RESUMO
Signaling pathways play a key role in HIV-1 latency. In this study, we used the 24ST1NLESG cell line of HIV-1 latency to screen a library of structurally diverse, medicinally active, cell permeable kinase inhibitors, which target a wide range of signaling pathways, to identify inhibitors of HIV-1 latency reversal. The screen was carried out in the absence or presence of three mechanistically distinct latency-reversing agents (LRAs), namely, prostratin, panobinostat, and JQ-1. We identified inhibitors that only blocked the activity of a specific LRA, as well as inhibitors that blocked the activity of all LRAs. For example, we identified 12 inhibitors targeted toward protein kinase C or downstream kinases that blocked the activity of prostratin. We also identified 12 kinase inhibitors that blocked the reversal of HIV-1 latency irrespective of the LRA used in the screen. Of these, danusertib, an Aurora kinase inhibitor, and PF-3758309, a PAK4 inhibitor, were the most potent. The 50% inhibitory concentrations in the 24ST1NLESG cells ranged from 40 to 147 nM for danusertib (selectivity indices, >150) and from 0.1 to 1 nM for PF-3758309 (selectivity indices, >3,300). Both danusertib and PF-3758309 inhibited latency reversal in CD4+ T cells isolated from HIV-1-infected donors. Collectively, our study describes a chemical approach that can be applied to elucidate the role of signaling pathways involved in LRA activity or the maintenance of HIV-1 latency and also identifies inhibitors of latent HIV-1 reactivation that could be used with antiretroviral therapy to reduce residual viremia.
Assuntos
Fármacos Anti-HIV/uso terapêutico , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Benzamidas/uso terapêutico , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Infecções por HIV/virologia , Humanos , Pirazóis/uso terapêutico , Pirróis/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Ativação Viral/efeitos dos fármacosRESUMO
Although antiretroviral therapy can suppress HIV-1 infection to undetectable levels of plasma viremia, integrated latent HIV-1 genomes that encode replication-competent virus persist in resting CD4+ T cells. This latent HIV-1 reservoir represents a major barrier to a cure. Currently, there are substantial efforts to identify therapeutic approaches that will eliminate or reduce the size of this latent HIV-1 reservoir. In this regard, a sensitive assay that can accurately and rapidly quantify inducible, replication-competent latent HIV-1 from resting CD4+ T cells is essential for HIV-1 eradication studies. Here we describe a reporter cell-based assay to quantify inducible, replication-competent latent HIV-1. This assay has several advantages over existing technology in that it (i) is sensitive; (ii) requires only a small blood volume; (iii) is faster, less labor intensive, and less expensive; and (iv) can be readily adapted into a high-throughput format. Using this assay, we show that the size of the inducible latent HIV-1 reservoir in aviremic participants on therapy is approximately 70-fold larger than previous estimates.
Assuntos
Linfócitos T CD4-Positivos/virologia , DNA Viral/análise , Infecções por HIV/virologia , HIV-1/genética , RNA Viral/análise , Carga Viral/métodos , Adulto , Idoso , Fármacos Anti-HIV/uso terapêutico , Feminino , Proteínas de Fusão gag-pol/genética , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Latência ViralRESUMO
OBJECTIVE: HIV-1 viral proteins and host inflammatory factors have a direct role in neuronal toxicity in vitro; however, the contribution of these factors in vivo in HIV-1-associated neurocognitive disorder (HAND) is not fully understood. We applied novel Systems Biology approaches to identify specific cellular and viral factors and their related pathways that are associated with different stages of HAND. DESIGN: A cross-sectional study of individuals enrolled in the Multicenter AIDS Cohort Study including HIV-1-seronegative (Nâ=â36) and HIV-1-seropositive individuals without neurocognitive symptoms (Nâ=â16) or with mild neurocognitive disorder (MND) (Nâ=â8) or HIV-associated dementia (HAD) (Nâ=â16). METHODS: A systematic evaluation of global transcriptome of peripheral blood mononuclear cells (PBMCs) obtained from HIV-1-seronegative individuals and from HIV-1-positive men without neurocognitive symptoms, or MND or HAD was performed. RESULTS: MND and HAD were associated with specific changes in mRNA transcripts and microRNAs in PBMCs. Comparison of upstream regulators and TimePath analyses identified specific cellular factors associated with MND and HAD, whereas HIV-1 viral proteins played a greater role in HAD. In addition, expression of specific microRNAs - miR-let-7a, miR-124, miR-15a and others - were found to correlate with mRNA gene expression and may have a potential protective role in asymptomatic HIV-1-seropositive individuals by regulating cellular signal transduction pathways downstream of chemokines and cytokines. CONCLUSION: These results identify signature transcriptome changes in PBMCs associated with stages of HAND and shed light on the potential contribution of host cellular factors and viral proteins in HAND development.
