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1.
Viruses ; 12(2)2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31991737

RESUMO

Combination antiretroviral therapy (cART) controls but does not eradicate HIV infection; HIV persistence is the principal obstacle to curing infections. The proportion of defective proviruses increases during cART, but the dynamics of this process are not well understood, and a quantitative analysis of how the proviral landscape is reshaped after cART is initiated is critical to understanding how HIV persists. Here, we studied longitudinal samples from HIV infected individuals undergoing long term cART using multiplexed Droplet Digital PCR (ddPCR) approaches to quantify the proportion of deleted proviruses in lymphocytes. In most individuals undergoing cART, HIV proviruses that contain gag are lost more quickly than those that lack gag. Increases in the fraction of gag-deleted proviruses occurred only after 1-2 years of therapy, suggesting that the immune system, and/or toxicity of viral re-activation helps to gradually shape the proviral landscape. After 10-15 years on therapy, there were as many as 3.5-5 times more proviruses in which gag was deleted or highly defective than those containing intact gag. We developed a provirus-specific ddPCR approach to quantify individual clones. Investigation of a clone of cells containing a deleted HIV provirus integrated in the HORMAD2 gene revealed that the cells underwent a massive expansion shortly after cART was initiated until the clone, which was primarily in effector memory cells, dominated the population of proviruses for over 6 years. The expansion of this HIV-infected clone had substantial effects on the overall proviral population.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/genética , HIV-1/isolamento & purificação , Leucócitos Mononucleares/virologia , Provírus/isolamento & purificação , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/virologia , Proteínas de Ciclo Celular/genética , DNA Viral/sangue , DNA Viral/genética , Vírus Defeituosos/genética , Genes gag , Repetição Terminal Longa de HIV , HIV-1/efeitos dos fármacos , Humanos , Memória Imunológica , Reação em Cadeia da Polimerase Multiplex , Provírus/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Fatores de Tempo , Resultado do Tratamento , Carga Viral
2.
Infect Immun ; 88(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31740528

RESUMO

Influenza A virus (H1N1) is an acute, highly contagious respiratory virus. The use of lactic acid bacteria (LAB) to deliver mucosal vaccines against influenza virus infection is a research hot spot. In this study, two recombinant Lactobacillus plantarum strains expressing hemagglutinin (HA) alone or coexpressing aCD11c-HA to target HA protein to dendritic cells (DCs) by fusion to an anti-CD11c single-chain antibody (aCD11c) were constructed. The activation of bone marrow dendritic cells (BMDCs) by recombinant strains and the interaction of activated BMDCs and sorted CD4+ or CD8+ T cells were evaluated through flow cytometry in vitro, and cellular supernatants were assessed by using an enzyme-linked immunosorbent assay kit. The results demonstrated that, compared to the HA strain, the aCD11c-HA strain significantly increased the activation of BMDCs and increased the production of CD4+ gamma interferon-positive (IFN-γ+) T cells, CD8+ IFN-γ+ T cells, and IFN-γ in the cell culture supernatant in vitro Consistent with these results, the aCD11c-HA strain clearly increased the activation and maturation of DCs, the HA-specific responses of CD4+ IFN-γ+ T cells, CD8+ IFN-γ+ T cells, and CD8+ CD107a+ T cells, and the proliferation of T cells in the spleen, finally increasing the levels of specific antibodies and neutralizing antibodies in mice. In addition, the protection of immunized mice was observed after viral infection, as evidenced by improved weight loss, survival, and lung pathology. The adoptive transfer of CD8+ T cells from the aCD11c-HA mice to NOD/Lt-SCID mice resulted in a certain level of protection after influenza virus infection, highlighting the efficacy of the aCD11c targeting strategy.


