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1.
Nat Commun ; 12(1): 1950, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782388

RESUMO

Human immunodeficiency virus-1 (HIV-1), the causative agent of AIDS, impacts millions of people. Entry into target cells is mediated by the HIV-1 envelope (Env) glycoprotein interacting with host receptor CD4, which triggers conformational changes allowing binding to a coreceptor and subsequent membrane fusion. Small molecule or peptide CD4-mimetic drugs mimic CD4's Phe43 interaction with Env by inserting into the conserved Phe43 pocket on Env subunit gp120. Here, we present single-particle cryo-EM structures of CD4-mimetics BNM-III-170 and M48U1 bound to a BG505 native-like Env trimer plus the CD4-induced antibody 17b at 3.7 Å and 3.9 Å resolution, respectively. CD4-mimetic-bound BG505 exhibits canonical CD4-induced conformational changes including trimer opening, formation of the 4-stranded gp120 bridging sheet, displacement of the V1V2 loop, and formation of a compact and elongated gp41 HR1C helical bundle. We conclude that CD4-induced structural changes on both gp120 and gp41 Env subunits are induced by binding to the gp120 Phe43 pocket.


Assuntos
Antígenos CD4/química , Guanidinas/química , Proteína gp120 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/química , HIV-1/química , Indenos/química , Receptores Virais/química , Animais , Sítios de Ligação , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Antígenos CD4/antagonistas & inibidores , Antígenos CD4/genética , Antígenos CD4/metabolismo , Células CHO , Clonagem Molecular , Cricetulus , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanidinas/metabolismo , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Humanos , Indenos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores Virais/antagonistas & inibidores , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo
2.
Viruses ; 13(2)2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557422

RESUMO

Understanding of the construction and function of the HIV capsid has advanced considerably in the last decade. This is due in large part to the development of more sophisticated structural techniques, particularly cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). The capsid is known to be a pleomorphic fullerene cone comprised of capsid protein monomers arranged into 200-250 hexamers and 12 pentamers. The latter of these induce high curvature necessary to close the cone at both ends. CryoEM/cryoET, NMR, and X-ray crystallography have collectively described these interactions to atomic or near-atomic resolutions. Further, these techniques have helped to clarify the role the HIV capsid plays in several parts of the viral life cycle, from reverse transcription to nuclear entry and integration into the host chromosome. This includes visualizing the capsid bound to host factors. Multiple proteins have been shown to interact with the capsid. Cyclophilin A, nucleoporins, and CPSF6 promote viral infectivity, while MxB and Trim5α diminish the viral infectivity. Finally, structural insights into the intra- and intermolecular interactions that govern capsid function have enabled development of small molecules, peptides, and truncated proteins to disrupt or stabilize the capsid to inhibit HIV replication. The most promising of these, GS6207, is now in clinical trial.


Assuntos
Antivirais/metabolismo , Capsídeo/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Animais , Antivirais/química , Antivirais/farmacologia , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Infecções por HIV/virologia , HIV-1/química , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Replicação Viral/efeitos dos fármacos
3.
Viruses ; 13(2)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578999

RESUMO

Since the discovery of HIV-1, the viral capsid has been recognized to have an important role as a structural protein that holds the viral genome, together with viral proteins essential for viral life cycle, such as the reverse transcriptase (RT) and the integrase (IN). The reverse transcription process takes place between the cytoplasm and the nucleus of the host cell, thus the Reverse Transcription Complexes (RTCs)/Pre-integration Complexes (PICs) are hosted in intact or partial cores. Early biochemical assays failed to identify the viral CA associated to the RTC/PIC, possibly due to the stringent detergent conditions used to fractionate the cells or to isolate the viral complexes. More recently, it has been observed that some host partners of capsid, such as Nup153 and CPSF6, can only bind multimeric CA proteins organized in hexamers. Those host factors are mainly located in the nuclear compartment, suggesting the entrance of the viral CA as multimeric structure inside the nucleus. Recent data show CA complexes within the nucleus having a different morphology from the cytoplasmic ones, clearly highlighting the remodeling of the viral cores during nuclear translocation. Thus, the multimeric CA complexes lead the viral genome into the host nuclear compartment, piloting the intranuclear journey of HIV-1 in order to successfully replicate. The aim of this review is to discuss and analyze the main discoveries to date that uncover the viral capsid as a key player in the reverse transcription and PIC maturation until the viral DNA integration into the host genome.


