RESUMO
Quantifying viral growth rates is key to understanding evolutionary dynamics and the potential for mutants to escape antiviral drugs. Defining evolutionary escape paths and their impact on viral fitness allows for the development of drugs that are resistant to escape. In the case of HIV, combination antiretroviral therapy can successfully prevent or treat infection, but it relies on strict adherence to prevent escape. Here, we present a method termed QuickFit that enables the quantification of viral fitness by employing large numbers of parallel viral cultures to measure growth rates accurately. QuickFit consistently recapitulated HIV growth measurements obtained by traditional approaches, but with significantly higher throughput and lower rates of error. This method represents a promising tool for rapid and consistent evaluation of viral fitness.
Assuntos
Replicação Viral , Humanos , Ensaios de Triagem em Larga Escala/métodos , HIV-1/genética , HIV-1/fisiologia , HIV-1/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real/métodos , Aptidão Genética , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , HIV/genética , HIV/fisiologia , HIV/crescimento & desenvolvimento , Linhagem CelularRESUMO
Macrophages are important target cells for diverse viruses and thus represent a valuable system for studying virus biology. Isolation of primary human macrophages is done by culture of dissociated tissues or from differentiated blood monocytes, but these methods are both time consuming and result in low numbers of recovered macrophages. Here, we explore whether macrophages derived from human induced pluripotent stem cells (iPSCs)-which proliferate indefinitely and potentially provide unlimited starting material-could serve as a faithful model system for studying virus biology. Human iPSC-derived monocytes were differentiated into macrophages and then infected with HIV-1, dengue virus, or influenza virus as model human viruses. We show that iPSC-derived macrophages support the replication of these viruses with kinetics and phenotypes similar to human blood monocyte-derived macrophages. These iPSC-derived macrophages were virtually indistinguishable from human blood monocyte-derived macrophages based on surface marker expression (flow cytometry), transcriptomics (RNA sequencing), and chromatin accessibility profiling. iPSC lines were additionally generated from non-human primate (chimpanzee) fibroblasts. When challenged with dengue virus, human and chimpanzee iPSC-derived macrophages show differential susceptibility to infection, thus providing a valuable resource for studying the species-tropism of viruses. We also show that blood- and iPSC-derived macrophages both restrict influenza virus at a late stage of the virus lifecycle. Collectively, our results substantiate iPSC-derived macrophages as an alternative to blood monocyte-derived macrophages for the study of virus biology. IMPORTANCE: Macrophages have complex relationships with viruses: while macrophages aid in the removal of pathogenic viruses from the body, macrophages are also manipulated by some viruses to serve as vessels for viral replication, dissemination, and long-term persistence. Here, we show that iPSC-derived macrophages are an excellent model that can be exploited in virology.
Assuntos
Vírus da Dengue , HIV-1 , Células-Tronco Pluripotentes Induzidas , Macrófagos , Modelos Biológicos , Orthomyxoviridae , Virologia , Animais , Humanos , Diferenciação Celular/genética , HIV-1/crescimento & desenvolvimento , HIV-1/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/virologia , Orthomyxoviridae/crescimento & desenvolvimento , Orthomyxoviridae/fisiologia , Pan troglodytes , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/fisiologia , Fibroblastos/citologia , Monócitos/citologia , Replicação Viral , Citometria de Fluxo , Perfilação da Expressão Gênica , Montagem e Desmontagem da Cromatina , Tropismo Viral , Virologia/métodos , Biomarcadores/análise , Biomarcadores/metabolismoRESUMO
IMPORTANCE: The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.