Assuntos
Antígeno CD11c/imunologia , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Lactobacillus plantarum/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Células Dendríticas/virologia , Feminino , Vírus da Influenza A Subtipo H1N1/imunologia , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia
3.
Arch Virol ; 165(2): 321-330, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31828511

RESUMO

Persistence of human immunodeficiency virus 1 (HIV-1) latency and residual immune activation remain major barriers to treatment in patients receiving highly active antiretroviral therapy (HAART). In the present study, we investigated the molecular mechanisms of persistent HIV infection and residual immune activation in HAART-treated patients. We showed that the expression level of B-cell CLL/lymphoma 11B (BCL11B) was significantly increased in CD4+T cells from HIV-infected patients undergoing HAART, and this was accompanied by increased expression of BCL11B-associated chromatin modifiers and inflammatory factors in comparison to healthy controls and untreated patients with HIV. In vitro assays showed that BCL11B significantly inhibited HIV-1 long terminal repeat (LTR)-mediated transcription. Knockdown of BCL11B resulted in the activation of HIV latent cells, and dissociation of BCL11B and its related chromatin remodeling factors from the HIV LTR. Our findings suggested that increased expression of BCL11B and its related chromatin modifiers contribute to HIV-1 transcriptional silencing, and alteration of BCL11B levels might lead to abnormal transcription and inflammation.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Infecções por HIV/genética , HIV-1/genética , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Transcrição Genética/imunologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia , Adulto , Terapia Antirretroviral de Alta Atividade/métodos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Cromatina/genética , Cromatina/virologia , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/imunologia , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/genética , Repetição Terminal Longa de HIV/imunologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Masculino , Transcrição Genética/efeitos dos fármacos , Latência Viral/genética , Latência Viral/imunologia
4.
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
5.
PLoS Pathog ; 15(9): e1008043, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31568492

RESUMO

Immunization vectors based on cytomegalovirus (CMV) have attracted a lot of interest in recent years because of their high efficacy in the simian immunodeficiency virus (SIV) macaque model, which has been attributed to their ability to induce strong, unusually broad, and unconventionally restricted CD8+ T cell responses. To evaluate the ability of CMV-based vectors to mediate protection by other immune mechanisms, we evaluated a mouse CMV (MCMV)-based vector encoding Friend virus (FV) envelope (Env), which lacks any known CD8+ T cell epitopes, for its protective efficacy in the FV mouse model. When we immunized highly FV-susceptible mice with the Env-encoding MCMV vector (MCMV.env), we could detect high frequencies of Env-specific CD4+ T cells after a single immunization. While the control of an early FV challenge infection was highly variable, an FV infection applied later after immunization was tightly controlled by almost all immunized mice. Protection of mice correlated with their ability to mount a robust anamnestic neutralizing antibody response upon FV infection, but Env-specific CD4+ T cells also produced appreciable levels of interferon γ. Depletion and transfer experiments underlined the important role of antibodies for control of FV infection but also showed that while no Env-specific CD8+ T cells were induced by the MCMV.env vaccine, the presence of CD8+ T cells at the time of FV challenge was required. The immunity induced by MCMV.env immunization was long-lasting, but was restricted to MCMV naïve animals. Taken together, our results demonstrate a novel mode of action of a CMV-based vaccine for anti-retrovirus immunization that confers strong protection from retrovirus challenge, which is conferred by CD4+ T cells and antibodies.


Assuntos
Vírus da Leucemia Murina de Friend/imunologia , Muromegalovirus/imunologia , Vacinas Virais/imunologia , Transferência Adotiva , Animais , Anticorpos Antivirais/biossíntese , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Feminino , Vírus da Leucemia Murina de Friend/genética , Vírus da Leucemia Murina de Friend/patogenicidade , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Vetores Genéticos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Muromegalovirus/genética , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/prevenção & controle , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/genética
6.
PLoS Pathog ; 15(10): e1008068, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31648236

RESUMO

Ebola virus (EBOV) infections are characterized by a pronounced lymphopenia that is highly correlative with fatalities. However, the mechanisms leading to T-cell depletion remain largely unknown. Here, we demonstrate that both viral mRNAs and antigens are detectable in CD4+ T cells despite the absence of productive infection. A protein phosphatase 1 inhibitor, 1E7-03, and siRNA-mediated suppression of viral antigens were used to demonstrate de novo synthesis of viral RNAs and antigens in CD4+ T cells, respectively. Cell-to-cell fusion of permissive Huh7 cells with non-permissive Jurkat T cells impaired productive EBOV infection suggesting the presence of a cellular restriction factor. We determined that viral transcription is partially impaired in the fusion T cells. Lastly, we demonstrate that exposure of T cells to EBOV resulted in autophagy through activation of ER-stress related pathways. These data indicate that exposure of T cells to EBOV results in an abortive infection, which likely contributes to the lymphopenia observed during EBOV infections.