Assuntos
Capsídeo/metabolismo , Núcleo Celular/virologia , HIV-1/fisiologia , Transporte Ativo do Núcleo Celular , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , HIV-1/química , HIV-1/metabolismo , Modelos Biológicos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transcrição Reversa , Integração Viral , Replicação Viral
4.
Protein Expr Purif ; 181: 105837, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33529763

RESUMO

Due to the important pathological roles of the HIV-1 gp120, the protein has been intensively used in the research of HIV. However, recombinant gp120 preparation has proven to be difficult because of extremely low expression levels. In order to facilitate gp120 expression, previous methods predominantly involved the replacement of native signal peptide with a heterologous one, resulting in very limited improvement. Currently, preparation of recombinant gp120 with native glycans relies solely on transient expression systems, which are not amendable for large scale production. In this work, we employed a different approach for gp120 expression. Besides replacing the native gp120 signal peptide with that of rat serum albumin and optimizing its codon usage, we generated a stable gp120-expressing cell line in a glutamine synthetase knockout HEK293T cell line that we established for the purpose of amplification of recombinant gene expressions. The combined usage of these techniques dramatically increased gp120 expression levels and yielded a functional product with human cell derived glycan. This method may be applicable to large scale preparation of other viral envelope proteins, such as that of the emerging SARS-CoV-2, or other glycoproteins which require the presence of authentic human glycans.


Assuntos
Glutamato-Amônia Ligase/genética , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Animais , Células CHO , Sistemas CRISPR-Cas , Códon , Cricetulus , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/metabolismo
5.
Arch Virol ; 166(3): 755-766, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33420627

RESUMO

MicroRNAs (miRNAs) are important host molecules involved in human immunodeficiency virus type 1 (HIV-1) infection. Antiretroviral therapy (ART) can affect the miRNA expression profile, but differentially expressed miRNAs still remain to be identified. In this study, we used gene chips to analyze miRNA expression profiles in peripheral blood mononuclear cells from ART-naive HIV-1 patients and those receiving ART, as well as from uninfected individuals. We measured differences in miRNA expression by quantitative polymerase chain reaction (qPCR) in an expanded sample. We found significant differences in the expression of has-miR-191-5p among the three groups (P < 0.05). Furthermore, we showed that hsa-miR-191-5p has an inhibitory effect on HIV-1 replication in cell models in vitro. We identified CCR1 and NUP50 as target molecules of hsa-miR-191-5p and found that hsa-miR-191-5p inhibits the expression of CCR1 and NUP50. Knockdown of NUP50 resulted in significant inhibition of HIV-1 replication. In summary, our research shows that hsa-miR-191-5p expression is reduced in HIV-1-infected patients and acts an inhibitor of HIV-1 infection via a mechanism that may involve targeted repression of NUP50 expression.


Assuntos
Regulação da Expressão Gênica/genética , HIV-1/metabolismo , MicroRNAs/genética , Complexo de Proteínas Formadoras de Poros Nucleares/biossíntese , Proteínas Nucleares/biossíntese , Receptores CCR1/biossíntese , Adulto , Linhagem Celular , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Células Jurkat , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase em Tempo Real , Replicação Viral/genética , Adulto Jovem
6.
Methods Mol Biol ; 2167: 225-252, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712923