Assuntos
Núcleo Celular , Eucromatina , HIV-1 , Código das Histonas , Histonas , Empacotamento do Genoma Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Humanos , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Elementos Facilitadores Genéticos/genética , Eucromatina/genética , Eucromatina/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Células HeLa , Histonas/metabolismo , HIV-1/genética , HIV-1/crescimento & desenvolvimento , HIV-1/metabolismo , Regiões Promotoras Genéticas/genética , Linfócitos T/virologia , Transcrição Gênica , Ativação ViralRESUMO
HIV-1 remains a global health crisis1, highlighting the need to identify new targets for therapies. Here, given the disproportionate HIV-1 burden and marked human genome diversity in Africa2, we assessed the genetic determinants of control of set-point viral load in 3,879 people of African ancestries living with HIV-1 participating in the international collaboration for the genomics of HIV3. We identify a previously undescribed association signal on chromosome 1 where the peak variant associates with an approximately 0.3 log10-transformed copies per ml lower set-point viral load per minor allele copy and is specific to populations of African descent. The top associated variant is intergenic and lies between a long intergenic non-coding RNA (LINC00624) and the coding gene CHD1L, which encodes a helicase that is involved in DNA repair4. Infection assays in iPS cell-derived macrophages and other immortalized cell lines showed increased HIV-1 replication in CHD1L-knockdown and CHD1L-knockout cells. We provide evidence from population genetic studies that Africa-specific genetic variation near CHD1L associates with HIV replication in vivo. Although experimental studies suggest that CHD1L is able to limit HIV infection in some cell types in vitro, further investigation is required to understand the mechanisms underlying our observations, including any potential indirect effects of CHD1L on HIV spread in vivo that our cell-based assays cannot recapitulate.
Assuntos
DNA Helicases , Proteínas de Ligação a DNA , Variação Genética , Infecções por HIV , HIV-1 , Carga Viral , Humanos , Linhagem Celular , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Infecções por HIV/genética , HIV-1/crescimento & desenvolvimento , HIV-1/fisiologia , Carga Viral/genética , África , Cromossomos Humanos Par 1/genética , Alelos , RNA Longo não Codificante/genética , Replicação ViralRESUMO
Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA+/p24+ cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. IMPORTANCE Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.
Assuntos
Infecções por HIV , HIV-1 , RNA Viral , Tropismo Viral , Ativação Viral , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/uso terapêutico , Antígenos Virais/análise , Antígenos Virais/genética , Antígenos Virais/metabolismo , Linfócitos T CD4-Positivos , Proteína do Núcleo p24 do HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Humanos , Imunoensaio , Hibridização in Situ Fluorescente , RNA Mensageiro/análise , RNA Viral/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
Elimination of human immunodeficiency virus (HIV) reservoirs is a critical endpoint to eradicate HIV. One therapeutic intervention against latent HIV is "shock and kill." This strategy is based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) with the consequent killing of the reactivated cell by either the cytopathic effect of HIV or the immune system. We have previously found that the small molecule 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) acts as an LRA by increasing signal transducer and activator of transcription (STAT) factor activation mediated by interleukin-15 (IL-15) in cells isolated from aviremic participants. The IL-15 superagonist N-803 is currently under clinical investigation to eliminate latent reservoirs. IL-15 and N-803 share similar mechanisms of action by promoting the activation of STATs and have shown some promise in preclinical models directed toward HIV eradication. In this work, we evaluated the ability of HODHBt to enhance IL-15 signaling in natural killer (NK) cells and the biological consequences associated with increased STAT activation in NK cell effector and memory-like functions. We showed that HODHBt increased IL-15-mediated STAT phosphorylation in NK cells, resulting in increases in the secretion of CXCL-10 and interferon gamma (IFN-γ) and the expression of cytotoxic proteins, including granzyme B, granzyme A, perforin, granulysin, FASL, and TRAIL. This increased cytotoxic profile results in increased cytotoxicity against HIV-infected cells and different tumor cell lines. HODHBt also improved the generation of cytokine-induced memory-like NK cells. Overall, our data demonstrate that enhancing the magnitude of IL-15 signaling with HODHBt favors NK cell cytotoxicity and memory-like generation, and thus, targeting this pathway could be further explored for HIV cure interventions. IMPORTANCE Several clinical trials targeting the HIV latent reservoir with LRAs have been completed. In spite of a lack of clinical benefit, they have been crucial to elucidate hurdles that "shock and kill" strategies have to overcome to promote an effective reduction of the latent reservoir to lead to a cure. These hurdles include low reactivation potential mediated by LRAs, the negative influence of some LRAs on the activity of natural killer and effector CD8 T cells, an increased resistance to apoptosis of latently infected cells, and an exhausted immune system due to chronic inflammation. To that end, finding therapeutic strategies that can overcome some of these challenges could improve the outcome of shock and kill strategies aimed at HIV eradication. Here, we show that the LRA HODHBt also improves IL-15-mediated NK cell effector and memory-like functions. As such, pharmacological enhancement of IL-15-mediated STAT activation can open new therapeutic avenues toward an HIV cure.