Assuntos
Linfócitos T CD4-Positivos/virologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Linfopenia/imunologia , Replicação Viral/fisiologia , Animais , Antígenos Virais/biossíntese , Antígenos Virais/genética , Autofagia/fisiologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Estresse do Retículo Endoplasmático/fisiologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Indóis/farmacologia , Células Jurkat , Proteína Fosfatase 1/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Viral/biossíntese , RNA Viral/genética , Fatores de Transcrição/metabolismo , Ureia/análogos & derivados , Ureia/farmacologia , Células Vero , Proteínas Virais/metabolismo
7.
Nat Commun ; 10(1): 4739, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31628331

RESUMO

HIV viral reservoirs are established very early during infection. Resident memory T cells (TRM) are present in tissues such as the lower female genital tract, but the contribution of this subset of cells to the pathogenesis and persistence of HIV remains unclear. Here, we show that cervical CD4+TRM display a unique repertoire of clusters of differentiation, with enrichment of several molecules associated with HIV infection susceptibility, longevity and self-renewing capacities. These protein profiles are enriched in a fraction of CD4+TRM expressing CD32. Cervical explant models show that CD4+TRM preferentially support HIV infection and harbor more viral DNA and protein than non-TRM. Importantly, cervical tissue from ART-suppressed HIV+ women contain high levels of viral DNA and RNA, being the TRM fraction the principal contributor. These results recognize the lower female genital tract as an HIV sanctuary and identify CD4+TRM as primary targets of HIV infection and viral persistence. Thus, strategies towards an HIV cure will need to consider TRM phenotypes, which are widely distributed in tissues.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Memória Imunológica/imunologia , Adulto , Idoso , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Colo do Útero/efeitos dos fármacos , Colo do Útero/virologia , Reservatórios de Doenças/virologia , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Pessoa de Meia-Idade , Membrana Mucosa/efeitos dos fármacos , Membrana Mucosa/virologia , Carga Viral/efeitos dos fármacos , Carga Viral/genética , Carga Viral/imunologia
8.
PLoS Pathog ; 15(10): e1007903, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584995

RESUMO

HIV-1 gene expression is regulated by host and viral factors that interact with viral motifs and is influenced by proviral integration sites. Here, expression variation among integrants was followed for hundreds of individual proviral clones within polyclonal populations throughout successive rounds of virus and cultured cell replication, with limited findings using CD4+ cells from donor blood consistent with observations in immortalized cells. Tracking clonal behavior by proviral "zip codes" indicated that mutational inactivation during reverse transcription was rare, while clonal expansion and proviral expression states varied widely. By sorting for provirus expression using a GFP reporter in the nef open reading frame, distinct clone-specific variation in on/off proportions were observed that spanned three orders of magnitude. Tracking GFP phenotypes over time revealed that as cells divided, their progeny alternated between HIV transcriptional activity and non-activity. Despite these phenotypic oscillations, the overall GFP+ population within each clone was remarkably stable, with clones maintaining clone-specific equilibrium mixtures of GFP+ and GFP- cells. Integration sites were analyzed for correlations between genomic features and the epigenetic phenomena described here. Integrants inserted in the sense orientation of genes were more frequently found to be GFP negative than those in the antisense orientation, and clones with high GFP+ proportions were more distal to repressive H3K9me3 peaks than low GFP+ clones. Clones with low frequencies of GFP positivity appeared to expand more rapidly than clones for which most cells were GFP+, even though the tested proviruses were Vpr-. Thus, much of the increase in the GFP- population in these polyclonal pools over time reflected differential clonal expansion. Together, these results underscore the temporal and quantitative variability in HIV-1 gene expression among proviral clones that are conferred in the absence of metabolic or cell-type dependent variability, and shed light on cell-intrinsic layers of regulation that affect HIV-1 population dynamics.


Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Provírus/genética , Integração Viral/genética , Replicação Viral , Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/genética , Ensaios de Triagem em Larga Escala , Humanos , Células Jurkat , Transdução Genética
9.
Nat Commun ; 10(1): 4059, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492853

RESUMO

HIV-1 recurrently targets active genes and integrates in the proximity of the nuclear pore compartment in CD4+ T cells. However, the genomic features of these genes and the relevance of their transcriptional activity for HIV-1 integration have so far remained unclear. Here we show that recurrently targeted genes are proximal to super-enhancer genomic elements and that they cluster in specific spatial compartments of the T cell nucleus. We further show that these gene clusters acquire their location during the activation of T cells. The clustering of these genes along with their transcriptional activity are the major determinants of HIV-1 integration in T cells. Our results provide evidence of the relevance of the spatial compartmentalization of the genome for HIV-1 integration, thus further strengthening the role of nuclear architecture in viral infection.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Núcleo Celular/genética , Elementos Facilitadores Genéticos , HIV-1/genética , Integração Viral/genética , Sequência de Bases , Linfócitos T CD4-Positivos/virologia , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Cromatina/genética , Cromatina/virologia , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Poro Nuclear/genética , Poro Nuclear/virologia , Regiões Promotoras Genéticas/genética , Transcrição Genética
10.
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
11.
PLoS Pathog ; 15(7): e1007869, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31291371

RESUMO

Clonal expansion of HIV infected cells plays an important role in the formation and persistence of the reservoir that allows the virus to persist, in DNA form, despite effective antiretroviral therapy. We used integration site analysis to ask if there is a similar clonal expansion of SIV infected cells in macaques. We show that the distribution of HIV and SIV integration sites in vitro is similar and that both viruses preferentially integrate in many of the same genes. We obtained approximately 8000 integration sites from blood samples taken from SIV-infected macaques prior to the initiation of ART, and from blood, spleen, and lymph node samples taken at necropsy. Seven clones were identified in the pre-ART samples; one persisted for a year on ART. An additional 100 clones were found only in on-ART samples; a number of these clones were found in more than one tissue. The timing and extent of clonal expansion of SIV-infected cells in macaques and HIV-infected cells in humans is quite similar. This suggests that SIV-infected macaques represent a useful model of the clonal expansion of HIV infected cells in humans that can be used to evaluate strategies intended to control or eradicate the viral reservoir.


Assuntos
Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Animais , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/virologia , Reservatórios de Doenças/virologia , Infecções por HIV/patologia , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Técnicas In Vitro , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Vírus da Imunodeficiência Símia/patogenicidade , Carga Viral/efeitos dos fármacos , Integração Viral/genética , Integração Viral/fisiologia , Replicação Viral/efeitos dos fármacos
12.
BMC Infect Dis ; 19(1): 588, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277590

