RESUMO
Despite the success of combination antiretroviral therapy (cART), HIV persists in long lived latently infected cells in the blood and tissue, and treatment is required lifelong. Recent clinical studies have trialed latency-reversing agents (LRA) as a method to eliminate latently infected cells; however, the effects of LRA on the central nervous system (CNS), a well-known site of virus persistence on cART, are unknown. In this study, we evaluated the toxicity and potency of a panel of commonly used and well-known LRA (panobinostat, romidepsin, vorinostat, chaetocin, disulfiram, hexamethylene bisacetamide [HMBA], and JQ-1) in primary fetal astrocytes (PFA) as well as monocyte-derived macrophages as a cellular model for brain perivascular macrophages. We show that most LRA are non-toxic in these cells at therapeutic concentrations. Additionally, romidepsin, JQ-1, and panobinostat were the most potent at inducing viral transcription, with greater magnitude observed in PFA. In contrast, vorinostat, chaetocin, disulfiram, and HMBA all demonstrated little or no induction of viral transcription. Together, these data suggest that some LRA could potentially activate transcription in latently infected cells in the CNS. We recommend that future trials of LRA also examine the effects of these agents on the CNS via examination of cerebrospinal fluid.
Assuntos
HIV-1/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Neurônios/efeitos dos fármacos , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Acetamidas/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/virologia , Azepinas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Depsipeptídeos/farmacologia , Dissulfiram/farmacologia , Feto , HIV-1/genética , HIV-1/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/virologia , Neurônios/metabolismo , Neurônios/virologia , Panobinostat , Piperazinas/farmacologia , Cultura Primária de Células , Transcrição Gênica/efeitos dos fármacos , Triazóis/farmacologia , Ativação Viral/genética , Latência Viral/genética , Replicação Viral/genética , VorinostatRESUMO
Human immunodeficiency virus type-1 (HIV-1) invades the central nervous system (CNS) during acute infection which can result in HIV-associated neurocognitive disorders in up to 50% of patients, even in the presence of combination antiretroviral therapy (cART). Within the CNS, productive HIV-1 infection occurs in the perivascular macrophages and microglia. Astrocytes also become infected, although their infection is restricted and does not give rise to new viral particles. The major barrier to the elimination of HIV-1 is the establishment of viral reservoirs in different anatomical sites throughout the body and viral persistence during long-term treatment with cART. While the predominant viral reservoir is believed to be resting CD4(+) T cells in the blood, other anatomical compartments including the CNS, gut-associated lymphoid tissue, bone marrow, and genital tract can also harbour persistently infected cellular reservoirs of HIV-1. Viral latency is predominantly responsible for HIV-1 persistence and is most likely governed at the transcriptional level. Current clinical trials are testing transcriptional activators, in the background of cART, in an attempt to purge these viral reservoirs and reverse viral latency. These strategies aim to activate viral transcription in cells constituting the viral reservoir, so they can be recognised and cleared by the immune system, while new rounds of infection are blocked by co-administration of cART. The CNS has several unique characteristics that may result in differences in viral transcription and in the way latency is established. These include CNS-specific cell types, different transcription factors, altered immune surveillance, and reduced antiretroviral drug bioavailability. A comprehensive understanding of viral transcription and latency in the CNS is required in order to determine treatment outcomes when using transcriptional activators within the CNS.
Assuntos
Encéfalo/virologia , Reservatórios de Doenças/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Ativação Transcricional/fisiologia , Humanos , Latência Viral/fisiologiaRESUMO
This investigation aimed to assess whether inhibition of cathecol-O-methyl transferase (COMT) by tolcapone could provide neuroprotection against HIV-associated neurodegenerative effects. This study was conducted based on a previous work, which showed that a single nucleotide polymorphism (SNP) at position 158 (val158met) in COMT, resulted in 40 % lower COMT activity. Importantly, this reduction confers a protective effect against HIV-associated neurocognitive disorders (HAND), which have been linked to HIV-associated brain changes. SH-SY5Y-differentiated neurons were exposed to macrophage-propagated HIV (neurotropic MACS2-Br strain) in the presence or absence of tolcapone for 6 days. RNA was extracted, and qPCR was performed using Qiagen RT2 custom array consisting of genes for neuronal and synaptic integrity, COMT and pro-inflammatory markers. Immunofluorescence was conducted to validate the gene expression changes at the protein level. Our findings demonstrated that HIV significantly increased the messenger RNA (mRNA) expression of COMT while reducing the expression of microtubule-associated protein 2 (MAP2) (p = 0.0015) and synaptophysin (SYP) (p = 0.012) compared to control. A concomitant exposure of tolcapone ameliorated the perturbed expression of MAP2 (p = 0.009) and COMT (p = 0.024) associated with HIV. Immunofluorescence revealed a trend reduction of SYP and MAP2 with exposure to HIV and that concomitant exposure of tolcapone increased SYP (p = 0.016) compared to HIV alone. Our findings demonstrated in vitro that inhibition of COMT can ameliorate HIV-associated neurodegenerative changes that resulted in the decreased expression of the structural and synaptic components MAP2 and SYP. As HIV-associated dendritic and synaptic damage are contributors to HAND, inhibition of COMT may represent a potential strategy for attenuating or preventing some of the symptoms of HAND.
