RESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos , Metilación de ADN , Infecciones por VIH , VIH-1 , Compuestos Heterocíclicos de 4 o más Anillos , Humanos , VIH-1/efectos de los fármacos , VIH-1/fisiología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Infecciones por VIH/genética , Metilación de ADN/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Transcripción Genética/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Células TH1/inmunología , Células TH1/efectos de los fármacos , Células TH1/metabolismo , IsoquinolinasRESUMEN
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.
Asunto(s)
VIH-1 , Receptor de Manosa , Proteínas Proto-Oncogénicas , Transactivadores , Humanos , VIH-1/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
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.
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Retrovirus Endógenos , Infecciones por VIH , Seropositividad para VIH , VIH-1 , Humanos , VIH-1/genética , Latencia del Virus , Provirus/genética , Seropositividad para VIH/genética , Linfocitos T CD4-PositivosRESUMEN
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.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , VIH-1/genética , Latencia del Virus/genética , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Provirus , Linfocitos T CD4-PositivosRESUMEN
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.
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Infecciones por VIH , Seropositividad para VIH , VIH-1 , Ratones , Animales , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Replicación Viral , Duplicado del Terminal Largo de VIHRESUMEN
Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2. The virus binds to angiotensinogen converting enzyme 2 (ACE2), which mediates viral entry into mammalian cells. COVID-19 is notably severe in the elderly and in those with underlying chronic conditions. The cause of selective severity is not well understood. Here we show cholesterol and the signaling lipid phosphatidyl-inositol 4,5 bisphosphate (PIP2) regulate viral infectivity through the localization of ACE2's into nanoscopic (<200 nm) lipid clusters. Uptake of cholesterol into cell membranes (a condition common to chronic disease) causes ACE2 to move from PIP2 lipids to endocytic ganglioside (GM1) lipids, where the virus is optimally located for viral entry. In mice, age and high-fat diet increase lung tissue cholesterol by up to 40%. And in smokers with chronic disease, cholesterol is elevated 2-fold, a magnitude of change that dramatically increases infectivity of virus in cell culture. We conclude increasing the ACE2 location near endocytic lipids increases viral infectivity and may help explain the selective severity of COVID-19 in aged and diseased populations.
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COVID-19 , Hipercolesterolemia , Animales , Ratones , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2 , Peptidil-Dipeptidasa A/metabolismo , Colesterol/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Mamíferos/metabolismoRESUMEN
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.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , VIH-1/fisiología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Chaperonas Moleculares/metabolismo , Infecciones por VIH/genética , Transcripción Genética , Duplicado del Terminal Largo de VIH/genéticaRESUMEN
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.
Asunto(s)
Infecciones por VIH , VIH-1 , Antirretrovirales/uso terapéutico , Linfocitos T CD4-Positivos , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Humanos , Provirus/genética , Latencia del VirusRESUMEN
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.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Tratamiento Farmacológico de COVID-19 , Animales , Técnicas de Cultivo de Célula , Colesterol , Células HEK293 , Humanos , Hidroxicloroquina/farmacología , Lípidos , Mamíferos , Peptidil-Dipeptidasa A , SARS-CoV-2RESUMEN
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.
Asunto(s)
Fármacos Anti-VIH , Infecciones por VIH , Profilaxis Pre-Exposición , Femenino , Humanos , Fármacos Anti-VIH/farmacología , Administración Intravaginal , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/prevención & controlRESUMEN
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.