RESUMO

Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNA (shRNA) molecules for targeted gene silencing has become a benchmark technology. Plasmid and viral vector systems can be used to express shRNA precursor transcripts that are processed by the cellular RNAi pathway to trigger sequence-specific gene knockdown. Intensive RNAi investigations documented that only a small percentage of computationally predicted target sequences can be used for efficient gene silencing, in part because not all shRNA designs are active. Many factors influence the shRNA activity and guidelines for optimal shRNA design have been proposed. We recently described an alternatively processed shRNA molecule termed AgoshRNA with a ~18 base pairs (bp) stem and a 3-5 nucleotides (nt) loop. This molecule is alternatively processed by the Argonaute (Ago) protein into a single guide RNA strand that efficiently induces the RNAi mechanism. The design rules proposed for regular shRNAs do not apply to AgoshRNA molecules and therefore new rules had to be defined. We optimized the AgoshRNA design and managed to create a set of active AgoshRNAs targeted against the human immunodeficiency virus (HIV). In an attempt to enhance the silencing activity of the AgoshRNA molecules, we included the hepatitis delta virus (HDV) ribozyme at the 3' terminus, which generates a uniform 3' end instead of a 3' U-tail of variable length. We evaluated the impact of this 3'-end modification on AgoshRNA processing and its gene silencing activity and we demonstrate that this novel AgoshRNA-HDV design exhibits enhanced antiviral activity.


Assuntos
Proteínas Argonauta/genética , Inativação Gênica , Infecções por HIV/genética , HIV-1/genética , Vírus Delta da Hepatite/genética , RNA Catalítico/genética , RNA Guia/genética , RNA Interferente Pequeno/genética , Proteínas Argonauta/metabolismo , Northern Blotting , Clonagem Molecular/métodos , Ensaios Enzimáticos/métodos , Vetores Genéticos , Células HEK293 , HIV-1/metabolismo , Humanos , Sequências Repetidas Invertidas/genética , Luciferases/metabolismo , Interferência de RNA , RNA Catalítico/metabolismo , RNA Guia/metabolismo , RNA Interferente Pequeno/metabolismo , Linfócitos T/virologia , Transfecção/métodos
7.
PLoS Biol ; 18(12): e3001010, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370289

RESUMO

An often-returning question for not only HIV-1, but also other organisms, is how predictable evolutionary paths are. The environment, mutational history, and random processes can all impact the exact evolutionary paths, but to which extent these factors contribute to the evolutionary dynamics of a particular system is an open question. Especially in a virus like HIV-1, with a large mutation rate and large population sizes, evolution is expected to be highly predictable if the impact of environment and history is low, and evolution is not neutral. We investigated the effect of environment and mutational history by analyzing sequences from a long-term evolution experiment, in which HIV-1 was passaged on 2 different cell types in 8 independent evolutionary lines and 8 derived lines, 4 of which involved a switch of the environment. The experiments lasted for 240-300 passages, corresponding to approximately 400-600 generations or almost 3 years. The sequences show signs of extensive parallel evolution-the majority of mutations that are shared between independent lines appear in both cell types, but we also find that both environment and mutational history significantly impact the evolutionary paths. We conclude that HIV-1 evolution is robust to small changes in the environment, similar to a transmission event in the absence of an immune response or drug pressure. We also find that the fitness landscape of HIV-1 is largely smooth, although we find some evidence for both positive and negative epistatic interactions between mutations.


Assuntos
Evolução Molecular , HIV-1/genética , HIV-1/metabolismo , Linhagem Celular Tumoral , Evolução Molecular Direcionada/métodos , Aptidão Genética/genética , Infecções por HIV/virologia , Soropositividade para HIV , Humanos , Modelos Genéticos , Mutação/genética , Taxa de Mutação
8.
PLoS Biol ; 18(12): e3001015, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33332391

RESUMO

Reverse transcription, an essential event in the HIV-1 life cycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral capsid. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics (MD) simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP6) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import, and reverse transcription.


Assuntos
Capsídeo/metabolismo , HIV-1/metabolismo , Replicação Viral/fisiologia , Capsídeo/química , Capsídeo/fisiologia , Proteínas do Capsídeo/genética , Replicação do DNA/fisiologia , DNA Viral/metabolismo , Células HEK293 , HIV-1/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Simulação de Dinâmica Molecular , Nucleotídeos/metabolismo , Permeabilidade , Ácido Fítico/análise , Ácido Fítico/metabolismo , Vírion/genética , Montagem de Vírus/fisiologia , Replicação Viral/genética
9.
Phys Rev Lett ; 125(12): 128101, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-33016741