Assuntos
Benzofenonas/farmacologia , Inibidores de Catecol O-Metiltransferase/farmacologia , Catecol O-Metiltransferase/metabolismo , HIV/enzimologia , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Nitrofenóis/farmacologia , Sinaptofisina/metabolismo , Linhagem Celular , Imunofluorescência , HIV/efeitos dos fármacos , Humanos , Neurônios/virologia , Reação em Cadeia da Polimerase em Tempo Real , Tolcapona , TranscriptomaRESUMO
Several critical events of apoptosis occur in the cell nucleus, including inter-nucleosomal DNA fragmentation (apoptotic DNA) and eventual chromatin condensation. The generation of apoptotic DNA has become a biochemical hallmark of apoptosis because it is a late 'point of no return' step in both the extrinsic (cell-death receptor) and intrinsic (mitochondrial) apoptotic pathways. Despite investigators observing apoptotic DNA and understanding its decisive role as a marker of apoptosis for over 20 years, measuring it has proved elusive. We have integrated ligation-mediated PCR and qPCR to design a new way of measuring apoptosis, termed ApoqPCR, which generates an absolute value for the amount (picogram) of apoptotic DNA per cell population. ApoqPCR's advances over current methods include a 1000-fold linear dynamic range yet sensitivity to distinguish subtle low-level changes, measurement with a 3- to 4-log improvement in sample economy, and capacity for archival or longitudinal studies combined with high-throughput capability. We demonstrate ApoqPCR's utility in both in vitro and in vivo contexts. Considering the fundamental role apoptosis has in vertebrate and invertebrate health, growth and disease, the reliable measurement of apoptotic nucleic acid by ApoqPCR will be of value in cell biology studies in basic and applied science.
Assuntos
Apoptose , Fragmentação do DNA , Reação em Cadeia da Polimerase/métodos , DNA/química , Infecções por HIV/patologia , Humanos , Células Jurkat , Peso Molecular , Nucleossomos/metabolismo , Reação em Cadeia da Polimerase/normas , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
BACKGROUND: The majority of HIV-1 subjects worldwide are infected with HIV-1 subtype C (C-HIV). Although C-HIV predominates in developing regions of the world such as Southern Africa and Central Asia, C-HIV is also spreading rapidly in countries with more developed economies and health care systems, whose populations are more likely to have access to wider treatment options, including the CCR5 antagonist maraviroc (MVC). The ability to reliably determine C-HIV coreceptor usage is therefore becoming increasingly more important. In silico V3 sequence based coreceptor usage prediction algorithms are a relatively rapid and cost effective method for determining HIV-1 coreceptor specificity. In this study, we elucidated the V3 sequence determinants of C-HIV coreceptor usage, and used this knowledge to develop and validate a novel, user friendly, and highly sensitive C-HIV specific coreceptor usage prediction algorithm. RESULTS: We characterized every phenotypically-verified C-HIV gp120 V3 sequence available in the Los Alamos HIV Database. Sequence analyses revealed that compared to R5 C-HIV V3 sequences, CXCR4-using C-HIV V3 sequences have significantly greater amino acid variability, increased net charge, increased amino acid length, increased frequency of insertions and substitutions within the GPGQ crown motif, and reduced frequency of glycosylation sites. Based on these findings, we developed a novel C-HIV specific coreceptor usage prediction algorithm (CoRSeqV3-C), which we show has superior sensitivity for determining CXCR4 usage by C-HIV strains compared to all other available algorithms and prediction rules, including Geno2pheno[coreceptor] and WebPSSMSINSI-C, which has been designed specifically for C-HIV. CONCLUSIONS: CoRSeqV3-C is now openly available for public use at http://www.burnet.edu.au/coreceptor. Our results show that CoRSeqV3-C is the most sensitive V3 sequence based algorithm presently available for predicting CXCR4 usage of C-HIV strains, without compromising specificity. CoRSeqV3-C may be potentially useful for assisting clinicians to decide the best treatment options for patients with C-HIV infection, and will be helpful for basic studies of C-HIV pathogenesis.