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Infecciones por VIH , VIH-1 , Linfocitos T CD4-Positivos , VIH-1/genética , Humanos , Provirus/genética , Latencia del VirusRESUMEN
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.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Inhibidores de Cisteína Proteinasa/farmacología , Dipéptidos/farmacología , Acoplamiento Viral/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Catepsina L/antagonistas & inhibidores , Línea Celular , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/crecimiento & desarrollo , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células VeroRESUMEN
Synaptic structural plasticity, key to long-term memory storage, requires translation of localized RNAs delivered by long-distance transport from the neuronal cell body. Mechanisms and regulation of this system remain elusive. Here, we explore the roles of KIF5C and KIF3A, two members of kinesin superfamily of molecular motors (Kifs), and find that loss of function of either kinesin decreases dendritic arborization and spine density whereas gain of function of KIF5C enhances it. KIF5C function is a rate-determining component of local translation and is associated with â¼650 RNAs, including EIF3G, a regulator of translation initiation, and plasticity-associated RNAs. Loss of function of KIF5C in dorsal hippocampal CA1 neurons constrains both spatial and contextual fear memory, whereas gain of function specifically enhances spatial memory and extinction of contextual fear. KIF5C-mediated long-distance transport of local translation substrates proves a key mechanism underlying structural plasticity and memory.
Asunto(s)
Cinesinas/metabolismo , Memoria a Largo Plazo , Proteínas Motoras Moleculares/metabolismo , Plasticidad Neuronal , Biosíntesis de Proteínas , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Espinas Dendríticas/metabolismo , Potenciales Postsinápticos Excitadores , Miedo , Femenino , Mutación con Ganancia de Función , Células HEK293 , Hipocampo/metabolismo , Humanos , Aprendizaje , Masculino , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/patología , Trastornos de la Memoria/fisiopatología , Ratones Endogámicos C57BL , Transporte de ARN , Transducción de Señal , Sinapsis/metabolismo , Transmisión SinápticaRESUMEN
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.
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Encéfalo/virología , VIH-1/patogenicidad , Organoides/virología , Animales , Infecciones por VIH/virología , Humanos , Técnicas In Vitro , Ratones , InvestigaciónRESUMEN
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.
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Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Isoquinolinas/farmacología , Animales , Regulación Viral de la Expresión Génica/efectos de los fármacos , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , Humanos , Regiones Promotoras Genéticas , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
In many species, ejaculation is followed by a state of decreased sexual activity, the post-ejaculatory refractory period. Several lines of evidence have suggested prolactin, a pituitary hormone released around the time of ejaculation in humans and other animals, to be a decisive player in the establishment of the refractory period. However, data supporting this hypothesis is controversial. We took advantage of two different strains of house mouse, a wild derived and a classical laboratory strain that differ substantially in their sexual performance, to investigate prolactin's involvement in sexual activity and the refractory period. First, we show that there is prolactin release during sexual behavior in male mice. Second, using a pharmacological approach, we show that acute manipulations of prolactin levels, either mimicking the natural release during sexual behavior or inhibiting its occurrence, do not affect sexual activity or shorten the refractory period, respectively. Therefore, we show compelling evidence refuting the idea that prolactin released during copulation is involved in the establishment of the refractory period, a long-standing hypothesis in the field of behavioral endocrinology.
Asunto(s)
Prolactina/fisiología , Conducta Sexual Animal/fisiología , Animales , Femenino , Masculino , Ratones Endogámicos C57BLRESUMEN
Coronavirus disease 2019 (COVID19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originating in Wuhan, China in 2019. The disease is notably severe in elderly and those with underlying chronic conditions. A molecular mechanism that explains why the elderly are vulnerable and why children are resistant is largely unknown. Here we show loading cells with cholesterol from blood serum using the cholesterol transport protein apolipoprotein E (apoE) enhances the entry of pseudotyped SARS-CoV-2 and the infectivity of the virion. Super resolution imaging of the SARS-CoV-2 entry point with high cholesterol shows almost twice the total number of endocytic entry points. Cholesterol concomitantly traffics angiotensinogen converting enzyme (ACE2) to the endocytic entry site where SARS-CoV-2 presumably docks to efficiently exploit entry into the cell. Furthermore, in cells producing virus, cholesterol optimally positions furin for priming SARS-CoV-2, producing a more infectious virion with improved binding to the ACE2 receptor. In vivo, age and high fat diet induces cholesterol loading by up to 40% and trafficking of ACE2 to endocytic entry sites in lung tissue from mice. We propose a component of COVID19 severity based on tissue cholesterol level and the sensitivity of ACE2 and furin to cholesterol. Molecules that reduce cholesterol or disrupt ACE2 localization with viral entry points or furin localization in the producer cells, may reduce the severity of COVID19 in obese patients.