Assuntos
HIV-1 , Memória Imunológica , Interleucina-15 , Células Matadoras Naturais , Fatores de Transcrição STAT , Triazinas , Latência Viral , Humanos , Linhagem Celular Tumoral , Quimiocina CXCL10 , Testes Imunológicos de Citotoxicidade , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Memória Imunológica/efeitos dos fármacos , Interferon gama , Interleucina-15/imunologia , Interleucina-15/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Fatores de Transcrição STAT/metabolismo , Ativação Transcricional/efeitos dos fármacos , Triazinas/farmacologia , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacosRESUMO
HIV-1 infection remains a public health problem with no cure. Anti-retroviral therapy (ART) is effective but requires lifelong drug administration owing to a stable reservoir of latent proviruses integrated into the genome of CD4+ T cells1. Immunotherapy with anti-HIV-1 antibodies has the potential to suppress infection and increase the rate of clearance of infected cells2,3. Here we report on a clinical study in which people living with HIV received seven doses of a combination of two broadly neutralizing antibodies over 20 weeks in the presence or absence of ART. Without pre-screening for antibody sensitivity, 76% (13 out of 17) of the volunteers maintained virologic suppression for at least 20 weeks off ART. Post hoc sensitivity analyses were not predictive of the time to viral rebound. Individuals in whom virus remained suppressed for more than 20 weeks showed rebound viraemia after one of the antibodies reached serum concentrations below 10 µg ml-1. Two of the individuals who received all seven antibody doses maintained suppression after one year. Reservoir analysis performed after six months of antibody therapy revealed changes in the size and composition of the intact proviral reservoir. By contrast, there was no measurable decrease in the defective reservoir in the same individuals. These data suggest that antibody administration affects the HIV-1 reservoir, but additional larger and longer studies will be required to define the precise effect of antibody immunotherapy on the reservoir.
Assuntos
Antirretrovirais , Anticorpos Anti-HIV , Infecções por HIV , HIV-1 , Carga Viral , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/virologia , Anticorpos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Humanos , Provírus/efeitos dos fármacos , Carga Viral/efeitos dos fármacos , Viremia/tratamento farmacológico , Latência Viral/efeitos dos fármacosRESUMO
The viral polyprotein Gag plays a central role for HIV-1 assembly, release and maturation. Proteolytic processing of Gag by the viral protease is essential for the structural rearrangements that mark the transition from immature to mature, infectious viruses. The timing and kinetics of Gag processing are not fully understood. Here, fluorescence lifetime imaging microscopy and single virus tracking are used to follow Gag processing in nascent HIV-1 particles in situ. Using a Gag polyprotein labelled internally with eCFP, we show that proteolytic release of the fluorophore from Gag is accompanied by an increase in its fluorescence lifetime. By tracking nascent virus particles in situ and analyzing the intensity and fluorescence lifetime of individual traces, we detect proteolytic cleavage of eCFP from Gag in a subset (6.5%) of viral particles. This suggests that for the majority of VLPs, Gag processing occurs with a delay after particle assembly.
Assuntos
Infecções por HIV/virologia , HIV-1/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , HIV-1/química , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Humanos , Cinética , Microscopia de Fluorescência , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
ABSTRACTHIV-1 latency posts a major obstacle for HIV-1 eradication. Currently, no desirable latency reversing agents (LRAs) have been implicated in the "Shock and Kill" strategy to mobilize the latently infected cells to be susceptible for clearance by immune responses. Identification of key cellular pathways that modulate HIV-1 latency helps to develop efficient LRAs. In this study, we demonstrate that the Wnt downstream ß-catenin/TCF1 pathway is a crucial modulator for HIV-1 latency. The pharmacological activation of the ß-catenin/TCF1 pathway with glycogen synthase kinase-3 (GSK3) inhibitors promoted transcription of HIV-1 proviral DNA and reactivated latency in CD4+ T cells; the GSK3 kinase inhibitor 6-bromoindirubin-3'-oxime (6-BIO)-induced HIV-1 reactivation was subsequently confirmed in resting CD4+ T cells from cART-suppressed patients and SIV-infected rhesus macaques. These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.
