RESUMO
HIV gene expression is modulated by the combinatorial activity of the HIV transcriptional activator, Tat, host transcription factors, and chromatin remodeling complexes. To identify host factors regulating HIV transcription, we used specific single-guide RNAs and endonuclease-deficient Cas9 to perform chromatin affinity purification of the integrated HIV promoter followed by mass spectrometry. The scaffold protein, p32, also called ASF/SF2 splicing factor-associated protein, was identified among the top enriched factors present in actively transcribing HIV promoters but absent in silenced ones. Chromatin immunoprecipitation analysis confirmed the presence of p32 on active HIV promoters and its enhanced recruitment by Tat. HIV uses Tat to efficiently recruit positive transcription elongation factor b (p-TEFb) (CDK9/CCNT1) to TAR, an RNA secondary structure that forms from the first 59 bp of HIV transcripts, to enhance RNAPII transcriptional elongation. The RNA interference of p32 significantly reduced HIV transcription in primary CD4+T cells and in HIV chronically infected cells, independently of either HIV splicing or p32 anti-splicing activity. Conversely, overexpression of p32 specifically increased Tat-dependent HIV transcription. p32 was found to directly interact with Tat's basic domain enhancing Tat stability and half-life. Conversely, p32 associates with Tat via N- and C-terminal domains. Likely due its scaffold properties, p32 also promoted Tat association with TAR, p-TEFb, and RNAPII enhancing Tat-dependent HIV transcription. In sum, we identified p32 as a host factor that interacts with and stabilizes Tat protein, promotes Tat-dependent transcriptional regulation, and may be explored for HIV-targeted transcriptional inhibition.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , HIV-1/fisiologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Chaperonas Moleculares/metabolismo , Infecções por HIV/genética , Transcrição Gênica , Repetição Terminal Longa de HIV/genéticaRESUMO
HIV-1 infection of human macrophages leads to the downmodulation of human mannose receptor 1 (hMRC1), a cell-surface glycoprotein that is involved in the host innate immune response. We previously reported that downmodulation of hMRC1 involves the transactivator of transcription (Tat)-dependent transcriptional silencing of the hMRC1 promoter. However, the inhibitory effect of Tat on hMRC1 transcription was indirect and involved inhibition of the transcriptional activator PU.1, which normally upregulates hMRC1 expression in macrophages and other myeloid cells. We cloned a 284-bp fragment of the hMRC1 promoter, and within it, we identified four PU.1 box elements. We assessed the relative contribution of each of the four PU.1 boxes to PU.1-dependent transcriptional regulation and, surprisingly, found that only one of the four PU.1 boxes [PU.1(b)] was critically required for PU.1-mediated upregulation of luciferase expression. Transfer of this PU.1 box to a heterologous promoter conferred PU.1 responsiveness to an otherwise PU.1 insensitive promoter. Electrophoretic mobility shift assays identified this PU.1 box as a direct binding site for PU.1 both in the context of the hMRC1 promoter and the heterologous promoter. Furthermore, mutational analysis of the PU.1 protein identified the C-terminal DNA-binding domain in PU.1 as the region responsible for interaction with the PU.1 box. Recombinant HIV-1 Tat protein did not bind to the hMRC1 promoter element but efficiently interfered with the binding of PU.1 protein to the hMRC1 promoter. Thus, Tat is likely to inhibit the formation of active PU.1 transcription complexes, presumably by binding to and depleting common transcriptional cofactors.IMPORTANCEHIV-1 infection of cells results in the modulation of cellular gene expression by virus-encoded proteins in a manner that benefits the virus. We reported that HIV-1 transactivator of transcription (Tat) dysregulates the expression of the human mannose receptor 1 (hMRC1). hMRC1 is involved in the innate immune response of macrophages to foreign pathogens. Tat does not act directly on the hMRC1 promoter but instead inhibits PU.1, a cellular transcription factor regulating hMRC1 gene expression. Here, we characterize the PU.1-dependent regulation of hMRC1 expression. We identified four potential PU.1 binding sites in the hMRC1 promoter region but found that only one, PU.1(b), functioned as a true binding site for PU.1. Transfer of the PU.1(b) box to a heterologous promoter did not activate this promoter per se but rendered it responsive to PU.1. Our results support the view that PU.1 acts as a transcriptional co-factor whose activity can be regulated by HIV-1 Tat.
