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1.
PLoS Pathog ; 15(12): e1008156, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790497

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human cancers, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). Current treatment options for KSHV infection and virus associated diseases are sometimes ineffective, therefore, more effectively antiviral agents are urgently needed. As a herpesvirus, lytic replication is critical for KSHV pathogenesis and oncogenesis. In this study, we have established a high-throughput screening assay by using an inducible KSHV+ cell-line, iSLK.219. After screening a compound library that consisted of 1280 Food and Drug Administration (FDA)-approved drugs, 15 hit compounds that effectively inhibited KSHV virion production were identified, most of which have never been reported with anti-KSHV activities. Interestingly, 3 of these drugs target histamine receptors or signaling. Our data further confirmed that antagonists targeting different histamine receptors (HxRs) displayed excellent inhibitory effects on KSHV lytic replication from induced iSLK.219 or BCBL-1 cells. In contrast, histamine and specific agonists of HxRs promoted viral lytic replication from induced iSLK.219 or KSHV-infected primary cells. Mechanistic studies indicated that downstream MAPK and PI3K/Akt signaling pathways were required for histamine/receptors mediated promotion of KSHV lytic replication. Direct knockdown of HxRs in iSLK.219 cells effectively blocked viral lytic gene expression during induction. Using samples from a cohort of HIV+ patients, we found that the KSHV+ group has much higher levels of histamine in their plasma and saliva than the KSHV- group. Taken together, our data have identified new anti-KSHV agents and provided novel insights into the molecular bases of host factors that contribute to lytic replication and reactivation of this oncogenic herpesvirus.


Assuntos
Antivirais/farmacologia , Herpesvirus Humano 8/efeitos dos fármacos , Histamina/metabolismo , Sarcoma de Kaposi/virologia , Ativação Viral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Herpesvirus Humano 8/fisiologia , Ensaios de Triagem em Larga Escala , Humanos , Receptores Histamínicos/metabolismo , Transdução de Sinais/fisiologia , Ativação Viral/fisiologia , Latência Viral/efeitos dos fármacos , Latência Viral/fisiologia
2.
PLoS Pathog ; 15(12): e1008174, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31830143

RESUMO

Primary effusion lymphoma (PEL) is an aggressive B-cell malignancy without effective treatment, and caused by the infection of Kaposi's sarcoma-associated herpesvirus (KSHV), predominantly in its latent form. Previously we showed that the SUMO2-interacting motif within the viral latency-associated nuclear antigen (LANASIM) is essential for establishment and maintenance of KSHV latency. Here, we developed a luciferase based live-cell reporter system to screen inhibitors selectively targeting the interaction between LANASIM and SUMO2. Cambogin, a bioactive natural product isolated from the Garcinia genus (a traditional herbal medicine used for cancer treatment), was obtained from the reporter system screening to efficiently inhibit the association of SUMO2 with LANASIM, in turn reducing the viral episome DNA copy number for establishment and maintenance of KSHV latent infection at a low concentration (nM). Importantly, Cambogin treatments not only specifically inhibited proliferation of KSHV-latently infected cells in vitro, but also induced regression of PEL tumors in a xenograft mouse model. This study has identified Cambogin as a novel therapeutic agent for treating PEL as well as eliminating persistent infection of oncogenic herpesvirus.


Assuntos
Antineoplásicos/farmacologia , Linfoma de Efusão Primária/virologia , Terpenos/farmacologia , Latência Viral/efeitos dos fármacos , Animais , Antígenos Virais/efeitos dos fármacos , Antígenos Virais/metabolismo , Células HEK293 , Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 8 , Humanos , Camundongos , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Extratos Vegetais/farmacologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
PLoS Pathog ; 15(10): e1008074, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31609991