Assuntos
Linfócitos T CD4-Positivos/virologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , HIV-1/crescimento & desenvolvimento , Indóis/farmacologia , Oximas/farmacologia , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , HIV-1/efeitos dos fármacos , HIV-1/genética , Células HeLa , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Macaca mulatta , Transcrição Gênica/efeitos dos fármacos , Células U937 , Ativação Viral/genética , Latência Viral/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Among CD4+ T cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier, resulting in a microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long term in the gastrointestinal lymphatic tract where a low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence. It is, however, unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on the expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T cells. The depletion of RORC2 inhibited HIV-1 infection, whereas its overexpression enhanced it. RORC2 was also found to promote HIV-1 gene expression by binding to the nuclear receptor responsive element in the HIV-1 long terminal repeats (LTR). In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2- cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T cells from antiretroviral-treated patients. Altogether, these results provide an explanation as to why Th17 cells are highly susceptible to HIV-1 infection and suggest that RORC2 may be a cell-specific target for HIV-1 therapy.
Assuntos
Regulação Viral da Expressão Gênica/genética , HIV-1/crescimento & desenvolvimento , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Adulto , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Citocinas/metabolismo , Feminino , Expressão Gênica/genética , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Cultura Primária de Células , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/metabolismo , Células Th17/fisiologia , Fatores de Transcrição/metabolismo , Viremia/imunologia , Viremia/virologia , Replicação Viral/fisiologiaRESUMO
HIV-1 entry into host cells leads to one of the following three alternative fates: (1) HIV-1 elimination by restriction factors, (2) establishment of HIV-1 latency, or (3) active viral replication in target cells. Here, we report the development of an improved system for monitoring HIV-1 fate at single-cell and population levels and show the diverse applications of this system to study specific aspects of HIV-1 fate in different cell types and under different environments. An analysis of the transcriptome of infected, primary CD4+ T cells that support alternative fates of HIV-1 identifies differential gene expression signatures in these cells. Small molecules are able to selectively target cells that support viral replication with no significant effect on viral latency. In addition, HIV-1 fate varies in different tissues following infection of humanized mice in vivo. Altogether, these studies indicate that intra- and extra-cellular environments contribute to the fate of HIV-1 infection.
Assuntos
Linfócitos T CD4-Positivos/virologia , Microambiente Celular , Infecções por HIV/virologia , HIV-1/patogenicidade , Animais , Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Interações Hospedeiro-Patógeno , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Células THP-1 , Transcriptoma , Internalização do Vírus , Latência Viral , Replicação ViralRESUMO
Removal of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed co-translationally, we report two additional post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves folding fidelity by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine. Simultaneously, the signal peptide delays folding by tethering the N terminus to the membrane, until assembly with the C terminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine are both highly conserved and important for viral fitness. Considering the â¼15% proteins with signal peptides and the frequency of N-to-C contacts in protein structures, these regulatory roles of signal peptides are bound to be more common in secretory-protein biogenesis.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Processamento de Proteína Pós-Traducional , Cisteína , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/genética , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Células HeLa , Humanos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Sinais Direcionadores de Proteínas , Estabilidade Proteica , Relação Estrutura-Atividade , Carga Viral , Replicação ViralRESUMO
Viral invasion of target cells triggers an immediate intracellular host defense system aimed at preventing further propagation of the virus. Viral genomes or early products of viral replication are sensed by a number of pattern recognition receptors, leading to the synthesis and production of type I interferons (IFNs) that, in turn, activate a cascade of IFN-stimulated genes (ISGs) with antiviral functions. Among these, several members of the tripartite motif (TRIM) family are antiviral executors. This article will focus, in particular, on TRIM22 as an example of a multitarget antiviral member of the TRIM family. The antiviral activities of TRIM22 against different DNA and RNA viruses, particularly human immunodeficiency virus type 1 (HIV-1) and influenza A virus (IAV), will be discussed. TRIM22 restriction of virus replication can involve either direct interaction of TRIM22 E3 ubiquitin ligase activity with viral proteins, or indirect protein-protein interactions resulting in control of viral gene transcription, but also epigenetic effects exerted at the chromatin level.