Assuntos
Biologia Computacional/métodos , Proteína gp120 do Envelope de HIV/genética , HIV-1/fisiologia , Biologia Molecular/métodos , Receptores de HIV/análise , Tropismo Viral , Virologia/métodos , Genótipo , HIV-1/genética , HumanosRESUMO
BACKGROUND: The CCR5 antagonist maraviroc (MVC) inhibits human immunodeficiency virus type 1 (HIV-1) entry by altering the CCR5 extracellular loops (ECL), such that the gp120 envelope glycoproteins (Env) no longer recognize CCR5. The mechanisms of HIV-1 resistance to MVC, the only CCR5 antagonist licensed for clinical use are poorly understood, with insights into MVC resistance almost exclusively limited to knowledge obtained from in vitro studies or from studies of resistance to other CCR5 antagonists. To more precisely understand mechanisms of resistance to MVC in vivo, we characterized Envs isolated from 2 subjects who experienced virologic failure on MVC. RESULTS: Envs were cloned from subjects 17 and 24 before commencement of MVC (17-Sens and 24-Sens) and after virologic failure (17-Res and 24-Res). The Envs cloned during virologic failure showed broad divergence in resistance levels, with 17-Res Env exhibiting a relatively high maximal percent inhibition (MPI) of ~90% in NP2-CD4/CCR5 cells and peripheral blood mononuclear cells (PBMC), and 24-Res Env exhibiting a very low MPI of ~0 to 12% in both cell types, indicating relatively "weak" and "strong" resistance, respectively. Resistance mutations were strain-specific and mapped to the gp120 V3 loop. Affinity profiling by the 293-Affinofile assay and mathematical modeling using VERSA (Viral Entry Receptor Sensitivity Analysis) metrics revealed that 17-Res and 24-Res Envs engaged MVC-bound CCR5 inefficiently or very efficiently, respectively. Despite highly divergent phenotypes, and a lack of common gp120 resistance mutations, both resistant Envs exhibited an almost superimposable pattern of dramatically increased reliance on sulfated tyrosine residues in the CCR5 N-terminus, and on histidine residues in the CCR5 ECLs. This altered mechanism of CCR5 engagement rendered both the resistant Envs susceptible to neutralization by a sulfated peptide fragment of the CCR5 N-terminus. CONCLUSIONS: Clinical resistance to MVC may involve divergent Env phenotypes and different genetic alterations in gp120, but the molecular mechanism of resistance of the Envs studied here appears to be related. The increased reliance on sulfated CCR5 N-terminus residues suggests a new avenue to block HIV-1 entry by CCR5 N-terminus sulfopeptidomimetic drugs.
Assuntos
Fármacos Anti-HIV/farmacologia , Cicloexanos/farmacologia , Proteína gp120 do Envelope de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação de Sentido Incorreto , Triazóis/farmacologia , Internalização do Vírus/efeitos dos fármacos , Fármacos Anti-HIV/uso terapêutico , Cicloexanos/uso terapêutico , Variação Genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/isolamento & purificação , HIV-1/fisiologia , Humanos , Maraviroc , Dados de Sequência Molecular , Análise de Sequência de DNA , Falha de Tratamento , Triazóis/uso terapêuticoRESUMO
Macrophage tropism of human immunodeficiency virus type 1 (HIV-1) is distinct from coreceptor specificity of the viral envelope glycoproteins (Env), but the virus-cell interactions that contribute to efficient HIV-1 entry into macrophages, particularly via CXCR4, are not well understood. Here, we characterized a panel of HIV-1 Envs that use CCR5 (n = 14) or CXCR4 (n = 6) to enter monocyte-derived macrophages (MDM) with various degrees of efficiency. Our results show that efficient CCR5-mediated MDM entry by Env-pseudotyped reporter viruses is associated with increased tolerance of several mutations within the CCR5 N terminus. In contrast, efficient CXCR4-mediated MDM entry was associated with reduced tolerance of a large deletion within the CXCR4 N terminus. Env sequence analysis and structural modeling identified amino acid variants at positions 261 and 263 within the gp41-interactive region of gp120 and a variant at position 326 within the gp120 V3 loop that were associated with efficient CXCR4-mediated MDM entry. Mutagenesis studies showed that the gp41 interaction domain variants exert a significant but strain-specific influence on CXCR4-mediated MDM entry, suggesting that the structural integrity of the gp120-gp41 interface is important for efficient CXCR4-mediated MDM entry of certain HIV-1 strains. However, the presence of Ile326 in the gp120 V3 loop stem, which we show by molecular