RESUMO

Studies have demonstrated that intensive ART alone is not capable of eradicating HIV-1, as the virus rebounds within a few weeks upon treatment interruption. Viral rebound may be induced from several cellular subsets; however, the majority of proviral DNA has been found in antigen experienced resting CD4+ T cells. To achieve a cure for HIV-1, eradication strategies depend upon both understanding mechanisms that drive HIV-1 persistence as well as sensitive assays to measure the frequency of infected cells after therapeutic interventions. Assays such as the quantitative viral outgrowth assay (QVOA) measure HIV-1 persistence during ART by ex vivo activation of resting CD4+ T cells to induce latency reversal; however, recent studies have shown that only a fraction of replication-competent viruses are inducible by primary mitogen stimulation. Previous studies have shown a correlation between the acquisition of effector memory phenotype and HIV-1 latency reversal in quiescent CD4+ T cell subsets that harbor the reservoir. Here, we apply our mechanistic understanding that differentiation into effector memory CD4+ T cells more effectively promotes HIV-1 latency reversal to significantly improve proviral measurements in the QVOA, termed differentiation QVOA (dQVOA), which reveals a significantly higher frequency of the inducible HIV-1 replication-competent reservoir in resting CD4+ T cells.


Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/imunologia , HIV-1/fisiologia , Memória Imunológica/imunologia , Latência Viral/imunologia , Idoso , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Infecções por HIV/imunologia , HIV-1/crescimento & desenvolvimento , Humanos , Masculino , Pessoa de Meia-Idade , Provírus/crescimento & desenvolvimento , Carga Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
4.
PLoS Pathog ; 15(8): e1007991, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31425551

RESUMO

Latency reversal agents (LRAs) have proven to induce HIV-1 transcription in vivo but are ineffective at decreasing the size of the latent reservoir in antiretroviral treated patients. The capacity of the LRAs to perturb the viral reservoir present in distinct subpopulations of cells is currently unknown. Here, using a new RNA FISH/flow ex vivo viral reactivation assay, we performed a comprehensive assessment of the viral reactivation capacity of different families of LRAs, and their combinations, in different CD4+ T cell subsets. We observed that a median of 16.28% of the whole HIV-reservoir induced HIV-1 transcripts after viral reactivation, but only 10.10% of these HIV-1 RNA+ cells produced the viral protein p24. Moreover, none of the LRAs were powerful enough to reactivate HIV-1 transcription in all CD4+ T cell subpopulations. For instance, the combination of Romidepsin and Ingenol was identified as the best combination of drugs at increasing the proportion of HIV-1 RNA+ cells, in most, but not all, CD4+ T cell subsets. Importantly, memory stem cells were identified as highly resistant to HIV-1 reactivation, and only the combination of Panobinostat and Bryostatin-1 significantly increased the number of cells transcribing HIV within this subset. Overall, our results validate the use of the RNA FISH/flow technique to assess the potency of LRAs among different CD4+ T cell subsets, manifest the intrinsic differences between cells that encompass the latent HIV reservoir, and highlight the difficulty to significantly impact the latent infection with the currently available drugs. Thus, our results have important implications for the rational design of therapies aimed at reversing HIV latency from diverse cellular reservoirs.


Assuntos
Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Ativação Viral/imunologia , Latência Viral/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Depsipeptídeos/farmacologia , Diterpenos/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Carga Viral , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos
5.
Nat Commun ; 10(1): 3193, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324762

RESUMO

The HIV-1 reservoir is the major hurdle to a cure. We here evaluate viral and host characteristics associated with reservoir size and long-term dynamics in 1,057 individuals on suppressive antiretroviral therapy for a median of 5.4 years. At the population level, the reservoir decreases with diminishing differences over time, but increases in 26.6% of individuals. Viral blips and low-level viremia are significantly associated with slower reservoir decay. Initiation of ART within the first year of infection, pretreatment viral load, and ethnicity affect reservoir size, but less so long-term dynamics. Viral blips and low-level viremia are thus relevant for reservoir and cure studies.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Reservatórios de Doenças , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Adulto , Feminino , Infecções por HIV/sangue , HIV-1/genética , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , RNA Viral/sangue , Carga Viral , Viremia , Latência Viral/efeitos dos fármacos
6.
PLoS Comput Biol ; 15(7): e1007229, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31339888