Assuntos
Infecções por HIV/virologia , HIV-1/patogenicidade , Imunidade Inata , Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Repressoras/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Replicação Viral , Animais , Regulação Viral da Expressão Gênica , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/genética , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Influenza Humana/metabolismo , Transdução de SinaisRESUMO
We determined social and behavioral factors associated with virologic non-suppression among pregnant women receiving Option B+ antiretroviral treatment (ART). Baseline data was used from women in Mobile WAChX trial from 6 public maternal child health (MCH) clinics in Kenya. Virologic non-suppression was defined as HIV viral load (VL) ≥1000 copies/ml. Antiretroviral resistance testing was performed using oligonucleotide ligation (OLA) assay. ART adherence information, motivation and behavioral skills were assessed using Lifewindows IMB tool, depression using PHQ-9, and food insecurity with the Household Food Insecurity Access Scale. Correlates of virologic non-suppression were assessed using Poisson regression. Among 470 pregnant women on ART ≥4 months, 57 (12.1%) had virologic non-suppression, of whom 65% had HIV drug resistance mutations. In univariate analyses, risk of virologic non-suppression was associated with moderate-to-severe food insecurity (RR 1.80 [95% CI 1.06-3.05]), and varied significantly by clinic site (range 2%-22%, p <0.001). In contrast, disclosure (RR 0.36 [95% CI 0.17-0.78]) and having higher adherence skills (RR 0.70 [95% CI 0.58-0.85]) were associated with lower risk of virologic non-suppression. In multivariate analysis adjusting for clinic site, disclosure, depression symptoms, adherence behavior skills and food insecurity, disclosure and food insecurity remained associated with virologic non-suppression. Age, side-effects, social support, physical or emotional abuse, and distance were not associated with virologic non-suppression. Prevalence of virologic non-suppression among pregnant women on ART was appreciable and associated with food insecurity, disclosure and frequent drug resistance. HIV VL and resistance monitoring, and tailored counseling addressing food security and disclosure, may improve virologic suppression in pregnancy.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral/genética , Insegurança Alimentar , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Adulto , Fatores Etários , Terapia Antirretroviral de Alta Atividade , Confidencialidade , Feminino , Infecções por HIV/psicologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Humanos , Quênia , Adesão à Medicação/psicologia , Adesão à Medicação/estatística & dados numéricos , Gravidez , RNA Viral/antagonistas & inibidores , RNA Viral/genética , Apoio Social , Carga Viral/efeitos dos fármacos , Carga Viral/genéticaRESUMO
Polymorphonuclear neutrophils (PMNs), the most abundant white blood cells, are recruited rapidly to sites of infection to exert potent anti-microbial activity. Information regarding their role in infection with human immunodeficiency virus (HIV) is limited. Here we report that addition of PMNs to HIV-infected cultures of human tonsil tissue or peripheral blood mononuclear cells causes immediate and long-lasting suppression of HIV-1 spread and virus-induced depletion of CD4 T cells. This inhibition of HIV-1 spread strictly requires PMN contact with infected cells and is not mediated by soluble factors. 2-Photon (2PM) imaging visualized contacts of PMNs with HIV-1-infected CD4 T cells in tonsil tissue that do not result in lysis or uptake of infected cells. The anti-HIV activity of PMNs also does not involve degranulation, formation of neutrophil extracellular traps, or integrin-dependent cell communication. These results reveal that PMNs efficiently blunt HIV-1 replication in primary target cells and tissue by an unconventional mechanism.