RESUMO

The efficiency of a virus to establish its infection in host cells varies broadly among viruses. It remains unclear if there is a key step in this process that controls viral infectivity. To address this question, we use single-particle tracking and Brownian dynamics simulation to examine human immunodeficiency virus type 1 (HIV-1) infection in cell culture. We find that the frequency of viral-cell encounters is consistent with diffusion-limited interactions. However, even under the most favorable conditions, only 1% of the viruses can become immobilized on cell surface and subsequently enter the cell. This is a result of weak interaction between viral surface gp120 and CD4 receptor, which is insufficient to form a stable complex the majority of the time. We provide the first direct quantitation for efficiencies of these events relevant to measured HIV-1 infectivity and demonstrate that immobilization on host cell surface post-virion-diffusion is the key step in viral infection. Variation of its probability controls the efficiency of a virus to infect its host cells. These results explain the low infectivity of cell-free HIV-1 in vitro and offer a potential rationale for the pervasive high efficiency of cell-to-cell transmission of animal viruses.


Assuntos
HIV-1/patogenicidade , Antígenos CD4/metabolismo , Linhagem Celular , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Humanos , Imagem Óptica , Imagem com Lapso de Tempo , Vírion/metabolismo , Vírion/patogenicidade
10.
PLoS One ; 15(9): e0238975, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32956380

RESUMO

BACKGROUND: Predicting long-term care trajectories at the time of HIV diagnosis may allow targeted interventions. Our objective was to uncover distinct CD4-based trajectories and determine baseline demographic, clinical, and contextual factors associated with trajectory membership. METHODS: We used data from the Sizanani trial (NCT01188941), in which adults were enrolled prior to HIV testing in Durban, South Africa from August 2010-January 2013. We ascertained CD4 counts from the National Health Laboratory Service over 5y follow-up. We used group-based statistical modeling to identify groups with similar CD4 count trajectories and Bayesian information criteria to determine distinct CD4 trajectories. We evaluated baseline factors that predict membership in specific trajectories using multinomial logistic regression. We examined calendar year of participant enrollment, age, gender, cohabitation, TB positivity, self-identified barriers to care, and ART initiation within 3 months of diagnosis. RESULTS: 688 participants had longitudinal data available. Group-based trajectory modeling identified four distinct trajectories: one with consistently low CD4 counts (21%), one with low CD4 counts that increased over time (22%), one with moderate CD4 counts that remained stable (41%), and one with high CD4 counts that increased over time (16%). Those with higher CD4 counts at diagnosis were younger, less likely to have TB, and less likely to identify barriers to care. Those in the least favorable trajectory (consistently low CD4 count) were least likely to start ART within 3 months. CONCLUSIONS: One-fifth of people newly-diagnosed with HIV presented with low CD4 counts that failed to rise over time. Less than 40% were in a trajectory characterized by increasing CD4 counts. Patients in more favorable trajectories were younger, less likely to have TB, and less likely to report barriers to healthcare. Better understanding barriers to early care engagement and ART initiation will be necessary to improve long-term clinical outcomes.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Previsões/métodos , Infecções por HIV/tratamento farmacológico , Adulto , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Teorema de Bayes , Biomarcadores Farmacológicos , Contagem de Linfócito CD4 , Assistência à Saúde , Feminino , Infecções por HIV/epidemiologia , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , África do Sul/epidemiologia
11.
PLoS Med ; 17(9): e1003325, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32936795