RESUMO

Antiretroviral therapy (ART) effectively controls HIV infection, suppressing HIV viral loads. Suspension of therapy is followed by rebound of viral loads to high, pre-therapy levels. However, there is significant heterogeneity in speed of rebound, with some rebounds occurring within days, weeks, or sometimes years. We present a stochastic mathematical model to gain insight into these post-treatment dynamics, specifically characterizing the dynamics of short term viral rebounds (≤ 60 days). Li et al. (2016) report that the size of the expressed HIV reservoir, i.e., cell-associated HIV RNA levels, and drug regimen correlate with the time between ART suspension and viral rebound to detectable levels. We incorporate this information and viral rebound times to parametrize our model. We then investigate insights offered by our model into the underlying dynamics of the latent reservoir. In particular, we refine previous estimates of viral recrudescence after ART interruption by accounting for heterogeneity in infection rebound dynamics, and determine a recrudescence rate of once every 2-4 days. Our parametrized model can be used to aid in design of clinical trials to study viral dynamics following analytic treatment interruption. We show how to derive informative personalized testing frequencies from our model and offer a proof-of-concept example. Our results represent first steps towards a model that can make predictions on a person living with HIV (PLWH)'s rebound time distribution based on biomarkers, and help identify PLWH with long viral rebound delays.


Assuntos
Fármacos Anti-HIV/administração & dosagem , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Modelos Biológicos , Algoritmos , Biomarcadores/sangue , Biologia Computacional , Infecções por HIV/imunologia , Humanos , Funções Verossimilhança , RNA Viral/sangue , Processos Estocásticos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/virologia , Fatores de Tempo , Carga Viral/efeitos dos fármacos , Viremia/tratamento farmacológico , Viremia/imunologia , Viremia/virologia , Latência Viral/efeitos dos fármacos , Latência Viral/imunologia , Suspensão de Tratamento
7.
PLoS One ; 14(5): e0217578, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31125383

RESUMO

Cellular sumoylation processes are proposed targets for anti-viral and anti-cancer therapies. We reported that Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) dysregulates cellular sumoylation processes, contributing to its oncogenic potential in EBV-associated malignancies. Ginkgolic acid and anacardic acid, known inhibitors of sumoylation, inhibit LMP1-induced protein sumoylation; however, both drugs have adverse effects in hosts. Here we test the effects of glycyrrhizic acid, a medicinal botanical extract with anti-inflammatory, anti-carcinogenic, and anti-viral properties, on cellular sumoylation processes. While glycyrrhizic acid is known to inhibit EBV penetration, its affect on cellular sumoylation processes remains to be documented. We hypothesized that glycyrrhizic acid inhibits cellular sumoylation processes and may be a viable treatment for Epstein-Barr virus-associated malignancies. Results showed that glycyrrhizic acid inhibited sumoylation processes (without affecting ubiquitination processes), limited cell growth, and induced apoptosis in multiple cell lines. Similar to ginkgolic acid; glycyrrhizic acid targeted the first step of the sumoylation process and resulted in low levels of spontaneous EBV reactivation. Glycyrrhizic acid did not affect induced reactivation of the virus, but the presence of the extract did reduce the ability of the produced virus to infect additional cells. Therefore, we propose that glycyrrhizic acid may be a potential therapeutic drug to augment the treatment of EBV-associated lymphoid malignancies.


Assuntos
Antivirais/farmacologia , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Ácido Glicirrízico/farmacologia , Herpesvirus Humano 4/efeitos dos fármacos , Sumoilação/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Linhagem Celular , Infecções por Vírus Epstein-Barr/metabolismo , Células HEK293 , Herpesvirus Humano 4/fisiologia , Humanos
8.
Biochem Pharmacol ; 164: 237-251, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30991051

RESUMO

The persistence of latent human immunodeficiency virus type 1 (HIV-1) reservoirs remains a major hurdle for HIV-1 eradication. The "shock and kill" strategy relies on the drug-mediated reversion of HIV-1 latency and the subsequent death of HIV-producing cells. Unfortunately, none of the agents currently in use possess a sufficient potency to reactivate latent virus or eliminate the latent HIV-1 reservoir in vivo. Here, we demonstrated that a promising specific bromodomain and extraterminal domain inhibitor, CPI-203, could potently reactivate latent HIV-1 in different latently infected cell lines with minimal cytotoxicity by activating the positive transcription elongation factor b signaling pathway. Notably, CPI-203 exhibited synergism in latent HIV-1 reactivation and alleviated the HIV-1-induced "cytokine storm" when used in combination with the protein kinase C (PKC) agonist prostratin. These findings highlight that CPI-203 shows promise as a novel, safe candidate for the design of targeted strategies to "shock and kill" HIV-1 and thus represents a potential functional cure.