Assuntos
Linfócitos T CD4-Positivos/imunologia , HIV-1/genética , Leucócitos Mononucleares/imunologia , Neutrófilos/imunologia , Tonsila Palatina/imunologia , Linfócitos T CD4-Positivos/virologia , Comunicação Celular , Armadilhas Extracelulares , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , HIV-1/patogenicidade , Humanos , Integrinas/genética , Integrinas/imunologia , Leucócitos Mononucleares/virologia , Neutrófilos/virologia , Tonsila Palatina/citologia , Cultura Primária de Células , Carga Viral , Replicação ViralRESUMO
Genital mucosal transmission is the most common route of HIV spread. The initial responses triggered at the site of viral entry are reportedly affected by host factors, especially complement components present at the site, and this will have profound consequences on the outcome and pathogenesis of HIV infection. We studied the initial events associated with host-pathogen interactions by exposing cervical biopsies to free or complement-opsonized HIV. Opsonization resulted in higher rates of HIV acquisition/infection in mucosal tissues and emigrating dendritic cells. Transcriptomic and proteomic data showed a significantly more pathways and higher expression of genes and proteins associated with viral replication and pathways involved in different aspects of viral infection including interferon signaling, cytokine profile and dendritic cell maturation for the opsonized HIV. Moreover, the proteomics data indicate a general suppression by the HIV exposure. This clearly suggests that HIV opsonization alters the initial signaling pathways in the cervical mucosa in a manner that promotes viral establishment and infection. Our findings provide a foundation for further studies of the role these early HIV induced events play in HIV pathogenesis.
Assuntos
Colo do Útero/virologia , Proteínas do Sistema Complemento/imunologia , Perfilação da Expressão Gênica , Infecções por HIV/virologia , HIV-1/patogenicidade , Mucosa/virologia , Proteoma , Proteômica , Transcriptoma , Colo do Útero/imunologia , Colo do Útero/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Feminino , Regulação da Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Mucosa/imunologia , Mucosa/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Fatores de Tempo , Técnicas de Cultura de Tecidos , Internalização do Vírus , Replicação ViralAssuntos
Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Vacinas contra COVID-19/administração & dosagem , Infecções por HIV/patologia , HIV-1/patogenicidade , Adenina/análogos & derivados , Adenina/uso terapêutico , Adulto , Alanina , Amidas , Terapia Antirretroviral de Alta Atividade/métodos , Vacina BNT162 , Contagem de Linfócito CD4 , Relação CD4-CD8 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Emtricitabina/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Compostos Heterocíclicos com 3 Anéis , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , Testes de Neutralização , Piperazinas , Piridonas , RNA Viral/sangue , RNA Viral/imunologia , SARS-CoV-2/imunologia , Tenofovir/análogos & derivados , Falha de TratamentoRESUMO
HIV-1 infection affects approximately 37 million individuals, and approximately 50% of seropositive individuals will develop symptoms of clinical depression and/or apathy. Dysfunctions of both serotonergic and dopaminergic neurotransmission have been implicated in the pathogenesis of motivational alterations. The present study evaluated the efficacy of a SSRI (escitalopram) in the HIV-1 transgenic (Tg) rat. Behavioral, neurochemical, and neuroanatomical outcomes with respect to HIV-1 and sex were evaluated to determine the efficacy of chronic escitalopram treatment. Escitalopram treatment restored function in each of the behavioral tasks that were sensitive to HIV-1-induced impairments. Further, escitalopram treatment restored HIV-1-mediated synaptodendritic damage in the nucleus accumbens; treatment with escitalopram significantly increased dendritic proliferation in HIV-1 Tg rats. However, restoration did not consistently occur with the neurochemical analysis in the HIV-1 rat. Taken together, these results suggest a role for SSRI therapies in repairing long-term HIV-1 protein-mediated neuronal damage and restoring function.
