novel classes of antiviral drugs. This review summarizes the current understanding of the HIV restriction mechanism. We discuss the ambiguous role of HIV restriction factors in viral infection and counteraction mediated by HIV-1 accessory proteins.


Assuntos
Fármacos Anti-HIV/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Desenvolvimento de Medicamentos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos
15.
Phys Chem Chem Phys ; 21(20): 10300-10310, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31070638

RESUMO

The trans-acting activator of transcription (TAT) peptide, which is derived from human immunodeficiency virus-1 (HIV-1), has been widely used as an effective nanocarrier to transport extracellular substances into cells. However, the underlying translocation mechanism of TAT peptide across cell membranes still remains controversial. Besides, the molecular process of TAT peptide facilitating the transport of extracellular substances into cells is largely unknown. In this study, we explore the interactions of TAT peptides and their conjugated gold nanoparticles with lipid membranes by coarse-grained molecular dynamics simulations. It is found that the TAT peptides can hardly penetrate through the membrane at low peptide concentrations; after the concentration increases to a threshold value, they can cross the membrane through an induced nanopore due to the transmembrane electrostatic potential difference. The translocation of TAT peptides is mainly caused by the overall structural changes of membranes. Furthermore, we demonstrate that the translocation of gold nanoparticles (AuNPs) across the membrane is significantly affected by the number of grafted TAT peptides on the particle surface. The transmembrane efficiency of AuNPs may even be reduced when a small number of peptides modify them; whereas, when the number of grafted peptides increases to a certain value, the TAT-AuNP complex can translocate across the membrane in a pore-mediated way. Based on our findings, an effective strategy has been proposed to enhance the delivery efficiency of AuNPs. The present study can improve our understanding of the interactions between TAT peptides and cell membranes; it may also give some insightful suggestions on the design and development of nanocarriers with high efficiency for the delivery of nanoparticles and drugs.


Assuntos
Membrana Celular/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Peptídeos/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , HIV-1/metabolismo , Humanos , Simulação de Dinâmica Molecular , Peptídeos/química
16.
J Biol Chem ; 294(20): 8286-8295, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30971426

RESUMO

Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catalyzed by the retroviral integrase. To date, the only substrate described to be involved in this critical reaction is the linear viral DNA produced in reverse transcription. However, during HIV-1 infection, two-long terminal repeat DNA circles (2-LTRcs) are also generated through the ligation of the viral DNA ends by the host cell's nonhomologous DNA end-joining pathway. These DNAs contain all the genetic information required for viral replication, but their role in HIV-1's life cycle remains unknown. We previously showed that both linear and circular DNA fragments containing the 2-LTR palindrome junction can be efficiently cleaved in vitro by recombinant integrases, leading to the formation of linear 3'-processed-like DNA. In this report, using in vitro experiments with purified proteins and DNAs along with DNA endonuclease and in vivo integration assays, we show that this circularized genome can also be efficiently used as a substrate in HIV-1 integrase-mediated integration both in vitro and in eukaryotic cells. Notably, we demonstrate that the palindrome cleavage occurs via a two-step mechanism leading to a blunt-ended DNA product, followed by a classical 3'-processing reaction; this cleavage leads to integrase-dependent integration, highlighted by a 5-bp duplication of the host genome. Our results suggest that 2-LTRc may constitute a reserve supply of HIV-1 genomes for proviral integration.


Assuntos
DNA Circular/química , DNA Viral/química , Integrase de HIV/química , Repetição Terminal Longa de HIV , HIV-1/química , Integração Viral , DNA Circular/genética , DNA Viral/genética , DNA Viral/metabolismo , Integrase de HIV/genética , Integrase de HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Humanos
17.
Molecules ; 24(8)2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013646

RESUMO

Small-molecule HIV-1 entry inhibitors are an extremely attractive therapeutic modality. We have previously demonstrated that the entry inhibitor class can be optimized by using computational means to identify and extend the chemotypes available. Here we demonstrate unique and differential effects of previously published antiviral compounds on the gross structure of the HIV-1 Env complex, with an azabicyclohexane scaffolded inhibitor having a positive effect on glycoprotein thermostability. We demonstrate that modification of the methyltriazole-azaindole headgroup of these entry inhibitors directly effects the potency of the compounds, and substitution of the methyltriazole with an amine-oxadiazole increases the affinity of the compound 1000-fold over parental by improving the on-rate kinetic parameter. These findings support the continuing exploration of compounds that shift the conformational equilibrium of HIV-1 Env as a novel strategy to improve future inhibitor and vaccine design efforts.