Assuntos
Acetamidas/farmacologia , Azepinas/farmacologia , HIV-1/efeitos dos fármacos , Fator B de Elongação Transcricional Positiva/metabolismo , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Acetamidas/química , Adulto , Animais , Azepinas/química , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/virologia , Feminino , HIV-1/fisiologia , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Domínios Proteicos/efeitos dos fármacos , Domínios Proteicos/fisiologia , Ativação Viral/fisiologia , Latência Viral/fisiologia
9.
Clin Epigenetics ; 11(1): 55, 2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30917875

RESUMO

Epigenetics is defined as the science that studies the modifications of gene expression that are not owed to mutations or changes in the genetic sequence. Recently, strong evidences are pinpointing toward a solid interplay between such epigenetic alterations and the outcome of human cytomegalovirus (HCMV) infection. Guided by the previous possibly promising experimental trials of human immunodeficiency virus (HIV) epigenetic reprogramming, the latter is paving the road toward two major approaches to control viral gene expression or latency. Reactivating HCMV from the latent phase ("shock and kill" paradigm) or alternatively repressing the virus lytic and reactivation phases ("block and lock" paradigm) by epigenetic-targeted therapy represent encouraging options to overcome latency and viral shedding or otherwise replication and infectivity, which could lead eventually to control the infection and its complications. Not limited to HIV and HCMV, this concept is similarly studied in the context of hepatitis B and C virus, herpes simplex virus, and Epstein-Barr virus. Therefore, epigenetic manipulations stand as a pioneering research area in modern biology and could constitute a curative methodology by potentially consenting the development of broad-spectrum antivirals to control viral infections in vivo.


Assuntos
Antivirais/farmacologia , Epigênese Genética/efeitos dos fármacos , Proteínas Virais/genética , Viroses/tratamento farmacológico , Antivirais/uso terapêutico , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular , Proteínas Virais/efeitos dos fármacos , Ativação Viral/efeitos dos fármacos , Viroses/genética , Latência Viral/efeitos dos fármacos
10.
Sci Transl Med ; 11(482)2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842315

RESUMO

Epstein-Barr virus (EBV) is a DNA tumor virus responsible for 1 to 2% of human cancers including subtypes of Burkitt's lymphoma, Hodgkin's lymphoma, gastric carcinoma, and nasopharyngeal carcinoma (NPC). Persistent latent infection drives EBV-associated tumorigenesis. Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all EBV-associated tumors and is therefore an attractive target for therapeutic intervention. It is a multifunctional DNA binding protein critical for viral replication, genome maintenance, viral gene expression, and host cell survival. Using a fragment-based approach and x-ray crystallography, we identify a 2,3-disubstituted benzoic acid series that selectively inhibits the DNA binding activity of EBNA1. We characterize these inhibitors biochemically and in cell-based assays, including chromatin immunoprecipitation and DNA replication assays. In addition, we demonstrate the potency of EBNA1 inhibitors to suppress tumor growth in several EBV-dependent xenograft models, including patient-derived xenografts for NPC. These inhibitors selectively block EBV gene transcription and alter the cellular transforming growth factor-ß (TGF-ß) signaling pathway in NPC tumor xenografts. These EBNA1-specific inhibitors show favorable pharmacological properties and have the potential to be further developed for the treatment of EBV-associated malignancies.


Assuntos
DNA Viral/metabolismo , Desenho de Drogas , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiologia , Neoplasias Nasofaríngeas/virologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Latência Viral/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos Nus , Neoplasias Nasofaríngeas/patologia , Relação Estrutura-Atividade
11.
Curr HIV/AIDS Rep ; 16(2): 129-140, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30835045

RESUMO

PURPOSE OF REVIEW: Gallant efforts are ongoing to achieve sustained antiretroviral therapy (ART)-free HIV remission in the HIV-infected person; however, most, if not all, current human clinical studies have primarily focused these efforts on targeting viral persistence in CD4 T cells in blood and tissue sanctuaries. The lack of myeloid centered HIV clinical trials, either as primary or secondary end points, has hindered our understanding of the contribution of myeloid cells in unsuccessful trials but may also guide successes in future HIV eradication clinical strategies. RECENT FINDINGS: Recent advances have highlighted the importance of myeloid reservoirs as sanctuaries of HIV persistence and therefore may partially be responsible for viral recrudescence following ART treatment interruption in several clinical trials where HIV was not detectable or recovered from CD4 T cells. Given these findings, novel innovative therapeutic approaches specifically focused on HIV clearance in myeloid cell populations need to be vigorously pursued if we are to achieve additional cases of sustained ART-free remission. This review will highlight new research efforts defining myeloid persistence and recent advances in HIV remission and cure trials that would be relevant in targeting this compartment and make an argument as to their clinical relevancy as we progress towards sustained ART-free HIV remission in all HIV-infected persons.