RESUMO

BACKGROUND: HIV controllers (HICs) are a rare group of HIV-1-infected individuals able to naturally control viral replication. Several studies have identified the occurrence of HIV dual infections in seropositive individuals leading to disease progression. In HICs, however, dual infections with divergent outcomes in pathogenesis have been described. CASE PRESENTATION: Here, we present a case report of a HIC diagnosed in late 1999 who displayed stable CD4+ T cell levels and low plasmatic viral load across 12 years of follow-up. In early 2013, the patient started to present an increase in viral load, reaching a peak of 10,000 copies/ml in early 2014, followed by an oscillation of viremia at moderate levels in the following years. The genetic diversity of env proviral quasispecies from peripheral blood mononuclear cells (PBMCs) was studied by single genome amplification (SGA) at six timepoints across 2009-2017. Phylogenetic analyses of env sequences from 2009 and 2010 samples showed the presence of a single subtype B variant (called B1). Analyses of sequences from 2011 and after revealed an additional subtype B variant (called B2) and a subsequent dominance shift in the proviral quasispecies frequencies, with the B2 variant becoming the most frequent from 2014 onwards. Latent syphilis related to unprotected sexual intercourse was diagnosed a year before the first detection of B2, evidencing risk behavior and supporting the superinfection hypothesis. Immunologic analyses revealed an increase in CD8+ and CD4+ T cell immune activation following viremia increase and minor T cell subset alterations during follow-up. HIV-specific T cell responses remained low throughout the follow-up period. CONCLUSIONS: Altogether, these results show that loss of viremia control in the HIC was associated with superinfection. These data alert to the negative consequences of reinfection on HIV pathogenesis, even in patients with a long history of viremia control and an absence of disease progression, reinforcing the need for continued use of adequate prevention strategies.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Superinfecção/virologia , Replicação Viral/fisiologia , Adulto , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Feminino , Seguimentos , Infecções por HIV/tratamento farmacológico , HIV-1/isolamento & purificação , HIV-1/patogenicidade , Antígenos HLA-B/genética , Humanos , Leucócitos Mononucleares/virologia , Masculino , Filogenia , RNA Viral/sangue , Sífilis/diagnóstico , Carga Viral , Viremia/tratamento farmacológico , Viremia/virologia
13.
Rev Soc Bras Med Trop ; 52: e20190101, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31340370

RESUMO

INTRODUCTION: Tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM) is a disease caused by human T-cell lymphotropic virus type 1 (HTLV-I) that mainly infects CD4 T cells-for example, those of the CD4+CD25hiFOXP3+ [Treg] phenotype-where it inhibits forkhead box protein P3 (FOXP3) expression and promotes interferon-γ (IFN-γ) expression. However, the role it exerts on regulatory B cells (CD19+CD24hiCD38hi; Breg) is unknown. METHODS: The frequencies of Treg and Breg cells was evaluated and the Th1 profiles were assessed in TSP/HAM patients and healthy control subjects. RESULTS: Low percentages of Breg cells and high production of IFN-γ were observed in patients compared to those in healthy control subjects. CONCLUSIONS: The low percentage of Breg cells in patients and the increase in the frequency of Th1 cells suggest an imbalance in the control of the inflammatory response that contributes to the immunopathogenesis of TSP/HAM.


Assuntos
Linfócitos B Reguladores/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Interferon gama/imunologia , Paraparesia Espástica Tropical/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Linfócitos B Reguladores/virologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Feminino , Humanos , Masculino , Paraparesia Espástica Tropical/virologia , Linfócitos T Reguladores/virologia , Carga Viral
14.
PLoS Pathog ; 15(6): e1007868, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31220191