modeling is located at the gp120-coreceptor interface and predicted to interact with the CXCR4 N terminus, was found to be critical for efficient CXCR4-mediated MDM entry of divergent CXCR4-using Envs. Together, the results of our study provide novel insights into alternative mechanisms of Env-coreceptor engagement that are associated with efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Macrófagos/virologia , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Receptores de HIV/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Linhagem Celular , Proteína gp120 do Envelope de HIV/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Receptores CCR5/genética , Receptores CXCR4/genéticaRESUMO
Human immunodeficiency virus type 1 (HIV-1) demonstrates a high degree of viral diversity which has an impact on viral fitness. Genetic compartmentalization of HIV-1 proteins between central nervous system (CNS) and lymphoid tissues is well established and reflects altered requirements for HIV-1 replication in macrophages/microglia, brain-specific immune selection pressures and possibly the timing of virus invasion of the CNS. Tat-encoding mRNA has been detected in the CNS of HIV-1 infected individuals and its neurotoxic effects in the CNS are well documented. However, while CNS-derived tat sequences have demonstrated significant diversity, the effect of this molecular diversity on transcriptional regulation and its impact on the pathogenesis of HIV-associated dementia (HAD) remains unclear. In this study, we cloned and characterised 44 unique tat alleles from brain, cerebral spinal fluid, spinal cord and blood/lymphoid tissue-derived HIV-1 isolates from five subjects with HAD. While phylogenetic analyses revealed tissue-specific compartmentalization of Tat variants for two patients, broad compartmentalization across the panel of tissue-derived viruses was not observed. Despite the lack of consistent tissue-specific compartmentalization, sequence variations within patients segregated CNS and non-CNS tat alleles. These amino acid alterations predominated within the transactivation domain of Tat and could account for alterations in the ability of particular Tat proteins to transactivate the LTR. Although a subset of patients demonstrated reduced transactivation capacity among CNS-derived Tat proteins compared to those from matched lymphoid tissues, overall Tat proteins from the CNS to lymphoid compartments maintained similar levels of transactivation function. Together, these data suggest that despite the observed heterogeneity in tat alleles isolated from matched lymphoid to CNS compartments, Tat function is maintained, highlighting the importance of Tat function in HIV-1 neuropathogenesis.
Assuntos
Complexo AIDS Demência/virologia , Alelos , Sistema Nervoso Central/virologia , Infecções por HIV/virologia , HIV-1/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Encéfalo/metabolismo , Encéfalo/virologia , Linhagem Celular , Sistema Nervoso Central/metabolismo , Genes tat , HIV-1/patogenicidade , Humanos , Tecido Linfoide/metabolismo , Tecido Linfoide/virologia , Dados de Sequência Molecular , Filogenia , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Human immunodeficiency virus type 1 (HIV-1) nef undergoes adaptive evolution in the central nervous system (CNS), reflecting altered requirements for HIV-1 replication in macrophages/microglia and brain-specific immune selection pressures. The role of Nef in HIV-1 neurotropism and pathogenesis of HIV-associated dementia (HAD) is unclear. In this study, we characterized 82 nef alleles cloned from brain, cerebral spinal fluid, spinal cord, and blood/lymphoid tissue-derived HIV-1 isolates from seven subjects with HAD. CNS isolate-derived nef alleles were genetically compartmentalized and had reduced sequence diversity compared to those from lymphoid tissue isolates. Defective nef alleles predominated in a brain-derived isolate from one of the seven subjects (MACS2-br). The ability of Nef to down-modulate CD4 and MHC class 1 (MHC-1) was generally conserved among nef alleles from both CNS and lymphoid tissues. However, the potency of CD4 and MHC-1 down-modulation was variable, which was associated with sequence alterations known to influence these Nef functions. These results suggest that CD4 and MHC-1 down-modulations are highly conserved functions among nef alleles from CNS- and lymphoid tissue-derived HIV-1 isolates that may contribute to viral replication and escape from immune surveillance in the CNS.