Assuntos
Antidepressivos/farmacologia , Apatia/efeitos dos fármacos , Depressão/tratamento farmacológico , Escitalopram/farmacologia , Infecções por HIV/tratamento farmacológico , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Animais , Comportamento de Escolha/efeitos dos fármacos , Dendritos/efeitos dos fármacos , Dendritos/patologia , Dendritos/virologia , Depressão/complicações , Depressão/fisiopatologia , Depressão/virologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Neurônios Dopaminérgicos/virologia , Feminino , Infecções por HIV/complicações , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , HIV-1/patogenicidade , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/patologia , Núcleo Accumbens/virologia , Ratos , Ratos Transgênicos , Neurônios Serotoninérgicos/efeitos dos fármacos , Neurônios Serotoninérgicos/patologia , Neurônios Serotoninérgicos/virologia , Sinapses/efeitos dos fármacos , Sinapses/patologia , Sinapses/virologia , Transmissão Sináptica/efeitos dos fármacos , Resultado do TratamentoRESUMO
Type I interferons (IFNs) are a family of cytokines that represent a first line of defense against virus infections. The 12 different IFN-α subtypes share a receptor on target cells and trigger similar signaling cascades. Several studies have collectively shown that this apparent redundancy conceals qualitatively different responses induced by individual subtypes, which display different efficacies of inhibition of HIV replication. Some studies, however, provided evidence that the disparities are quantitative rather than qualitative. Since RNA expression analyses show a large but incomplete overlap of the genes induced, they may support both models. To explore if the IFN-α subtypes induce functionally relevant different anti-HIV activities, we have compared the efficacies of inhibition of all 12 subtypes on HIV spread and on specific steps of the viral replication cycle, including viral entry, reverse transcription, protein synthesis, and virus release. Finding different hierarchies of inhibition would validate the induction of qualitatively different responses. We found that while most subtypes similarly inhibit virus entry, they display distinctive potencies on other early steps of HIV replication. In addition, only some subtypes were able to target effectively the late steps. The extent of induction of known anti-HIV factors helps to explain some, but not all differences observed, confirming the participation of additional IFN-induced anti-HIV effectors. Our findings support the notion that different IFN-α subtypes can induce the expression of qualitatively different antiviral activities. IMPORTANCE The initial response against viruses relies in large part on type I interferons, which include 12 subtypes of IFN-α. These cytokines bind to a common receptor on the cell surface and trigger the expression of incompletely overlapping sets of genes. Whether the anti-HIV responses induced by IFN-α subtypes differ in the extent of expression or in the nature of the genes involved remains debated. Also, RNA expression profiles led to opposite conclusions, depending on the importance attributed to the induction of common or distinctive genes. To explore if relevant anti-HIV activities can be differently induced by the IFN-α subtypes, we compared their relative efficacies on specific steps of the replication cycle. We show that the hierarchy of IFN potencies depends on the step analyzed, supporting qualitatively different responses. This work will also prompt the search for novel IFN-induced anti-HIV factors acting on specific steps of the replication cycle.
Assuntos
HIV-1/crescimento & desenvolvimento , Interferon-alfa/classificação , Interferon-alfa/imunologia , Receptor de Interferon alfa e beta/metabolismo , Replicação Viral/fisiologia , Linhagem Celular , Células HEK293 , HIV-1/imunologia , Humanos , Imunidade Inata/imunologia , Transdução de Sinais/imunologia , Internalização do VírusRESUMO
HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T cells, indicating increased tension between proapoptotic and prosurvival family members-and suggesting that inhibition of prosurvival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-XL due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T cells with HIV resulted in increased BCL-XL protein expression, and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4+ T cells. In a primary cell model of latency, both BCL-XL antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination phorbol myristate acetate (PMA) + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T cells from antiretroviral therapy (ART)-suppressed donors. Our results add to growing evidence that bona fide reservoir-harboring cells are resistant to multiple "kick and kill" modalities-relative to latency models. We also interpret our results as encouraging further exploration of BCL-XL antagonists for cure, where combination approaches, including with immune effectors, may unlock the ability to eliminate ex vivo reservoirs. IMPORTANCE Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in "reservoirs" of infected cells that reinitiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a "prosurvival" factor, BCL-XL, that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-XL antagonists. These antagonists also reduced reservoirs in a primary-cell latency model but were insufficient to reduce "natural" reservoirs in ex vivo CD4+ T cells-adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-XL antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.