RESUMO

We previously reported the presence of memory CD4+ T cells that express low levels of SAMHD1 (SAMHD1low) in peripheral blood and lymph nodes from both HIV-1 infected and uninfected individuals. These cells are enriched in Th17 and Tfh subsets, two populations known to be preferentially targeted by HIV-1. Here we investigated whether SAMHD1low CD4+ T-cells harbour replication-competent virus and compartimentalized HIV-1 genomes. We sorted memory CD4+CD45RO+SAMHD1low, CD4+CD45RO+SAMHD1+ and naive CD4+CD45RO-SAMHD1+ cells from HIV-1-infected patients on anti-retroviral therapy (c-ART) and performed HIV-1 DNA quantification, ultra-deep-sequencing of partial env (C2/V3) sequences and phenotypic characterization of the cells. We show that SAMHD1low cells include novel Th17 CCR6+ subsets that lack CXCR3 and CCR4 (CCR6+DN). There is a decrease of the % of Th17 in SAMHD1low compartment in infected compared to uninfected individuals (41% vs 55%, p<0.05), whereas the % of CCR6+DN increases (7.95% vs 3.8%, p<0.05). Moreover, in HIV-1 infected patients, memory SAMHD1low cells harbour high levels of HIV-1 DNA compared to memory SAMHD1+ cells (4.5 vs 3.8 log/106cells, respectively, p<0.001), while naïve SAMHD1+ showed significantly lower levels (3.1 log/106cells, p<0.0001). Importantly, we show that SAMHD1low cells contain p24-producing cells. Moreover, phylogenetic analyses revealed well-segregated HIV-1 DNA populations with compartmentalization between SAMHD1low and SAMHD1+ memory cells, and limited viral exchange. As expected, the % of Ki67+ cells was significantly higher in SAMHD1low compared to SAMHD1+ cells. There was positive association between levels of HIV-1 DNA and Ki67+ in memory SAMHD1low cells, but not in memory and naïve SAMHD1+ CD4+ T-cells. Altogether, these data suggest that proliferative memory SAMHD1low cells contribute to viral persistence.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Memória Imunológica , Proteína 1 com Domínio SAM e Domínio HD/imunologia , Adulto , Idoso , Antirretrovirais/administração & dosagem , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , Humanos , Masculino , Pessoa de Meia-Idade
15.
EBioMedicine ; 45: 624-629, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31227439

RESUMO

Latent HIV reservoir is the main obstacle that prevents a cure for HIV-1 (HIV). While antiretroviral therapy is effective in controlling viral replication, it cannot eliminate latent HIV reservoirs in patients. Several strategies have been proposed to combat HIV latency, including bone marrow transplantation to replace blood cells with CCR5-mutated stem cells, gene editing to disrupt the HIV genome, and "Shock and Kill" to reactivate latent HIV followed by an immune clearance. However, high risks and limitations to scale-up in clinics, off-target effects in human genomes or failure to reduce reservoir sizes in patients hampered our current efforts to achieve an HIV cure. This necessitates alternative strategies to control the latent HIV reservoirs. This review will discuss an emerging strategy aimed to deeply silence HIV reservoirs, the development of this concept, its potential and caveats for HIV remission/cure, and prospective directions for silencing the latent HIV, thereby preventing viruses from rebound.


Assuntos
Reservatórios de Doenças/virologia , Infecções por HIV/genética , HIV-1/genética , Latência Viral/genética , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/epidemiologia , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Receptores CCR5/genética , Ativação Viral/genética , Replicação Viral/genética
16.
mBio ; 10(3)2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213558

RESUMO

The HIV-1 accessory protein Vpu enhances viral release by counteracting the restriction factor BST-2. Furthermore, Vpu promotes NK cell evasion by downmodulating cell surface NTB-A and PVR, known ligands of the NK cell receptors NTB-A and DNAM-1, respectively. While it has been established that Vpu's transmembrane domain (TMD) is required for the interaction and intracellular sequestration of BST-2, NTB-A, and PVR, it remains unclear how Vpu manages to target these proteins simultaneously. In this study, we show that upon upregulation, BST-2 is preferentially downregulated by Vpu over its other TMD substrates. We found that type I interferon (IFN)-mediated BST-2 upregulation greatly impairs the ability of Vpu to downregulate NTB-A and PVR. Our results suggest that occupation of Vpu by BST-2 affects its ability to downregulate other TMD substrates. Accordingly, knockdown of BST-2 increases Vpu's potency to downmodulate NTB-A and PVR in the presence of type I IFN treatment. Moreover, we show that expression of human BST-2, but not that of the macaque orthologue, decreases Vpu's capacity to downregulate NTB-A. Importantly, we show that type I IFNs efficiently sensitize HIV-1-infected cells to NTB-A- and DNAM-1-mediated direct and antibody-dependent NK cell responses. Altogether, our results reveal that type I IFNs decrease Vpu's polyfunctionality, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses.IMPORTANCE The restriction factor BST-2 and the NK cell ligands NTB-A and PVR are among a growing list of membrane proteins found to be downregulated by HIV-1 Vpu. BST-2 antagonism enhances viral release, while NTB-A and PVR downmodulation contributes to NK cell evasion. However, it remains unclear how Vpu can target multiple cellular factors simultaneously. Here we provide evidence that under physiological conditions, BST-2 is preferentially targeted by Vpu over NTB-A and PVR. Specifically, we show that type I IFNs decrease Vpu's polyfunctionality by upregulating BST-2, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses. This indicates that there is a hierarchy of Vpu substrates upon IFN treatment, revealing that for the virus, targeting BST-2 as part of its resistance to IFN takes precedence over evading NK cell responses. This reveals a potential weakness in HIV-1's immunoevasion mechanisms that may be exploited therapeutically to harness NK cell responses against HIV-1.