Assuntos
HIV-1 , Receptor de Manose , Proteínas Proto-Oncogênicas , Transativadores , Humanos , HIV-1/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
Antiretroviral therapy effectively controls human immunodeficiency virus (HIV) infection. However, a reservoir of latently infected cells persists under suppressive therapy, constituting a major barrier to an HIV cure. The block-and-lock approach to a functional cure aims at the transcriptional and epigenetic silencing of proviruses, blocking viral reactivation in the absence of therapy, preventing disease progression and transmission, despite the presence of detectable integrated proviruses. This approach has been put forward for exploration based on the activity of didehydro-cortistatin A, an inhibitor of the HIV transcriptional activator Tat. Here we review the mechanisms by which didehydro-cortistatin A inhibition of Tat's feedback loop transcriptional amplification results in epigenetic silencing of the HIV promoter, and we discuss the benefits and limitations of the block-and-lock approach for an HIV cure.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Isoquinolinas/farmacologia , Animais , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/virologia , HIV-1/genética , HIV-1/metabolismo , Humanos , Regiões Promotoras Genéticas , Produtos do Gene tat do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The HIV-1 transactivation protein (Tat) binds the HIV mRNA transactivation responsive element (TAR), regulating transcription and reactivation from latency. Drugs against Tat are unfortunately not clinically available. We reported that didehydro-cortistatin A (dCA) inhibits HIV-1 Tat activity. In human CD4+ T cells isolated from aviremic individuals and in the humanized mouse model of latency, combining dCA with antiretroviral therapy accelerates HIV-1 suppression and delays viral rebound upon treatment interruption. This drug class is amenable to block-and-lock functional cure approaches, aimed at a durable state of latency. Simian immunodeficiency virus (SIV) infection of rhesus macaques (RhMs) is the best-characterized model for AIDS research. Here, we demonstrate, using in vitro and cell-based assays, that dCA directly binds to SIV Tat's basic domain. dCA specifically inhibits SIV Tat binding to TAR, but not a Tat-Rev fusion protein, which activates transcription when Rev binds to its cognate RNA binding site replacing the apical region of TAR. Tat-TAR inhibition results in loss of RNA polymerase II recruitment to the SIV promoter. Importantly, dCA potently inhibits SIV reactivation from latently infected Hut78 cells and from primary CD4+ T cells explanted from SIVmac239-infected RhMs. In sum, dCA's remarkable breadth of activity encourages SIV-infected RhM use for dCA preclinical evaluation.-Mediouni, S., Kessing, C. F., Jablonski, J. A., Thenin-Houssier, S., Clementz, M., Kovach, M. D., Mousseau, G., de Vera, I.M.S., Li, C., Kojetin, D. J., Evans, D. T., Valente, S. T. The Tat inhibitor didehydro-cortistatin A suppresses SIV replication and reactivation.
Assuntos
Linfócitos T CD4-Positivos/virologia , Produtos do Gene tat/antagonistas & inibidores , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Vírus da Imunodeficiência Símia/fisiologia , Ativação Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Produtos do Gene tat/metabolismo , Células HEK293 , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis , Humanos , Isoquinolinas , Macaca mulatta , Regiões Promotoras Genéticas , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Sequências Repetidas TerminaisRESUMO
A genetic screen previously identified the N-terminal 91 amino acids of the eukaryotic initiation factor 3 subunit f (N91-eIF3f) as a potent inhibitor of HIV-1 replication. Overexpression of N91-eIF3f or full-length eIF3f reduced the level of HIV-1 mRNAs in the infected cell. Here we show that N91-eIF3f and eIF3f act by specifically blocking the 3' end processing of the HIV-1 pre-mRNA both in vivo and in vitro. Furthermore, the results suggest that eIF3f mediates this restriction of HIV-1 expression through the previously unsuspected involvement of a set of factors that includes eIF3f, the SR protein 9G8, and the cyclin-dependent kinase 11 (CDK11). eIF3f affects HIV-1 3' end processing by modulating the sequence-specific recognition of the HIV-1 pre-mRNA by 9G8.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , HIV-1/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Processamento de Terminações 3' de RNA/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Sítios de Ligação , Extratos Celulares , Núcleo Celular/metabolismo , Fator de Iniciação 3 em Eucariotos/química , Repetição Terminal Longa de HIV/genética , HIV-1/fisiologia , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Nucleares , Poli A/metabolismo , Ligação Proteica , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-Arginina , Replicação ViralRESUMO
The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development.