RESUMO

BACKGROUND: Current World Health Organization (WHO) antiretroviral therapy (ART) guidelines define virologic failure as two consecutive viral load (VL) measurements ≥1,000 copies/mL, triggering empiric switch to next-line ART. This trial assessed if patients with sustained low-level HIV-1 viremia on first-line ART benefit from a switch to second-line treatment. METHODS AND FINDINGS: This multicenter, parallel-group, open-label, superiority, randomized controlled trial enrolled patients on first-line ART containing non-nucleoside reverse transcriptase inhibitors (NNRTI) with two consecutive VLs ≥100 copies/mL, with the second VL between 100-999 copies/mL, from eight clinics in Lesotho. Consenting participants were randomly assigned (1:1), stratified by facility, demographic group, and baseline VL, to either switch to second-line ART (switch group) or continued first-line ART (control group; WHO guidelines). The primary endpoint was viral suppression (<50 copies/mL) at 36 weeks. Analyses were by intention to treat, using logistic regression models, adjusted for demographic group and baseline VL. Between August 1, 2017, and August 7, 2019, 137 individuals were screened, of whom 80 were eligible and randomly assigned to switch (n = 40) or control group (n = 40). The majority of participants were female (54 [68%]) with a median age of 42 y (interquartile range [IQR] 35-51), taking tenofovir disoproxil fumarate/lamivudine/efavirenz (49 [61%]) and on ART for a median of 5.9 y (IQR 3.3-8.6). At 36 weeks, 22/40 (55%) participants in the switch versus 10/40 (25%) in the control group achieved viral suppression (adjusted difference 29%, 95% CI 8%-50%, p = 0.009). The switch group had significantly higher probability of viral suppression across different VL thresholds (<20, <100, <200, <400, and <600 copies/mL) but not for <1,000 copies/mL. Thirty-four (85%) participants in switch group and 21 (53%) in control group experienced at least one adverse event (AE) (p = 0.002). No hospitalization or death or other serious adverse events were observed. Study limitations include a follow-up period too short to observe differences in clinical outcomes, missing values in CD4 cell counts due to national stockout of reagents during the study, and limited generalizability of findings to other than NNRTI-based first-line ART regimens. CONCLUSIONS: In this study, switching to second-line ART among patients with sustained low-level HIV-1 viremia resulted in a higher proportion of participants with viral suppression. These results endorse lowering the threshold for virologic failure in future WHO guidelines. TRIAL REGISTRATION: The trial is registered at ClinicalTrials.gov, NCT03088241.


Assuntos
Infecções por HIV/tratamento farmacológico , Viremia/tratamento farmacológico , Adulto , Fármacos Anti-HIV/uso terapêutico , Antirretrovirais/uso terapêutico , Terapia Antirretroviral de Alta Atividade/métodos , Quimioterapia Combinada , Feminino , Soropositividade para HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , Humanos , Lesoto/epidemiologia , Masculino , Pessoa de Meia-Idade , Carga Viral
12.
PLoS One ; 15(8): e0238114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822432

RESUMO

INTRODUCTION: Hypertension among HIV positive patients in low- and middle-income countries has got little attention and data on the problem is limited in Ethiopia. Hence, this study aims to determine the magnitude of hypertension and its associated factors among HIV-positive patients receiving care at referral hospitals of Northwest Ethiopia. MATERIALS AND METHODS: A cross-sectional study design was conducted to determine the burden of hypertension in patients living with HIV receiving care at referral hospitals of Northwest Ethiopia between November 2018 and May 2019. Four hundred seven randomly selected adult patients were included for the study. Using standardized questionnaire, sociodemographic, behavioral and clinical data were collected. Anthropometric parameters, fasting blood sugar as well as lipid profiles were determined. Bivariate and multivariate binary logistic regression analysis was performed. RESULT: A total of 407 study subjects with 98% response rate have been included in this study. The prevalence of hypertension was 14.0% (95% CI: 10.63,17.37). Elementary educational status as compared to no education [AOR (95% CI) 2.75 (1.12,6.75), p< 0.05], moderate monthly income compared to low [AOR (95% CI) 4.27 (2.09,8.73), p<0.01], waist circumference [AOR (95% CI) 4.27 (2.09,8.73), p<0.01], taking concomitant other drug therapy [AOR (95% CI) 5.72 (2.25,14.54), p<0.01] and duration of antiretroviral therapy [AOR (95% CI) 1.12 (1.04,1.20) were significantly associated with hypertension. CONCLUSION: Hypertension is not uncommon in patients living with HIV. Educational status, monthly income, waist circumference, concomitant drug therapy and duration of antiretroviral therapy are linked with hypertension. The finding pinpoints that health care providers should work up on risk factors to reduce the burden of hypertension among the patients.