Assuntos
Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Células Mieloides/virologia , Linfócitos T CD4-Positivos/imunologia , Ensaios Clínicos como Assunto , Humanos , Células Mieloides/efeitos dos fármacos , Latência Viral/efeitos dos fármacos
12.
J Acquir Immune Defic Syndr ; 80(5): 605-613, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30768485

RESUMO

OBJECTIVES: Few single latency-reversing agents (LRAs) have been tested in vivo, and only some of them have demonstrated an effect, albeit weak, on the decrease of latent reservoir. Therefore, other LRAs and combinations of LRAs need to be assessed. Here, we evaluated the potential of combined treatments of therapeutically promising LRAs, disulfiram and romidepsin. SETTING AND METHODS: We assessed the reactivation potential of individual disulfiram or simultaneous or sequential combined treatments with romidepsin in vitro in latently infected cell lines of T-lymphoid and myeloid origins and in ex vivo cultures of CD8-depleted peripheral blood mononuclear cells isolated from 18 HIV-1 combination antiretroviral therapy-treated individuals. RESULTS: We demonstrated heterogeneous reactivation effects of disulfiram in vitro in various cell lines of myeloid origin and no latency reversal neither in vitro in T-lymphoid cells nor ex vivo, even if doses corresponding to maximal plasmatic concentration or higher were tested. Disulfiram+romidepsin combined treatments produced distinct reactivation patterns in vitro. Ex vivo, the combined treatments showed a modest reactivation effect when used simultaneously as opposed to no viral reactivation for the corresponding sequential treatment. CONCLUSIONS: Exclusive reactivation effects of disulfiram in myeloid latency cell lines suggest that disulfiram could be a potential LRA for this neglected reservoir. Moreover, distinct reactivation profiles pinpoint heterogeneity of the latent reservoir and confirm that the mechanisms that contribute to HIV latency are diverse. Importantly, disulfiram+romidepsin treatments are not potent ex vivo and most likely do not represent an effective drug combination to achieve high levels of latency reversal in vivo.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Depsipeptídeos/uso terapêutico , Dissulfiram/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Fármacos Anti-HIV/administração & dosagem , Linhagem Celular , Depsipeptídeos/administração & dosagem , Dissulfiram/administração & dosagem , Quimioterapia Combinada , HIV-1/fisiologia , Humanos
13.
Nat Commun ; 10(1): 814, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30778080

RESUMO

HIV persists in latently infected CD4+ T cells during antiretroviral therapy (ART). Immune checkpoint molecules, including PD-1, are preferentially expressed at the surface of persistently infected cells. However, whether PD-1 plays a functional role in HIV latency and reservoir persistence remains unknown. Using CD4+ T cells from HIV-infected individuals, we show that the engagement of PD-1 inhibits viral production at the transcriptional level and abrogates T-cell receptor (TCR)-induced HIV reactivation in latently infected cells. Conversely, PD-1 blockade with the monoclonal antibody pembrolizumab enhances HIV production in combination with the latency reversing agent bryostatin without increasing T cell activation. Our results suggest that the administration of immune checkpoint blockers to HIV-infected individuals on ART may facilitate latency disruption.


Assuntos
Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Latência Viral/efeitos dos fármacos , Anticorpos Monoclonais Humanizados/farmacologia , Antígeno B7-H1/farmacologia , Briostatinas/farmacologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Receptor de Morte Celular Programada 1/metabolismo
14.
PLoS One ; 14(2): e0211746, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30716099