Assuntos
Fármacos Anti-HIV/farmacologia , Azóis/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Capsídeo/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Compostos Organosselênicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Fármacos Anti-HIV/química , Azóis/química , Sítios de Ligação , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Bases de Dados de Produtos Farmacêuticos , Transferência Ressonante de Energia de Fluorescência , HIV-1/fisiologia , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Isoindóis , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Vírus da Leucemia Murina de Moloney/efeitos dos fármacos , Vírus da Leucemia Murina de Moloney/fisiologia , Compostos Organosselênicos/química , Ligação Proteica , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/fisiologia , Bibliotecas de Moléculas Pequenas/química , Montagem de Vírus/efeitos dos fármacos , Montagem de Vírus/fisiologia , Replicação Viral/efeitos dos fármacosRESUMO
Antiretroviral therapy (ART) potently suppresses HIV-1 replication, but the virus persists in quiescent infected CD4(+)T cells as a latent integrated provirus, and patients must indefinitely remain on therapy. If ART is terminated, these integrated proviruses can reactivate, driving new rounds of infection. A functional cure for HIV requires eliminating low-level ongoing viral replication that persists in certain tissue sanctuaries and preventing viral reactivation. The HIV Tat protein plays an essential role in HIV transcription by recruiting the kinase activity of the P-TEFb complex to the viral mRNA's stem-bulge-loop structure, TAR, activating transcriptional elongation. Because the Tat-mediated transactivation cascade is critical for robust HIV replication, the Tat/TAR/P-TEFb complex is one of the most attractive targets for drug development. Importantly, compounds that interfere with transcription could impair viral reactivation, low-level ongoing replication, and replenishment of the latent reservoir, thereby reducing the size of the latent reservoir pool. Here, we discuss the potential importance of transcriptional inhibitors in the treatment of latent HIV-1 disease and review recent findings on targeting Tat, TAR, and P-TEFb individually or as part of a complex. Finally, we discuss the impact of extracellular Tat in HIV-associated neurocognitive disorders and cancers.
Assuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Fármacos Anti-HIV/farmacologia , Repetição Terminal Longa de HIV/efeitos dos fármacos , HIV-1 , Fator B de Elongação Transcricional Positiva/antagonistas & inibidores , Produtos do Gene tat do Vírus da Imunodeficiência Humana/antagonistas & inibidores , HumanosRESUMO
We present here an efficient alternative to N-methylation for the purpose of morphing protein-binding peptides into more serum-stable and cell-permeable compounds. This involves the incorporation of a cycloalanine (CyAla) into a peptide in a way that avoids difficult coupling steps. We demonstrate the utility of this chemistry in creating a cell-permeable derivative of a high-affinity HIV Rev protein-binding peptide.
Assuntos
Alanina/química , Peptídeos Penetradores de Células/química , Células HeLa , Humanos , Conformação MolecularRESUMO
Ongoing viral transcription from the reservoir of HIV-1 infected long-lived memory CD4+ T cells presents a barrier to cure and associates with poorer health outcomes for people living with HIV, including chronic immune activation and inflammation. We previously reported that didehydro-cortistatin A (dCA), an HIV-1 Tat inhibitor, blocks HIV-1 transcription. Here, we examine the impact of dCA on host immune CD4+ T-cell transcriptional and epigenetic states. We performed a comprehensive analysis of genome-wide transcriptomic and DNA methylation profiles upon long-term dCA treatment of primary human memory CD4+ T cells. dCA prompted specific transcriptional and DNA methylation changes in cell cycle, histone, interferon-response, and T-cell lineage transcription factor genes, through inhibition of both HIV-1 and Mediator kinases. These alterations establish a tolerogenic Treg/Th2 phenotype, reducing viral gene expression and mitigating inflammation in primary CD4+ T cells during HIV-1 infection. In addition, dCA suppresses the expression of lineage-defining transcription factors for Th17 and Th1 cells, critical HIV-1 targets, and reservoirs. dCA's benefits thus extend beyond viral transcription inhibition, modulating the immune cell landscape to limit HIV-1 acquisition and inflammatory environment linked to HIV infection.