Assuntos
Antígenos CD/genética , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Proteínas do Vírus da Imunodeficiência Humana/genética , Interferon Tipo I/farmacologia , Células Matadoras Naturais/imunologia , Proteínas Virais Reguladoras e Acessórias/genética , Linfócitos T CD4-Positivos/virologia , Regulação para Baixo , Proteínas Ligadas por GPI/genética , Células HEK293 , HIV-1 , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Humanos , Evasão da Resposta Imune , Receptores Virais/genética , Receptores Virais/imunologia , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia , Ativação Transcricional , Regulação para Cima , Proteínas Virais Reguladoras e Acessórias/imunologia
17.
Cell Host Microbe ; 26(1): 86-99.e7, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31253590

RESUMO

The Cullin-RING E3 ligase (CRL) family is commonly hijacked by pathogens to redirect the host ubiquitin proteasome machinery to specific targets. During HIV infection, CRL5 is hijacked by HIV Vif to target viral restriction factors of the APOBEC3 family for ubiquitination and degradation. Here, using a quantitative proteomics approach, we identify the E3 ligase ARIH2 as a regulator of CRL5-mediated APOBEC3 degradation. The CUL5Vif/CBFß complex recruits ARIH2 where it acts to transfer ubiquitin directly to the APOBEC3 targets. ARIH2 is essential for CRL5-dependent HIV infectivity in primary CD4+ T cells. Furthermore, we show that ARIH2 cooperates with CRL5 to prime other cellular substrates for polyubiquitination, suggesting this may represent a general mechanism beyond HIV infection and APOBEC3 degradation. Taken together, these data identify ARIH2 as a co-factor in the Vif-hijacked CRL5 complex that contributes to HIV infectivity and demonstrate the operation of the E1-E2-E3/E3-substrate ubiquitination mechanism in a viral infection context.


Assuntos
Desaminase APOBEC-3G/metabolismo , Proteínas Culina/metabolismo , Infecções por HIV/patologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Ubiquitina-Proteína Ligases/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Humanos , Modelos Teóricos , Proteólise , Proteoma/análise , Replicação Viral
18.
Nat Immunol ; 20(7): 824-834, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209403

RESUMO

Multiple genome-wide studies have identified associations between outcome of human immunodeficiency virus (HIV) infection and polymorphisms in and around the gene encoding the HIV co-receptor CCR5, but the functional basis for the strongest of these associations, rs1015164A/G, is unknown. We found that rs1015164 marks variation in an activating transcription factor 1 binding site that controls expression of the antisense long noncoding RNA (lncRNA) CCR5AS. Knockdown or enhancement of CCR5AS expression resulted in a corresponding change in CCR5 expression on CD4+ T cells. CCR5AS interfered with interactions between the RNA-binding protein Raly and the CCR5 3' untranslated region, protecting CCR5 messenger RNA from Raly-mediated degradation. Reduction in CCR5 expression through inhibition of CCR5AS diminished infection of CD4+ T cells with CCR5-tropic HIV in vitro. These data represent a rare determination of the functional importance of a genome-wide disease association where expression of a lncRNA affects HIV infection and disease progression.