RESUMO

Antiretroviral therapy (ART) suppresses HIV replication, but does not cure the infection because replication-competent virus persists within latently infected CD4+ T cells throughout years of therapy. These reservoirs contain integrated HIV-1 genomes and can resupply active virus. Thus, the development of strategies to eliminate the reservoir of latently infected cells is a research priority of global significance. In this study, we tested efficacy of a new inhibitor of apoptosis protein antagonist (IAPa) called Debio 1143 at reversing HIV latency and investigated its mechanisms of action. Debio 1143 activates HIV transcription via NF-kB signaling by degrading the ubiquitin ligase baculoviral IAP repeat-containing 2 (BIRC2), a repressor of the non-canonical NF-kB pathway. Debio 1143-induced BIRC2 degradation results in the accumulation of NF-κB-inducing kinase (NIK) and proteolytic cleavage of p100 into p52, leading to nuclear translocation of p52 and RELB. Debio 1143 greatly enhances the binding of RELB to the HIV-1 LTR. These data indicate that Debio 1143 activates the non-canonical NF-kB signaling pathway by promoting the binding of RELB:p52 complexes to the HIV-1 LTR, resulting in the activation of the LTR-dependent HIV-1 transcription. Importantly, Debio 1143 reverses viral latency in HIV-1 latent T cell lines. Using knockdown (siRNA BIRC2), knockout (CRIPSR NIK) and proteasome machinery neutralization (MG132) approaches, we found that Debio 1143-mediated HIV latency reversal is BIRC2 degradation- and NIK stabilization-dependent. Debio 1143 also reverses HIV-1 latency in resting CD4+ T cells derived from ART-treated patients or HIV-1-infected humanized mice under ART. Interestingly, daily oral administration of Debio 1143 in cancer patients at well-tolerated doses elicited BIRC2 target engagement in PBMCs and induced a moderate increase in cytokines and chemokines mechanistically related to NF-kB signaling. In conclusion, we provide strong evidences that the IAPa Debio 1143, by initially activating the non-canonical NF-kB signaling and subsequently reactivating HIV-1 transcription, represents a new attractive viral latency reversal agent (LRA).


Assuntos
Fármacos Anti-HIV/farmacologia , Azocinas/farmacologia , Compostos Benzidrílicos/farmacologia , Linfócitos T CD4-Positivos/metabolismo , HIV-1/fisiologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Latência Viral/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Feminino , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
15.
Curr HIV/AIDS Rep ; 16(1): 96-104, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30734905

RESUMO

PURPOSE OF REVIEW: In addition to preventive protocols and antiretroviral therapy, HIV-1 eradication has been considered as an additional strategy to help fight the AIDS epidemic. With the support of multiple funding agencies, research groups worldwide have been developing protocols to achieve either a sterilizing or a functional cure for HIV-infection. RECENT FINDINGS: Most of the studies focus on the elimination or suppression of circulating CD4+ T cells, the best characterized HIV-1 latent reservoir. The role of the central nervous system (CNS) as a latent reservoir is still controversial. Although brain macrophages and astrocytes are susceptible to HIV-1 infection, it has not been ascertained whether the CNS carries latent HIV-1 during cART and, if so, whether the virus can be reactivated and spread to other compartments after ART interruption. Here, we examine the implications of HIV-1 eradication strategies on the CNS, regardless of whether it is a true latent reservoir and, if so, whether it is present in all patients.


Assuntos
Encéfalo/virologia , Linfócitos T CD4-Positivos/virologia , Erradicação de Doenças/métodos , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Latência Viral/fisiologia , Astrócitos/virologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Reservatórios de Doenças/virologia , HIV-1/efeitos dos fármacos , Humanos , Macrófagos/virologia , Latência Viral/efeitos dos fármacos
16.
J Biol Chem ; 294(14): 5576-5589, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30745362