Assuntos
Fármacos Anti-HIV , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Internalização do Vírus/efeitos dos fármacos , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Humanos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade
18.
PLoS One ; 14(4): e0215353, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30986228

RESUMO

BACKGROUND: HIV-self-testing (HIVST) could be a strategy to get more people tested for HIV in resource limited settings. One of the prerequisites of a successful HIVST programme is the availability of an easy to use, valid HIV-test which is robust against field conditions and procedural errors by untrained lay users. METHODS AND FINDINGS: The primary objective of this study was to evaluate the ability of untrained persons to correctly interpret the OraQuick HIV Self-Test results with oral fluid compared with results obtained by trained users using the matched lot OraQuick Rapid HIV-1/2 Antibody Test and blinded to the results of the Self-Test. Sensitivity of the OraQuick HIV Self-Test in untrained users was 101 in 102 (99.02%; 95%CI = 93.88-99.95%)-and specificity- 1,241 in 1,241 (100.0%; 95%CI = 99.62-100.0%). Forty-eight Self-Tests were excluded in the accuracy analysis (due to a result read as invalid, not sure or ambiguous) resulting in a test system failure rate of 3.45% (95% CI 2.56%-4.55%). At least one observation of difficulty or error with one or more of the test steps were seen in 1,193 (84.6%) participants. Age, education and health literacy were independently associated with the sum score of procedural errors and difficulties. Four tests did not provide a valid result as determined by the trained user's interpretation of the Self-Test. CONCLUSIONS: The OraQuick HIV Self-Test provides reliable and repeatable results in a rural field environment in spite of procedural errors.


Assuntos
Sorodiagnóstico da AIDS/métodos , Anticorpos Anti-HIV/metabolismo , Infecções por HIV , HIV-1/metabolismo , HIV-2/metabolismo , Saliva/metabolismo , Autocuidado , Adolescente , Adulto , Feminino , Infecções por HIV/diagnóstico , Infecções por HIV/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , População Rural , África do Sul
19.
Molecules ; 24(7)2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30974914

RESUMO

The pharmacological relevance of ODNs forming G-quadruplexes as anti-HIV agents has been extensively reported in the literature over the last few years. Recent detailed studies have elucidated the peculiar arrangement adopted by many G-quadruplex-based aptamers and provided insight into their mechanism of action. In this review, we have reported the history of a strong anti-HIV agent: the 6-mer d(TGGGAG) sequence, commonly called "Hotoda's sequence". In particular, all findings reported on this sequence and its modified sequences have been discussed considering the following research phases: (i) discovery of the first 5'-modified active d(TGGGAG) sequences; (ii) synthesis of a variety of end-modified d(TGGGAG) sequences; (iii) biophysical and NMR investigations of natural and modified Hotoda's sequences; (iv); kinetic studies on the most active 5'-modified d(TGGGAG) sequences; and (v) extensive anti-HIV screening of G-quadruplexes formed by d(TGGGAG) sequences. This review aims to clarify all results obtained over the years on Hotoda's sequence, revealing its potentiality as a strong anti-HIV agent (EC50 = 14 nM).


Assuntos
Fármacos Anti-HIV , Quadruplex G , Infecções por HIV , HIV-1 , Oligonucleotídeos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , HIV-1/genética , HIV-1/metabolismo , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/uso terapêutico
20.
PLoS One ; 14(3): e0212888, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30889178

RESUMO

TRIM5α is an interferon inducible restriction factor which contributes to intrinsic defense against HIV infection by targeting the HIV capsid protein CA. Although human TRIM5α (huTRIM5α) does not potently inhibit HIV-1 infection, the ability of huTRIM5α to exhibit some control of HIV-1 infection is evidenced by a single nucleotide polymorphism in huTRIM5α which substitutes aspartic acid to glycine at position 249 (G249D) in the L2 region and is associated with higher susceptibility to HIV-1 infection. To understand the mechanistic basis for the reduced antiviral activity, we employed biophysical and cell biological methods coupled with molecular dynamics simulations to compare WT and the G249D polymorphism of huTRIM5α. We investigated the differences in conformational dynamics of rhesus and huTRIM5α Coiled Coil-Linker 2 (CC-L2) dimers utilizing circular dichroism and single molecule-Fluorescence Energy Transfer (sm-FRET). These methods revealed that the G249D dimer exhibits secondary structure and conformational dynamics similar to WT huTRIM5α. Homology modelling revealed that G249 was present on the hairpin of the antiparallel dimer, in a position which may act to stabilize the adjacent BBox2 domain which mediates the inter-dimeric contacts required for the formation of TRIM5 assemblies. We therefore asked if the G249D mutant forms assemblies in cells with the same efficiency as WT protein by expressing these proteins as YFP fusions and quantifying the number of assemblies in cells. In cells expressing comparable amounts of protein, the G249D mutant formed fewer assemblies than WT protein, in agreement with our homology modeling predictions and molecular dynamics simulations of dimers and higher oligomers of TRIM5α, providing a mechanistic explanation of the reduced antiviral activity of the G249D polymorphism.


Assuntos
Proteínas de Transporte/genética , Infecções por HIV/genética , HIV-1/imunologia , Animais , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Gatos , Predisposição Genética para Doença , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Simulação de Dinâmica Molecular , Polimorfismo de Nucleotídeo Único , Conformação Proteica em alfa-Hélice/genética , Domínios Proteicos/genética , Estrutura Quaternária de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo
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