Assuntos
Linfócitos T CD4-Positivos , Metilação de DNA , Infecções por HIV , HIV-1 , Compostos Heterocíclicos de 4 ou mais Anéis , Humanos , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , Infecções por HIV/genética , Metilação de DNA/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Transcrição Gênica/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células Th1/imunologia , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , IsoquinolinasRESUMO
PURPOSE OF REVIEW: This review highlights advances in HIV transcription and epigenetic latency mechanisms and outlines current therapeutic approaches to eliminate or block the HIV-1 latent reservoir. RECENT FINDINGS: Novel host factors have been reported to modulate HIV-1 transcription and latency. Chromatin affinity purification strategies followed by mass spectrometry (ChAP-MS) identified the chaperone protein p32 to play an important role in HIV-1 transcriptional regulation via interactions with the viral transcriptional activator Tat. Similarly, an shRNA screen identified the methyltransferase SMYD5 contributing to HIV-1 transcriptional activation also by modulating Tat activity. These new factors, among others, represent potential druggable targets that could be explored in the 'block-and-lock' or 'shock-and-kill' approaches. SUMMARY: The HIV-1 latent reservoir is established early after infection, persists during antiretroviral therapy, and is the source of viral rebound after treatment interruption. An HIV cure requires either eliminating this reservoir or blocking latent proviral reactivation in the absence of antiretroviral therapy (ART). Understanding the mechanisms and key-players modulating HIV transcriptional and reactivation may facilitate therapeutic advancements. Here we summarize, the latest findings on host factors' roles in HIV transcriptional regulation.
Assuntos
Infecções por HIV , HIV-1 , Humanos , HIV-1/genética , Latência Viral/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Provírus , Linfócitos T CD4-PositivosRESUMO
HIV resistance to the Tat inhibitor didehydro-cortistatin A (dCA) in vitro correlates with higher levels of Tat-independent viral transcription and a seeming inability to enter latency, which rendered resistant isolates more susceptible to CTL-mediated immune clearance. Here, we investigated the ability of dCA-resistant viruses to replicate in vivo using a humanized mouse model of HIV infection. Animals were infected with WT or two dCA-resistant HIV-1 isolates in the absence of dCA and followed for 5 weeks. dCA-resistant viruses exhibited lower replication rates compared to WT. Viral replication was suppressed early after infection, with viral emergence at later time points. Multiplex analysis of cytokine and chemokines from plasma samples early after infection revealed no differences in expression levels between groups, suggesting that dCA-resistance viruses did not elicit potent innate immune responses capable of blocking the establishment of infection. Viral single genome sequencing results from plasma samples collected at euthanasia revealed that at least half of the total number of mutations in the LTR region of the HIV genome considered essential for dCA evasion reverted to WT. These results suggest that dCA-resistant viruses identified in vitro suffer a fitness cost in vivo, with mutations in LTR and Nef pressured to revert to wild type.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Camundongos , Animais , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Replicação Viral , Repetição Terminal Longa de HIVRESUMO
Despite remarkable progress, a cure for HIV-1 infection remains elusive. Rebound competent latent and transcriptionally active reservoir cells persevere despite antiretroviral therapy and rekindle infection due to inefficient proviral silencing. We propose a novel "block-lock-stop" approach, entailing long term durable silencing of viral expression towards an irreversible transcriptionally inactive latent provirus to achieve long term antiretroviral free control of the virus. A graded transformation of remnant HIV-1 in PLWH from persistent into silent to permanently defective proviruses is proposed, emulating and accelerating the natural path that human endogenous retroviruses (HERVs) take over millions of years. This hypothesis was based on research into delineating the mechanisms of HIV-1 latency, lessons from latency reversing agents and advances of Tat inhibitors, as well as expertise in the biology of HERVs. Insights from elite controllers and the availability of advanced genome engineering technologies for the direct excision of remnant virus set the stage for a rapid path to an HIV-1 cure.
Assuntos
Retrovirus Endógenos , Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , HIV-1/genética , Latência Viral , Provírus/genética , Soropositividade para HIV/genética , Linfócitos T CD4-PositivosRESUMO
Viruses often use host machinery in unusual ways to execute different steps during their replication. To identify host factors critical for virus replication, we screened cDNA expression libraries for genes or gene fragments that could interfere with HIV-1 vector transduction. The DNA clone that most potently inhibited HIV-1 expression encoded the N-terminal 91 aa of the eukaryotic initiation factor 3 subunit f (N91-eIF3f). Overexpression of N91-eIF3f or full-length eIF3f drastically restricted HIV-1 replication by reducing nuclear and cytoplasmic viral mRNA levels. N91-eIF3f and eIF3f specifically targeted the 3' long terminal repeat (3'LTR) region in the viral mRNA. We show that the 3' end cleavage of HIV-1 mRNA precursors is specifically reduced in N91-eIF3f expressing cells. Our results suggest a role of eIF3f in mRNA maturation and that it can specifically interfere with the 3' end processing of HIV-1 mRNAs.