Assuntos
Encéfalo/virologia , Antígenos CD4/metabolismo , Genes MHC Classe I , HIV-1/genética , Tecido Linfoide/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Complexo AIDS Demência/genética , Complexo AIDS Demência/metabolismo , Complexo AIDS Demência/virologia , Alelos , Sequência de Aminoácidos , Encéfalo/metabolismo , Linhagem Celular , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/virologia , Regulação para Baixo , Genes nef , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Tecido Linfoide/metabolismo , Masculino , Dados de Sequência Molecular , Filogenia , Replicação Viral , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
Gene/cell therapies are promising strategies for the many presently incurable diseases. A key step in this process is the efficient delivery of genes and gene-editing enzymes to many cell types that may be resistant to lentiviral vector transduction. Herein we describe tuning of a lentiviral gene therapy platform to focus on genetic modifications of resting CD4+ T cells. The motivation for this was to find solutions for HIV gene therapy efforts. Through selection of the optimal viral envelope and further modification to its expression, lentiviral fusogenic delivery into resting CD4+ T cells exceeded 80%, yet Sterile Alpha Motif and HD domain 1 (SAMHD1) dependent and independent intracellular restriction factors within resting T cells then dominate delivery and integration of lentiviral cargo. Overcoming SAMHD1-imposed restrictions, only observed up to 6-fold increase in transduction, with maximal gene delivery and expression of 35%. To test if the biologically limiting steps of lentiviral delivery are reverse transcription and integration, we re-engineered lentiviral vectors to simply express biologically active mRNA to direct transgene expression in the cytoplasm. In this setting, we observed gene expression in up to 65% of resting CD4+ T cells using unconcentrated MS2 lentivirus-like particles (MS2-LVLPs). Taken together, our findings support a gene therapy platform that could be readily used in resting T cell gene editing.
Assuntos
Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Lentivirus/genética , Fase de Repouso do Ciclo Celular , Transgenes , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citometria de Fluxo , Genótipo , Humanos , Linfócitos T/metabolismo , Transdução GenéticaRESUMO
OBJECTIVE: To determine whether latency can be established and reversed in both proliferating and nonproliferating CD4+ T cells in the same model in vitro. METHODS: Activated CD4+ T cells were infected with either a nonreplication competent, luciferase reporter virus or wild-type full-length enhanced green fluorescent protein (EGFP) reporter virus and cultured for 12 days. The cells were then sorted by flow cytometry to obtain two distinct T-cell populations that did not express the T-cell activation markers, CD69, CD25 and human leukocyte antigen (HLA)-DR: CD69CD25HLA-DR small cells (nonblasts) that had not proliferated in vitro following mitogen stimulation and CD69CD25HLA-DR large cells (which we here call transitional blasts) that had proliferated. The cells were then reactivated with latency-reversing agents and either luciferase or EGFP quantified. RESULTS: Inducible luciferase expression, consistent with latent infection, was observed in nonblasts and transitional blasts following stimulation with either phorbol-myristate-acetate/phytohemagglutinin (3.8â±â1 and 2.9â±â0.5 fold above dimethyl sulfoxide, respectively) or romidepsin (2.1â±â0.6 and 1.8â±â0.2 fold above dimethyl sulfoxide, respectively). Constitutive expression of luciferase was higher in transitional blasts compared with nonblasts. Using wild-type full-length EGFP reporter virus, inducible virus was observed in nonblasts but not in transitional blasts. No significant difference was observed in the response to latency-reversing agents in either nonblasts or transitional blasts. CONCLUSION: HIV latency can be established in vitro in resting T cells that have not proliferated (nonblasts) and blasts that have proliferated (transitional blasts). This model could potentially be used to assess new strategies to eliminate latency.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/virologia , Proliferação de Células , HIV/fisiologia , Latência Viral , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/classificação , Células Cultivadas , Citometria de Fluxo , Antígenos HLA-DR/análise , Humanos , Subunidade alfa de Receptor de Interleucina-2/análise , Lectinas Tipo C/análise , Coloração e RotulagemRESUMO
BACKGROUND: CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively. RESULTS: Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure. CONCLUSION: Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.