RESUMEN
HIV transcription requires assembly of cellular transcription factors at the HIV-1promoter. The TFIIH general transcription factor facilitates transcription initiation by opening the DNA strands around the transcription start site and phosphorylating the C-terminal domain for RNA polymerase II (RNAPII) for activation. Spironolactone (SP), an FDA approved aldosterone antagonist, triggers the proteasomal degradation of the XPB subunit of TFIIH, and concurrently suppresses acute HIV infection in vitro Here we investigated SP as a possible block-and-lock agent for a functional cure aimed at the transcriptional silencing of the viral reservoir. The long-term activity of SP was investigated in primary and cell line models of HIV-1 latency and reactivation. We show that SP rapidly inhibits HIV-1 transcription by reducing RNAPII recruitment to the HIV-1 genome. shRNA knockdown of XPB confirmed XPB degradation as the mechanism of action. Unfortunately, long-term pre-treatment with SP does not result in epigenetic suppression of HIV upon SP treatment interruption, since virus rapidly rebounds when XPB reemerges; however, SP alone without ART maintains the transcriptional suppression. Importantly, SP inhibits HIV reactivation from latency in both cell line models and resting CD4+T cells isolated from aviremic infected individuals upon cell stimulation with latency reversing agents. Furthermore, long-term treatment with concentrations of SP that potently degrade XPB does not lead to global dysregulation of cellular mRNA expression. Overall, these results suggest that XPB plays a key role in HIV transcriptional regulation and XPB degradation by SP strengthens the potential of HIV transcriptional inhibitors in block-and-lock HIV cure approaches.IMPORTANCE Antiretroviral therapy (ART) effectively reduces an individual's HIV loads to below the detection limit, nevertheless rapid viral rebound immediately ensues upon treatment interruption. Furthermore, virally suppressed individuals experience chronic immune activation from ongoing low-level virus expression. Thus, the importance of identifying novel therapeutics to explore in block-and-lock HIV functional cure approaches, aimed at the transcriptional and epigenetic silencing of the viral reservoir to block reactivation from latency. We investigated the potential of repurposing the FDA-approved spironolactone (SP), as one such drug. SP treatment rapidly degrades a host transcription factor subunit, XPB, inhibiting HIV transcription and blocking reactivation from latency. Long-term SP treatment does not affect cellular viability, cell cycle progression or global cellular transcription. SP alone blocks HIV transcription in the absence of ART but does not delay rebound upon drug removal as XPB rapidly reemerges. This study highlights XPB as a novel drug target in block-and-lock therapeutic approaches.
RESUMEN
HIV-1/AIDS remains a global public health problem. The world health organization (WHO) reported at the end of 2019 that 38 million people were living with HIV-1 worldwide, of which only 67% were accessing antiretroviral therapy (ART). Despite great success in the clinical management of HIV-1 infection, ART does not eliminate the virus from the host genome. Instead, HIV-1 remains latent as a viral reservoir in any tissue containing resting memory CD4+ T cells. The elimination of these residual proviruses that can reseed full-blown infection upon treatment interruption remains the major barrier towards curing HIV-1. Novel approaches have recently been developed to excise or disrupt the virus from the host cells (e.g., gene editing with the CRISPR-Cas system) to permanently shut off transcription of the virus (block-and-lock and RNA interference strategies), or to reactivate the virus from cell reservoirs so that it can be eliminated by the immune system or cytopathic effects (shock-and-kill strategy). Here, we will review each of these approaches, with the major focus placed on the block-and-lock strategy.