Assuntos
Fator de Iniciação 3 em Eucariotos/fisiologia , HIV-1/imunologia , Replicação Viral/imunologia , Fator de Iniciação 3 em Eucariotos/imunologia , Biblioteca Gênica , Humanos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , RNA Viral/análise , RNA Viral/efeitos dos fármacos , Sequências Repetidas Terminais/efeitos dos fármacos , Transdução GenéticaRESUMO
The majority of virally suppressed individuals will experience rapid viral rebound upon antiretroviral therapy (ART) interruption, providing a strong rationale for the development of cure strategies. Moreover, despite ART virological control, HIV infection is still associated with chronic immune activation, inflammation, comorbidities, and accelerated aging. These effects are believed to be due, in part, to low-grade persistent transcription and trickling production of viral proteins from the pool of latent proviruses constituting the viral reservoir. In recent years there has been an increasing interest in developing what has been termed a functional cure for HIV. This approach entails the long-term, durable control of viral expression in the absence of therapy, preventing disease progression and transmission, despite the presence of detectable integrated proviruses. One such strategy, the block-and-lock approach for a functional cure, proposes the epigenetic silencing of proviral expression, locking the virus in a profound latent state, from which reactivation is very unlikely. The proof-of-concept for this approach was demonstrated with the use of a specific small molecule targeting HIV transcription. Here we review the principles behind the block-and-lock approach and some of the additional strategies proposed to silence HIV expression.
Assuntos
Infecções por HIV , HIV-1 , Linfócitos T CD4-Positivos , HIV-1/genética , Humanos , Provírus/genética , Latência ViralRESUMO
Current antiretroviral therapy (ART) increases the survival of HIV-infected individuals, yet it is not curative. The major barrier to finding a definitive cure for HIV is our inability to identify and eliminate long-lived cells containing the dormant provirus, termed viral reservoir. When ART is interrupted, the viral reservoir ensures heterogenous and stochastic HIV viral gene expression, which can reseed infection back to pre-ART levels. While strategies to permanently eradicate the virus have not yet provided significant success, recent work has focused on the management of this residual viral reservoir to effectively limit comorbidities associated with the ongoing viral transcription still observed during suppressive ART, as well as limit the need for daily ART. Our group has been at the forefront of exploring the viability of the block-and-lock remission approach, focused on the long-lasting epigenetic block of viral transcription such that without daily ART, there is no risk of viral rebound, transmission, or progression to AIDS. Numerous studies have reported inhibitors of both viral and host factors required for HIV transcriptional activation. Here, we highlight and review some of the latest HIV transcriptional inhibitor discoveries that may be leveraged for the clinical exploration of block-and-lock and revolutionize the way we treat HIV infections.
Assuntos
Infecções por HIV , HIV-1 , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Humanos , Provírus/genética , Latência ViralRESUMO
Locally delivered pre-exposure prophylaxis (PrEP) has proven to be a promising strategy to combat Human immunodeficiency virus (HIV) transmission but several findings encountered toxicities or proved to be marginally effective in clinical settings. Therefore, innovative, multifunctional, and safer alternatives are being progressively investigated. Herein, we explored negatively charged carbohydrate, anionic pullulan (AP) as a rapidly soluble film-former and novel anti-HIV agent. Additionally, Bictegravir (BCT), an HIV integrase inhibitor was co-delivered in the form of nanomicelles for sustained antiviral activity. BCT-loaded PLGA-PEG polymeric nanomicelles (BN) were incorporated into PVA/pullulan-based film matrix comprising of 2 % w/v AP (BN-AP film). In cell-based assays, biocompatibility and TEER values for BN-AP films were similar to control while the commercial vaginal contraceptive film (VCF®) showed severe cytotoxicity and drastically reduced the tight junction integrity. Rapid disintegration of BN-AP film with >85 % drug release was observed in simulated vaginal and seminal fluid. Most importantly, AP and BN-AP film significantly inhibited HIV-1 replication with IC50 at as low as 91 µg/mL and 0.708 nM, respectively. Therefore, this study entails successful development of BN-AP film that functioned as an effective, biocompatible dual-acting PrEP formulation.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , Profilaxia Pré-Exposição , Feminino , Humanos , Fármacos Anti-HIV/farmacologia , Administração Intravaginal , Infecções por HIV/tratamento farmacológico , Infecções por HIV/prevenção & controleRESUMO