Assuntos
Infecções por HIV/complicações , Hipertensão/epidemiologia , Adulto , Estudos Transversais , Etiópia/epidemiologia , Feminino , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Hipertensão/metabolismo , Modelos Logísticos , Masculino , Adesão à Medicação , Pessoa de Meia-Idade , Prevalência , Encaminhamento e Consulta
13.
Proc Natl Acad Sci U S A ; 117(35): 21637-21646, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817566

RESUMO

Understanding the molecular mechanisms involved in the assembly of viruses is essential for discerning how viruses transmit from cell to cell and host to host. Although molecular aspects of assembly have been studied for many viruses, we still have little information about these events in real time. Enveloped viruses such as HIV that assemble at, and bud from, the plasma membrane have been studied in some detail using live cell fluorescence imaging techniques; however, these approaches provide little information about the real-time morphological changes that take place as viral components come together to form individual virus particles. Here we used correlative scanning ion conductance microscopy and fluorescence confocal microscopy to measure the topological changes, together with the recruitment of fluorescently labeled viral proteins such as Gag and Vpr, during the assembly and release of individual HIV virus-like particles (VLPs) from the top, nonadherent surfaces of living cells. We show that 1) labeling of viral proteins with green fluorescent protein affects particle formation, 2) the kinetics of particle assembly on different plasma membrane domains can vary, possibly as a consequence of differences in membrane biophysical properties, and 3) VLPs budding from the top, unimpeded surface of cells can reach full size in 20 s and disappear from the budding site in 0.5 to 3 min from the moment curvature is initially detected, significantly faster than has been previously reported.


Assuntos
HIV-1/metabolismo , Vírion/metabolismo , Montagem de Vírus/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Liberação de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
14.
PLoS One ; 15(8): e0237013, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32818950

RESUMO

BACKGROUND: Highly Active Antiretroviral Therapy (HAART) is a standard of HIV management to suppress viral load and delay progression to AIDS. However, questions have been raised about the use of antiretroviral therapy and how it affects quality of life (QoL) of people living with HIV/AIDS (PLWHA). The study hence aimed to assess the QoL of PLWHA who were taking HAART at Mizan-Tepi University Teaching Hospital (MTUTH) and identify factors associated with QoL. METHODS: A cross sectional study was conducted among PLWHA receiving HAART at MTUTH from March 04-April 1, 2018. Patients were recruited consecutively and interviewed with structured questionnaire. A data abstraction tool was used to extract data from patient medical records. Quality of life was assessed using the World Health Organization Quality of Life HIV- BREF (WHOQOL-HIV-BREF) standard tool. Data was entered to Epi-Info version 3.5.3 and analyzed using SPSS version 22 for windows. A multivariable logistic regression analysis was fitted to identify factors associated with QoL. A statistical significance was established at a p value <0.05. RESULTS: A total of 240 participants with the mean age of 35.11 (SD = 9.08) years were included in the study. This study found that 57.1% of the patients had high global score of QoL. Patients with normal current health (AOR = 3.38, 95% CI = 1.56-7.31)) and having family support (AOR = 3.12, 95% CI = 1.51-6.46) were positively associated with high global score of QoL, while patients with low HAART adherence (AOR = 0.40, 95%, CI = 0.19-0.86) were negatively associated with high global score of QoL. CONCLUSION: The study revealed that more than half of the participants had high global score of QoL. Normal current health and family support were associated with better global score of QoL, while low HAART adherence was found to be associated with the lower global score of QoL.


Assuntos
Terapia Antirretroviral de Alta Atividade/psicologia , Infecções por HIV/psicologia , Qualidade de Vida/psicologia , Adulto , Terapia Antirretroviral de Alta Atividade/métodos , Estudos Transversais , Etiópia/epidemiologia , Feminino , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , Inquéritos e Questionários
15.
J Virol ; 94(21)2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32788194

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here, we characterized the use of a codon-optimized SARS-COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, murine leukemia virus (MLV), and vesicular stomatitis virus (VSV) particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail. Infection of 293FT target cells was possible only if the cells were engineered to stably express the human angiotensin-converting enzyme 2 (ACE2) receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5- to 10-fold. Replacement of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals.IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/fisiologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/genética , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , Células HEK293 , HIV-1/genética , HIV-1/metabolismo , Humanos , Vírus da Leucemia Murina , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/metabolismo , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/metabolismo , Vírion/genética , Vírion/imunologia , Vírion/metabolismo , Internalização do Vírus
16.
PLoS Pathog ; 16(8): e1008696, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760139