Assuntos
Síndrome da Imunodeficiência Adquirida/virologia , Asparagina/fisiologia , Proteína gp120 do Envelope de HIV/química , HIV-1/fisiologia , Receptores CCR5/metabolismo , Antígenos CD4/metabolismo , Fusão Celular , Células Cultivadas , HIV-1/química , HIV-1/patogenicidade , Humanos , Leucócitos Mononucleares , Modelos Moleculares , Virulência , Ligação ViralRESUMO
Maraviroc (MVC) is an allosteric inhibitor of human immunodeficiency virus type 1 (HIV-1) entry, and is the only CCR5 antagonist licensed for use as an anti-HIV-1 therapeutic. It acts by altering the conformation of the CCR5 extracellular loops, rendering CCR5 unrecognizable by the HIV-1 envelope (Env) glycoproteins. This study aimed to understand the mechanisms underlying the development of MVC resistance in HIV-1-infected patients. To do this, we obtained longitudinal plasma samples from eight subjects who experienced treatment failure with phenotypically verified, CCR5-tropic MVC resistance. We then cloned and characterized HIV-1 Envs (n = 77) from plasma of pretreatment (n = 36) and treatment failure (n = 41) samples. Our results showed variation in the magnitude of MVC resistance as measured by reductions in maximal percent inhibition of Env-pseudotyped viruses, which was more pronounced in 293-Affinofile cells compared to other cells with similar levels of CCR5 expression. Amino acid determinants of MVC resistance localized to the V3 Env region and were strain specific. We also observed minimal cross-resistance to other CCR5 antagonists by MVC-resistant strains. We conclude that 293-Affinofile cells are highly sensitive for detecting and measuring MVC resistance through a mechanism that is CCR5-dependent yet independent of CCR5 expression levels. The strain-specific nature of resistance mutations suggests that sequence-based diagnostics and prognostics will need to be more sophisticated than simple position scoring to be useful for managing resistance in subjects taking MVC. Finally, the minimal levels of cross-resistance suggests that recognition of the MVC-modified form of CCR5 does not necessarily lead to recognition of other antagonist-modified forms of CCR5.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Antagonistas dos Receptores CCR5/uso terapêutico , Cicloexanos/uso terapêutico , Farmacorresistência Viral/genética , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/tratamento farmacológico , Receptores CCR5/efeitos dos fármacos , Triazóis/uso terapêutico , Adulto , Contagem de Linfócito CD4 , Linhagem Celular , Feminino , Células HEK293 , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Masculino , Maraviroc , Pessoa de Meia-Idade , Falha de Tratamento , Internalização do Vírus/efeitos dos fármacosRESUMO
PURPOSE OF REVIEW: To review current knowledge of viral reservoirs in the central nervous system (CNS) and identify the CNS-specific barriers and strategies to cure human immunodeficiency virus type 1 (HIV-1) within the brain. RECENT FINDINGS: The cumulative data of HIV-1 infection of the CNS support the ability of the CNS to act as a viral reservoir for HIV-1. The HIV-1 viral strains found in the CNS are distinct to those found in other parts of the body. These differences have been well documented for env and also extend to the viral promoter, the long terminal repeat, and influence the ability of the virus to replicate, establish latency and respond to latency-reversing agents (LRAs). In addition, the bioavailability and activity of LRAs and antiretrovirals within the CNS suggest altered properties compared with the blood, which may influence their effectiveness. Selected LRAs were shown to have reduced effectiveness against CNS-derived viral strains compared with blood-derived strains from the same patients. Finally, altered immune surveillance within the CNS may also interfere with the efficiency of cure strategies within this compartment. SUMMARY: Together, these data suggest that the CNS viral reservoir is unique and presents a distinct set of challenges that need to be overcome to ensure successful viral elimination within this compartment. Future studies will need to develop CNS-active LRAs and biomarkers to enable monitoring and evaluation of treatment outcomes within the CNS during HIV-1 cure clinical trials.
Assuntos
Sistema Nervoso Central/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Latência Viral , Pesquisa Biomédica/tendências , Tratamento Farmacológico/métodos , HumanosRESUMO
Over the past decade antiretroviral drugs have dramatically improved the prognosis for HIV-1 infected individuals, yet achieving better access to vulnerable populations remains a challenge. The principal obstacle to the CCR5-antagonist, maraviroc, from being more widely used in anti-HIV-1 therapy regimens is that the pre-treatment genotypic "tropism tests" to determine virus susceptibility to maraviroc have been developed primarily for HIV-1 subtype B strains, which account for only 10% of infections worldwide. We therefore developed PhenoSeq, a suite of HIV-1 genotypic tropism assays that are highly sensitive and specific for establishing the tropism of HIV-1 subtypes A, B, C, D and circulating recombinant forms of subtypes AE and AG, which together account for 95% of HIV-1 infections worldwide. The PhenoSeq platform will inform the appropriate use of maraviroc and future CCR5 blocking drugs in regions of the world where non-B HIV-1 predominates, which are burdened the most by the HIV-1 pandemic.