RESUMO

Histone deacetylase (HDAC) inhibitors (HDACis) have been widely tested in clinical trials for their ability to reverse HIV latency but have yielded only limited success. One HDACi, suberoylanilide hydroxamic acid (SAHA), exhibits off-target effects on host gene expression predicted to interfere with induction of HIV transcription. Romidepsin (RMD) has higher potency and specificity for class I HDACs implicated in maintaining HIV provirus in the latent state. More robust HIV reactivation has indeed been achieved with RMD use ex vivo than with SAHA; however, reduction of viral reservoir size has not been observed in clinical trials. Therefore, using RNA-Seq, we sought to compare the effects of SAHA and RMD on gene expression in primary CD4+ T cells. Among the genes whose expression was modulated by both HDACi agents, we identified genes previously implicated in HIV latency. Two genes, SMARCB1 and PARP1, whose modulation by SAHA and RMD is predicted to inhibit HIV reactivation, were evaluated in the major maturation subsets of CD4+ T cells and were consistently either up- or down-regulated by both HDACi compounds. Our results indicate that despite having different potencies and HDAC specificities, SAHA and RMD modulate an overlapping set of genes, implicated in HIV latency regulation. Some of these genes merit exploration as additional targets to improve the therapeutic outcomes of "shock and kill" strategies. The overall complexity of HDACi-induced responses among host genes with predicted stimulatory or inhibitory effects on HIV expression likely contributes to differential HDACi potencies and dictates the outcome of HIV reactivation.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Depsipeptídeos/farmacologia , HIV-1/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Ativação Viral/efeitos dos fármacos , Vorinostat/farmacologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Poli(ADP-Ribose) Polimerase-1/biossíntese , Proteína SMARCB1/biossíntese , Transcrição Genética/efeitos dos fármacos , Latência Viral/efeitos dos fármacos
17.
Nat Commun ; 10(1): 728, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760706

RESUMO

After initiating antiretroviral therapy (ART), a rapid decline in HIV viral load is followed by a long period of undetectable viremia. Viral outgrowth assay suggests the reservoir continues to decline slowly. Here, we use full-length sequencing to longitudinally study the proviral landscape of four subjects on ART to investigate the selective pressures influencing the dynamics of the treatment-resistant HIV reservoir. We find intact and defective proviruses that contain genetic elements favoring efficient protein expression decrease over time. Moreover, proviruses that lack these genetic elements, yet contain strong donor splice sequences, increase relatively to other defective proviruses, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by the expansion of proviral clones. Paradoxically, clonal expansion may also be enhanced by HIV expression that leads to splicing between HIV donor splice sites and downstream human exons.


Assuntos
Terapia Antirretroviral de Alta Atividade , Antivirais/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Adulto , Feminino , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Mutação , Filogenia , Provírus/classificação , Provírus/efeitos dos fármacos , Provírus/genética , Carga Viral/efeitos dos fármacos , Viremia/prevenção & controle , Latência Viral/efeitos dos fármacos
18.
PLoS One ; 14(1): e0211112, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30682108

RESUMO

Blockade of the programmed cell death protein/ligand 1 (PD-1/PD-L1) pathway with monoclonal antibodies (mAb) is now commonly used for cancer immunotherapy and has therapeutic potential in chronic viral infections including HIV-1. PD-1/PD-L1 blockade could augment HIV-1-specific immune responses and reverse HIV-1 latency, but the latter effect has not been clearly shown. We tested the ability of the human anti-PD-L1 mAb BMS-936559 and the human anti-PD-1 mAb nivolumab to increase HIV-1 virion production ex vivo from different peripheral blood mononuclear cell populations obtained from donors on suppressive antiretroviral therapy (ART). Fresh peripheral blood mononuclear cells (PBMC), CD8-depleted PBMC, total CD4+ T cells, and resting CD4+ T cells were purified from whole blood of HIV-1-infected donors and cultured in varying concentrations of BMS-936559 (20, 5, or 1.25µg/mL) or nivolumab (5 or 1.25µg/mL), with or without anti-CD3/CD28 stimulatory antibodies. Culture supernatants were assayed for virion HIV-1 RNA by qRT-PCR. Ex vivo exposure to BMS-936559 or nivolumab, with or without anti-CD3/CD28 stimulation, did not consistently increase HIV-1 virion production from blood mononuclear cell populations. Modest (2-fold) increases in virus production were observed in a subset of donors and in some cell types but were not reproducible in longitudinal samples. Cell surface expression of PD-1 and PD-L1 were not associated with changes in virus production. Ex vivo blockade of the PD-1 axis alone has limited effects on HIV-1 latency.