RESUMO

HLA-B*35Px is associated with HIV-1 disease rapid progression to AIDS. However, the mechanism(s) underlying this deleterious effect of this HLA allele on HIV-1 infection outcome has not fully understood. CD8+ T cells play a crucial role to control the viral replication but impaired CD8+ T cells represent a major hallmark of HIV-1 infection. Here, we examined the effector functions of CD8+ T cells restricted by HLA-B*35Px (HLA-B*35:03 and HLA-B*35:02), HLA-B*27/B57 and non-HLA-B*27/B57 (e.g. HLA-A*01, A*02, A*03, A*11, A*24, A*26, B*40, B*08, B*38, B*44). CD8+ T cells restricted by HLA-B*35Px exhibited an impaired phenotype compared with those restricted by HLA-B*27/B57 and even non-HLA-B*27/B57. CD8+ T cells restricted by non-HLA-B*27/B57 when encountered their cognate epitopes upregulated TIM-3 and thus became suppressed by regulatory T cells (Tregs) via TIM-3: Galectin-9 (Gal-9). Strikingly, CD8+ T cells restricted by HLA-B*35Px expressed fewer TIM-3 and therefore did not get suppressed by Tregs, which was similar to CD8+ T cells restricted by HLA-B*27/B57. Instead, CD8+ T cells restricted by HLA-B*35Px upon recognition of their cognate epitopes upregulated CTLA-4. The transcriptional and impaired phenotype (e.g. poor effector functions) of HIV-specific CD8+ T cells restricted by HLA-B*35 was related to persistent CTLA-4, elevated Eomes and blimp-1 but poor T-bet expression. As such, anti-CTLA-4 antibody, Ipilimumab, reversed the impaired proliferative capacity of antigen-specific CD8+ T cells restricted by HLA-B*35Px but not others. This study supports the concept that CD8+ T resistance to Tregs-mediated suppression is related to allele restriction rather than the epitope specificity. Our results aid to explain a novel mechanism for the inability of HIV-specific CD8+ T cells restricted by HLA-B*35Px to control viral replication.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/metabolismo , Epitopos de Linfócito T/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Antígeno HLA-B35/imunologia , Replicação Viral , Antígeno CTLA-4/imunologia , Citocinas/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/metabolismo , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Fenótipo , Linfócitos T Reguladores/imunologia
17.
Proc Natl Acad Sci U S A ; 117(31): 18719-18728, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690692

RESUMO

CD4-based decoy approaches against HIV-1 are attractive options for long-term viral control, but initial designs, including soluble CD4 (sCD4) and CD4-Ig, were ineffective. To evaluate a therapeutic that more accurately mimics HIV-1 target cells compared with monomeric sCD4 and dimeric CD4-Ig, we generated virus-like nanoparticles that present clusters of membrane-associated CD4 (CD4-VLPs) to permit high-avidity binding of trimeric HIV-1 envelope spikes. In neutralization assays, CD4-VLPs were >12,000-fold more potent than sCD4 and CD4-Ig and >100-fold more potent than the broadly neutralizing antibody (bNAb) 3BNC117, with >12,000-fold improvements against strains poorly neutralized by 3BNC117. CD4-VLPs also neutralized patient-derived viral isolates that were resistant to 3BNC117 and other bNAbs. Intraperitoneal injections of CD4-CCR5-VLP produced only subneutralizing plasma concentrations in HIV-1-infected humanized mice but elicited CD4-binding site mutations that reduced viral fitness. All mutant viruses showed reduced sensitivity to sCD4 and CD4-Ig but remained sensitive to neutralization by CD4-VLPs in vitro. In vitro evolution studies demonstrated that CD4-VLPs effectively controlled HIV-1 replication at neutralizing concentrations, and viral escape was not observed. Moreover, CD4-VLPs potently neutralized viral swarms that were completely resistant to CD4-Ig, suggesting that escape pathways that confer resistance against conventional CD4-based inhibitors are ineffective against CD4-VLPs. These findings suggest that therapeutics that mimic HIV-1 target cells could prevent viral escape by exposing a universal vulnerability of HIV-1: the requirement to bind CD4 on a target cell. We propose that therapeutic and delivery strategies that ensure durable bioavailability need to be developed to translate this concept into a clinically feasible functional cure therapy.