Assuntos
HIV-1/fisiologia , Tropismo Viral/genética , Algoritmos , Sequência de Aminoácidos , Antagonistas dos Receptores CCR5/uso terapêutico , Biologia Computacional , Cicloexanos/uso terapêutico , Genótipo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/tratamento farmacológico , Humanos , Maraviroc , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fenótipo , Receptores CCR5/química , Receptores CCR5/metabolismo , Triazóis/uso terapêuticoRESUMO
PURPOSE OF REVIEW: To summarize the evidence in the literature that supports the central nervous system (CNS) as a viral reservoir for HIV-1 and to prioritize future research efforts. RECENT FINDINGS: HIV-1 DNA has been detected in brain tissue of patients with undetectable viral load or neurocognitive disorders, and is associated with long-lived cells such as astrocytes and microglia. In neurocognitively normal patients, HIV-1 can be found at high frequency in these cells (4% of astrocytes and 20% of macrophages). CNS cells have unique molecular mechanisms to suppress viral replication and induce latency, which include increased expression of dominant negative transcription factors and suppressive epigenetic factors. There is also evidence of continued inflammation in patients lacking a CNS viral load, suggesting the production and activity of viral neurotoxins (for example, Tat). SUMMARY: Together, these findings provide evidence that the CNS can potentially act as a viral reservoir of HIV-1. However, the majority of these studies were performed in historical cohorts (absence of combination antiretroviral therapy or presence of viral load), which do not reflect modern day patients (combination antiretroviral therapy-treated and undetectable viral load). Future studies will need to examine patient samples with these characteristics to conclusively determine whether the CNS represents a relevant and important viral reservoir.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Sistema Nervoso Central/citologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/virologia , Humanos , Especificidade de Órgãos , Carga Viral , Latência ViralRESUMO
Histone deacetylase inhibitors (HDACi) can induce human immunodeficiency virus (HIV) transcription from the HIV long terminal repeat (LTR). However, ex vivo and in vivo responses to HDACi are variable and the activity of HDACi in cells other than T-cells have not been well characterised. Here, we developed a novel assay to determine the activity of HDACi on patient-derived HIV LTRs in different cell types. HIV LTRs from integrated virus were amplified using triple-nested Alu-PCR from total memory CD4+ T-cells (CD45RO+) isolated from HIV-infected patients prior to and following suppressive antiretroviral therapy. NL4-3 or patient-derived HIV LTRs were cloned into the chromatin forming episomal vector pCEP4, and the effect of HDACi investigated in the astrocyte and epithelial cell lines SVG and HeLa, respectively. There were no significant differences in the sequence of the HIV LTRs isolated from CD4+ T-cells prior to and after 18 months of combination antiretroviral therapy (cART). We found that in both cell lines, the HDACi panobinostat, trichostatin A, vorinostat and entinostat activated patient-derived HIV LTRs to similar levels seen with NL4-3 and all patient derived isolates had similar sensitivity to maximum HDACi stimulation. We observed a marked difference in the maximum fold induction of luciferase by HDACi in HeLa and SVG, suggesting that the effect of HDACi may be influenced by the cellular environment. Finally, we observed significant synergy in activation of the LTR with vorinostat and the viral protein Tat. Together, our results suggest that the LTR sequence of integrated virus is not a major determinant of a functional response to an HDACi.
Assuntos
Infecções por HIV/sangue , Repetição Terminal Longa de HIV/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Linfócitos T/virologia , Adulto , Idoso , Fármacos Anti-HIV/uso terapêutico , Benzamidas/farmacologia , Linhagem Celular , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Estudos Observacionais como Assunto , Panobinostat , Filogenia , Piridinas/farmacologia , Linfócitos T/efeitos dos fármacos , Vorinostat , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologiaRESUMO
Astrocytes are extensively infected with HIV-1 in vivo and play a significant role in the development of HIV-1-associated neurocognitive disorders. Despite their extensive infection, little is known about how astrocytes become infected, since they lack cell surface CD4 expression. In the present study, we investigated the fate of HIV-1 upon infection of astrocytes. Astrocytes were found to bind and harbor virus followed by biphasic decay, with HIV-1 detectable out to 72 hours. HIV-1 was observed to associate with CD81-lined vesicle structures. shRNA silencing of CD81 resulted in less cell-associated virus but no loss of co-localization between HIV-1 and CD81. Astrocytes supported trans-infection of HIV-1 to T-cells without de novo virus production, and the virus-containing compartment required 37°C to form, and was trypsin-resistant. The CD81 compartment observed herein, has been shown in other cell types to be a relatively protective compartment. Within astrocytes, this compartment may be actively involved in virus entry and/or spread. The ability of astrocytes to transfer virus, without de novo viral synthesis suggests they are capable of sequestering and protecting virus and thus, they could potentially facilitate viral dissemination in the CNS.