Hydroxychloroquine (HCQ), a drug used to treat lupus and malaria, was proposed as a treatment for SARS-coronavirus-2 (SARS-CoV-2) infection, albeit with controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ's mechanism of actions in vitro is needed. Recently, anesthetics were shown to disrupt ordered clusters of monosialotetrahexosylganglioside1 (GM1) lipid. These same lipid clusters recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to endocytic lipids, away from phosphatidylinositol 4,5 bisphosphate (PIP2) clusters. Here we employed super-resolution imaging of cultured mammalian cells (VeroE6, A549, H1793, and HEK293T) to show HCQ directly perturbs clustering of ACE2 receptor with both endocytic lipids and PIP2 clusters. In elevated (high) cholesterol, HCQ moves ACE2 nanoscopic distances away from endocytic lipids. In cells with resting (low) cholesterol, ACE2 primarily associates with PIP2 clusters, and HCQ moves ACE2 away from PIP2 clusters-erythromycin has a similar effect. We conclude HCQ inhibits viral entry through two distinct mechanisms in high and low tissue cholesterol and does so prior to inhibiting cathepsin-L. HCQ clinical trials and animal studies will need to account for tissue cholesterol levels when evaluating dosing and efficacy.
Assuntos
Enzima de Conversão de Angiotensina 2 , Tratamento Farmacológico da COVID-19 , Animais , Técnicas de Cultura de Células , Colesterol , Células HEK293 , Humanos , Hidroxicloroquina/farmacologia , Lipídeos , Mamíferos , Peptidil Dipeptidase A , SARS-CoV-2RESUMO
The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Catepsina L/antagonistas & inibidores , Linhagem Celular , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células VeroRESUMO
Long-term effective use of antiretroviral therapy (ART) among people with HIV (PWH) has significantly reduced the burden of disease, yet a cure for HIV has not been universally achieved, likely due to the persistence of an HIV reservoir. The central nervous system (CNS) is an understudied HIV sanctuary. Importantly, due to viral persistence in the brain, cognitive disturbances persist to various degrees at high rates in PWH despite suppressive ART. Given the complexity and accessibility of the CNS compartment and that it is a physiologically and anatomically unique immune site, human studies to reveal molecular mechanisms of viral entry, reservoir establishment, and the cellular and structural interactions leading to viral persistence and brain injury to advance a cure and either prevent or limit cognitive impairments in PWH remain challenging. Recent advances in human brain organoids show that they can mimic the intercellular dynamics of the human brain and may recapitulate many of the events involved in HIV infection of the brain (neuroHIV). Human brain organoids can be produced, spontaneously or with addition of growth factors and at immature or mature states, and have become stronger models to study neurovirulent viral infections of the CNS. While organoids provide opportunities to study neuroHIV, obstacles such as the need to incorporate microglia need to be overcome to fully utilize this model. Here, we review the current achievements in brain organoid biology and their relevance to neuroHIV research efforts.
Assuntos
Encéfalo/virologia , HIV-1/patogenicidade , Organoides/virologia , Animais , Infecções por HIV/virologia , Humanos , Técnicas In Vitro , Camundongos , PesquisaRESUMO
HIV-1 establishes a life-long infection when proviral DNA integrates into the host genome. The provirus can then either actively transcribe RNA or enter a latent state, without viral production. The switch between these two states is governed in great part by the viral protein, Tat, which promotes RNA transcript elongation. Latency is also influenced by the availability of host transcription factors, integration site, and the surrounding chromatin environment. The latent reservoir is established in the first few days of infection and serves as the source of viral rebound upon treatment interruption. Despite effective suppression of HIV-1 replication by antiretroviral therapy (ART), to below the detection limit, ART is ineffective at reducing the latent reservoir size. Elimination of this reservoir has become a major goal of the HIV-1 cure field. However, aside from the ideal total HIV-1 eradication from the host genome, an HIV-1 remission or functional cure is probably more realistic. The "block-and-lock" approach aims at the transcriptional silencing of the viral reservoir, to render suppressed HIV-1 promoters extremely difficult to reactivate from latency. There are unfortunately no clinically available HIV-1 specific transcriptional inhibitors. Understanding the mechanisms that regulate latency is expected to provide novel targets to be explored in cure approaches.