Assuntos
Antirretrovirais/farmacologia , Anticorpos Monoclonais/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Nivolumabe/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Vírion/metabolismo , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Anticorpos Monoclonais Humanizados , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Humanos , Receptor de Morte Celular Programada 1/metabolismo
19.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30602611

RESUMO

The HIV accessory protein Nef modulates key immune evasion and pathogenic functions, and its encoding gene region exhibits high sequence diversity. Given the recent identification of early HIV-specific adaptive immune responses as novel correlates of HIV reservoir size, we hypothesized that viral factors that facilitate the evasion of such responses-namely, Nef genetic and functional diversity-might also influence reservoir establishment and/or persistence. We isolated baseline plasma HIV RNA-derived nef clones from 30 acute/early-infected individuals who participated in a clinical trial of early combination antiretroviral therapy (cART) (<6 months following infection) and assessed each Nef clone's ability to downregulate CD4 and human leukocyte antigen (HLA) class I in vitro We then explored the relationships between baseline clinical, immunological, and virological characteristics and the HIV reservoir size measured 48 weeks following initiation of suppressive cART (where the reservoir size was quantified in terms of the proviral DNA loads as well as the levels of replication-competent HIV in CD4+ T cells). Maximal within-host Nef-mediated downregulation of HLA, but not CD4, correlated positively with post-cART proviral DNA levels (Spearman's R = 0.61, P = 0.0004) and replication-competent reservoir sizes (Spearman's R = 0.36, P = 0.056) in univariable analyses. Furthermore, the Nef-mediated HLA downregulation function was retained in final multivariable models adjusting for established clinical and immunological correlates of reservoir size. Finally, HIV subtype B-infected persons (n = 25) harbored significantly larger viral reservoirs than non-subtype B-infected persons (2 infected with subtype CRF01_AE and 3 infected with subtype G). Our results highlight a potentially important role of viral factors-in particular, HIV subtype and accessory protein function-in modulating viral reservoir establishment and persistence.IMPORTANCE While combination antiretroviral therapies (cART) have transformed HIV infection into a chronic manageable condition, they do not act upon the latent HIV reservoir and are therefore not curative. As HIV cure or remission should be more readily achievable in individuals with smaller HIV reservoirs, achieving a deeper understanding of the clinical, immunological, and virological determinants of reservoir size is critical to eradication efforts. We performed a post hoc analysis of 30 participants of a clinical trial of early cART who had previously been assessed in detail for their clinical, immunological, and reservoir size characteristics. We observed that the HIV subtype and autologous Nef-mediated HLA downregulation function correlated with the viral reservoir size measured approximately 1 year post-cART initiation. Our findings highlight virological characteristics-both genetic and functional-as possible novel determinants of HIV reservoir establishment and persistence.


Assuntos
Infecções por HIV/imunologia , HIV/imunologia , Evasão da Resposta Imune/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Adulto , Antirretrovirais/farmacologia , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , HIV/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Antígenos HLA/imunologia , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Latência Viral/efeitos dos fármacos , Latência Viral/imunologia , Adulto Jovem
20.
PLoS Pathog ; 15(1): e1007498, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30645648

RESUMO

The establishment of HIV-1 latency gives rise to persistent chronic infection that requires life-long treatment. To reverse latency for viral eradiation, the HIV-1 Tat protein and its associated ELL2-containing Super Elongation Complexes (ELL2-SECs) are essential to activate HIV-1 transcription. Despite efforts to identify effective latency-reversing agents (LRA), avenues for exposing latent HIV-1 remain inadequate, prompting the need to identify novel LRA targets. Here, by conducting a CRISPR interference-based screen to reiteratively enrich loss-of-function genotypes that increase HIV-1 transcription in latently infected CD4+ T cells, we have discovered a key role of the proteasome in maintaining viral latency. Downregulating or inhibiting the proteasome promotes Tat-transactivation in cell line models. Furthermore, the FDA-approved proteasome inhibitors bortezomib and carfilzomib strongly synergize with existing LRAs to reactivate HIV-1 in CD4+ T cells from antiretroviral therapy-suppressed individuals without inducing cell activation or proliferation. Mechanistically, downregulating/inhibiting the proteasome elevates the levels of ELL2 and ELL2-SECs to enable Tat-transactivation, indicating the proteasome-ELL2 axis as a key regulator of HIV-1 latency and promising target for therapeutic intervention.


Assuntos
HIV-1/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Latência Viral/efeitos dos fármacos , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Soropositividade para HIV , HIV-1/patogenicidade , Humanos , Células Jurkat , Complexo de Endopeptidases do Proteassoma/fisiologia , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Fatores de Elongação da Transcrição , Ativação Viral/efeitos dos fármacos , Latência Viral/fisiologia
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