Assuntos
Astrócitos/metabolismo , HIV-1/metabolismo , Tetraspanina 28/metabolismo , Internalização do Vírus , Astrócitos/virologia , Linhagem Celular , Técnicas de Cocultura , Células HEK293 , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Microscopia de Fluorescência , Ligação Proteica , Interferência de RNA , Linfócitos T/virologia , Temperatura , Tetraspanina 28/genética , Fatores de Tempo , Vesículas Transportadoras/metabolismo , Replicação ViralRESUMO
New evidence indicates that astrocytes of the central nervous system (CNS) are extensively infected with human immunodeficiency virus type 1 (HIV-1) in vivo. Although no new virus is produced, this nonproductive or restricted infection contributes to the pathogenesis of HIV-associated dementia (HAD) and compromises virus eradication strategies. The HIV-1 long terminal repeat (LTR) plays a critical role in regulating virus production from infected cells. Here, we determined whether LTRs derived from CNS and non-CNS compartments are genetically and functionally distinct and contribute to the restricted nature of astrocyte infection. CNS- and/or non-CNS-derived LTRs (n=82) were cloned from primary HIV-1 viruses isolated from autopsy tissues of seven patients who died with HAD. Phylogenetic analysis showed interpatient and intrapatient clustering of LTR nucleotide sequences. Functional analysis showed reduced basal transcriptional activity of CNS-derived LTRs in both astrocytes and T cells compared to that of non-CNS-derived LTRs. However, LTRs were heterogeneous in their responsiveness to activation by Tat. Therefore, using a relatively large, independent panel of primary HIV-1 LTRs derived from clinically well-characterized subjects, we show that LTRs segregate CNS- from non-CNS-derived tissues both genetically and functionally. The reduced basal transcriptional activity of LTRs derived from the CNS may contribute to the restricted HIV-1 infection of astrocytes and latent infection within the CNS. These findings have significance for understanding the molecular basis of HIV-1 persistence within cellular reservoirs of the CNS that need to be considered for strategies aimed at eradicating HIV-1.
Assuntos
Complexo AIDS Demência/virologia , Astrócitos/virologia , Sistema Nervoso Central/virologia , HIV-1/isolamento & purificação , Sequências Repetidas Terminais , Transcrição Gênica , Replicação Viral , Clonagem Molecular , Variação Genética , Genótipo , HIV-1/genética , Humanos , Filogenia , Análise de Sequência de DNA , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
BR-derived HIV-1 strains have an exceptional ability to enter macrophages via mechanisms involving their gp120 Env that remain incompletely understood. Here, we used cell-based affinity-profiling methods and mathematical modeling to generate quantitative VERSA metrics that simultaneously measure Env-CD4 and Env-CCR5 interactions. These metrics were analyzed to distinguish the phenotypes of M-tropic and non-M-tropic CCR5-using HIV-1 variants derived from autopsy BRs and LNs, respectively. We show that highly M-tropic Env variants derived from brain can be defined by two distinct and simultaneously occurring phenotypes. First, BR-derived Envs demonstrated an enhanced ability to interact with CD4 compared with LN-derived Envs, permitting entry into cells expressing scant levels of CD4. Second, BR-derived Envs displayed an altered mechanism of engagement between CD4-bound gp120 and CCR5 occurring in tandem. With the use of epitope mapping, mutagenesis, and structural studies, we show that this altered mechanism is characterized by increased exposure of CD4-induced epitopes in gp120 and by a more critical interaction between BR-derived Envs and the CCR5 N-terminus, which was associated with the predicted presence of additional atomic contacts formed at the gp120-CCR5 N-terminus interface. Our results suggest that BR-derived HIV-1 variants with highly efficient macrophage entry adopt conformations in gp120 that simultaneously alter the way in which the Env interacts with CD4 and CCR5.