Assuntos
Antígenos CD4 , HIV-1 , Nanopartículas , Vírion , Fármacos Anti-HIV , Antígenos CD4/química , Antígenos CD4/metabolismo , Linhagem Celular , HIV-1/química , HIV-1/genética , HIV-1/metabolismo , Humanos , Mimetismo Molecular , Nanomedicina/métodos , Nanopartículas/química , Nanopartículas/metabolismo , Vírion/química , Vírion/metabolismo
18.
Nucleic Acids Res ; 48(14): 7801-7817, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32597987

RESUMO

HIV-1 persists lifelong in memory cells of the immune system as latent provirus that rebounds upon treatment interruption. Therefore, the latent reservoir is the main target for an HIV cure. Here, we studied the direct link between integration site and transcription using LEDGINs and Barcoded HIV-ensembles (B-HIVE). LEDGINs are antivirals that inhibit the interaction between HIV-1 integrase and the chromatin-tethering factor LEDGF/p75. They were used as a tool to retarget integration, while the effect on HIV expression was measured with B-HIVE. B-HIVE tracks insert-specific HIV expression by tagging a unique barcode in the HIV genome. We confirmed that LEDGINs retarget integration out of gene-dense and actively transcribed regions. The distance to H3K36me3, the marker recognized by LEDGF/p75, clearly increased. LEDGIN treatment reduced viral RNA expression and increased the proportion of silent provirus. Finally, silent proviruses obtained after LEDGIN treatment were located further away from epigenetic marks associated with active transcription. Interestingly, proximity to enhancers stimulated transcription irrespective of LEDGIN treatment, while the distance to H3K36me3 only changed after treatment with LEDGINs. The fact that proximity to these markers are associated with RNA expression support the direct link between provirus integration site and viral expression.


Assuntos
Regulação Viral da Expressão Gênica , Inativação Gênica , HIV-1/genética , Provírus/genética , Integração Viral , Linhagem Celular , Cromatina/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , RNA Viral/metabolismo , Integração Viral/efeitos dos fármacos
19.
PLoS Pathog ; 16(6): e1008378, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32492044

RESUMO

The HIV-1 reservoir consists of latently infected cells that persist despite antiretroviral therapy (ART). Elucidating the proviral genetic composition of the reservoir, particularly in the context of pre-therapy viral diversity, is therefore important to understanding reservoir formation and the persistence of latently infected cells. Here we investigate reservoir proviral variants from 13 Zambian acutely-infected individuals with additional pre-therapy sampling for a unique comparison to the ART-naïve quasispecies. We identified complete transmitted/founder (TF) viruses from seroconversion plasma samples, and additionally amplified and sequenced HIV-1 from plasma obtained one year post-infection and just prior to ART initiation. While the majority of proviral variants in the reservoir were most closely related to viral variants from the latest pre-therapy time point, we also identified reservoir proviral variants dating to or near the time of infection, and to intermediate time points between infection and treatment initiation. Reservoir proviral variants differing by five or fewer nucleotide changes from the TF virus persisted during treatment in five individuals, including proviral variants that exactly matched the TF in two individuals, one of whom had remained ART-naïve for more than six years. Proviral variants during treatment were significantly less divergent from the TF virus than plasma variants present at the last ART-naïve time point. These findings indicate that reservoir proviral variants are archived throughout infection, recapitulating much of the viral diversity that arises throughout untreated HIV-1 infection, and strategies to target and reduce the reservoir must therefore permit for the clearance of proviruses encompassing this extensive diversity.


Assuntos
Variação Genética , Infecções por HIV/genética , HIV-1/genética , Filogenia , Doença Aguda , Adulto , Antirretrovirais , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , HIV-1/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Zâmbia
20.
PLoS One ; 15(6): e0231679, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559193

RESUMO

The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145, which is currently in Phase I clinical trials, produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5-5.5 and 6.0-7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown.


Assuntos
Glicopeptídeos/análise , Anticorpos Anti-HIV/imunologia , HIV-1/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Adulto , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Reações Antígeno-Anticorpo , Células CHO , Cricetinae , Cricetulus , Feminino , Glicosilação , Células HEK293 , Humanos , Pessoa de Meia-Idade , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto Jovem , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
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