Assuntos
Regulação da Expressão Gênica , Variação Genética , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1 , RNA Antissenso/genética , RNA Longo não Codificante/genética , Receptores CCR5/genética , Regiões 3' não Traduzidas , Alelos , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Membrana Celular/metabolismo , Genes Reporter , Genótipo , Infecções por HIV/metabolismo , Humanos , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único , Grupos Populacionais/genética , Prognóstico , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR5/metabolismo , Carga Viral
19.
PLoS One ; 14(6): e0217502, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31206536

RESUMO

BACKGROUND: Residual HIV-1 replication among individuals under antiretroviral therapy (ART) relates to HIV micro-inflammation. OBJECTIVES: To determine the levels of residual HIV replication markers among distinct subgroups of antiretroviral-treated individuals. METHODS: One hundred sixteen patients were distributed into 5 treatment groups: first-line suppressive ART with a non-nucleoside analog reverse-transcriptase inhibitor (NNRTI) (n = 26), first-line suppressive ART with boosted protease inhibitors (PI-r) (n = 25), salvage therapy using PI-r (n = 27), salvage therapy with PI-r and raltegravir (n = 22) and virologic failure (n = 16). Episomal and total DNA quantitation was evaluated. ELISA was used for HIV antibody and LPS quantitation. RESULTS: Episomal DNA was positive in 26% to 38% of individuals under suppressive ART, and it was higher among individuals experiencing ART virologic failure (p = 0.04). The HIV proviral load was higher among patients with detectable episomal DNA (p = 0.01). Individuals receiving initial PI-r treatment presented lower HIV antibody (p = 0.027) and LPS (p = 0.029) levels than individuals receiving NNRTI. There was a negative correlation between episomal DNA quantitation and the duration of suppressive ART (p = 0.04), CD4+ T-cell count (p = 0.08), and CD8+ T-cell count (p = 0.07). CONCLUSIONS: Residual HIV replication has been inferred among individuals under suppressive ART according to episomal DNA detection. Residual replication may decrease with longer periods of suppressive ART and higher levels of CD4+ and CD8+ T cells. The relationship between episomal DNA and total DNA suggests there is a replenishment of the proviral reservoir with impacts on HIV persistence. Lower antibody and LPS levels among patients with initial PI-r ART suggest these regimens may more effectively suppress HIV and have a higher capacity to decrease the HIV antigenic component.


Assuntos
Anticorpos Anti-HIV/sangue , Infecções por HIV , Inibidores da Protease de HIV/administração & dosagem , HIV-1/fisiologia , Plasmídeos/metabolismo , Inibidores da Transcriptase Reversa/administração & dosagem , Replicação Viral/efeitos dos fármacos , Biomarcadores/sangue , Brasil , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Feminino , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Humanos , Lipopolissacarídeos/farmacologia , Masculino
20.
Cell Host Microbe ; 25(6): 858-872.e13, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31175045

RESUMO

The interferon γ-inducible protein 16 (IFI16) is known as immune sensor of retroviral DNA intermediates. We show that IFI16 restricts HIV-1 independently of immune sensing by binding and inhibiting the host transcription factor Sp1 that drives viral gene expression. This antiretroviral activity and ability to bind Sp1 require the N-terminal pyrin domain and nuclear localization of IFI16, but not the HIN domains involved in DNA binding. Highly prevalent clade C HIV-1 strains are more resistant to IFI16 and less dependent on Sp1 than other HIV-1 subtypes. Furthermore, inhibition of Sp1 by IFI16 or pharmacologically by Mithramycin A suppresses reactivation of latent HIV-1 in CD4+ T cells. Finally, IFI16 also inhibits retrotransposition of LINE-1, known to engage Sp1, and murine IFI16 homologs restrict Friend retrovirus replication in mice. Thus, IFI16 restricts retroviruses and retrotransposons by interfering with Sp1-dependent gene expression, and evasion from this restriction may facilitate spread of HIV-1 subtype C.


Assuntos
HIV-1/imunologia , Fatores Imunológicos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fator de Transcrição Sp1/antagonistas & inibidores , Transcrição Genética , Ativação Viral , Latência Viral , Animais , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Genótipo , HIV-1/classificação , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Camundongos
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