Assuntos
Complexo AIDS Demência/fisiopatologia , Perfilação da Expressão Gênica , Infecções por HIV/complicações , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno , Leucócitos Mononucleares/fisiologia , Leucócitos Mononucleares/virologia , Células Cultivadas , Estudos Transversais , Redes Reguladoras de Genes , Humanos , Masculino , Biologia de Sistemas/métodosRESUMO
MOTIVATION: Most methods for reconstructing response networks from high throughput data generate static models which cannot distinguish between early and late response stages. RESULTS: We present TimePath, a new method that integrates time series and static datasets to reconstruct dynamic models of host response to stimulus. TimePath uses an Integer Programming formulation to select a subset of pathways that, together, explain the observed dynamic responses. Applying TimePath to study human response to HIV-1 led to accurate reconstruction of several known regulatory and signaling pathways and to novel mechanistic insights. We experimentally validated several of TimePaths' predictions highlighting the usefulness of temporal models. AVAILABILITY AND IMPLEMENTATION: Data, Supplementary text and the TimePath software are available from http://sb.cs.cmu.edu/timepath CONTACT: zivbj@cs.cmu.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
HIV-1 , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Modelos Teóricos , SoftwareRESUMO
BACKGROUND: Latent HIV-1 reservoirs are identified as one of the major challenges to achieve HIV-1 cure. Currently available strategies are associated with wide variability in outcomes both in patients and CD4(+) T cell models. This underlines the critical need to develop innovative strategies to predict and recognize ways that could result in better reactivation and eventual elimination of latent HIV-1 reservoirs. RESULTS AND DISCUSSION: In this study, we combined genome wide transcriptome datasets post activation with Systems Biology approach (Signaling and Dynamic Regulatory Events Miner, SDREM analyses) to reconstruct a dynamic signaling and regulatory network involved in reactivation mediated by specific activators using a latent cell line. This approach identified several critical regulators for each treatment, which were confirmed in follow-up validation studies using small molecule inhibitors. Results indicate that signaling pathways involving JNK and related factors as predicted by SDREM are essential for virus reactivation by suberoylanilide hydroxamic acid. ERK1/2 and NF-κB pathways have the foremost role in reactivation with prostratin and TNF-α, respectively. JAK-STAT pathway has a central role in HIV-1 transcription. Additional evaluation, using other latent J-Lat cell clones and primary T cell model, also confirmed that many of the cellular factors associated with latency reversing agents are similar, though minor differences are identified. JAK-STAT and NF-κB related pathways are critical for reversal of HIV-1 latency in primary resting T cells. CONCLUSION: These results validate our combinatorial approach to predict the regulatory cellular factors and pathways responsible for HIV-1 reactivation in latent HIV-1 harboring cell line models. JAK-STAT have a role in reversal of latency in all the HIV-1 latency models tested, including primary CD4(+) T cells, with additional cellular pathways such as NF-κB, JNK and ERK 1/2 that may have complementary role in reversal of HIV-1 latency.
Assuntos
HIV-1/fisiologia , Ativação Viral/efeitos dos fármacos , Ativação Viral/genética , Latência Viral/genética , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/virologia , Perfilação da Expressão Gênica/métodos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Células Jurkat , Masculino , Ésteres de Forbol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Biologia de Sistemas/métodos , Fator de Necrose Tumoral alfa , Latência Viral/efeitos dos fármacos , VorinostatRESUMO
Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus recently isolated from human prostate cancer and peripheral blood mononuclear cells (PBMCs) of patients with chronic fatigue syndrome (CFS). We and others have shown that host restriction factors APOBEC3G (A3G) and APOBEC3F (A3F), which are expressed in human PBMCs, inhibit XMRV in transient-transfection assays involving a single cycle of viral replication. However, the recovery of infectious XMRV from human PBMCs suggested that XMRV can replicate in these cells despite the expression of APOBEC3 proteins. To determine whether XMRV can replicate and spread in cultured PBMCs even though it can be inhibited by A3G/A3F, we infected phytohemagglutinin-activated human PBMCs and A3G/A3F-positive and -negative cell lines (CEM and CEM-SS, respectively) with different amounts of XMRV and monitored virus production by using quantitative real-time PCR. We found that XMRV efficiently replicated in CEM-SS cells and viral production increased by >4,000-fold, but there was only a modest increase in viral production from CEM cells (<14-fold) and a decrease in activated PBMCs, indicating little or no replication and spread of XMRV. However, infectious XMRV could be recovered from the infected PBMCs by cocultivation with a canine indicator cell line, and we observed hypermutation of XMRV genomes in PBMCs. Thus, PBMCs can potentially act as a source of infectious XMRV for spread to cells that express low levels of host restriction factors. Overall, these results suggest that hypermutation of XMRV in human PBMCs constitutes one of the blocks to replication and spread of XMRV. Furthermore, hypermutation of XMRV proviruses at GG dinucleotides may be a useful and reliable indicator of human PBMC infection.
Assuntos
Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Replicação Viral , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/imunologia , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/patogenicidade , Desaminase APOBEC-3G , Células Cultivadas , Citidina Desaminase/genética , Citidina Desaminase/imunologia , Citosina Desaminase/genética , Citosina Desaminase/imunologia , HumanosRESUMO
Recent studies have shown that APOBEC3G (A3G), a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is localized to cytoplasmic mRNA-processing bodies (P bodies). However, the functional relevance of A3G colocalization with P body marker proteins has not been established. To explore the relationship between HIV-1, A3G, and P bodies, we analyzed the effects of overexpression of P body marker proteins Mov10, DCP1a, and DCP2 on HIV-1 replication. Our results show that overexpression of Mov10, a putative RNA helicase that was previously reported to belong to the DExD superfamily and was recently reported to belong to the Upf1-like group of helicases, but not the decapping enzymes DCP1a and DCP2, leads to potent inhibition of HIV-1 replication at multiple stages. Mov10 overexpression in the virus producer cells resulted in reductions in the steady-state levels of the HIV-1 Gag protein and virus production; Mov10 was efficiently incorporated into virions and reduced virus infectivity, in part by inhibiting reverse transcription. In addition, A3G and Mov10 overexpression reduced proteolytic processing of HIV-1 Gag. The inhibitory effects of A3G and Mov10 were additive, implying a lack of functional interaction between the two inhibitors. Small interfering RNA (siRNA)-mediated knockdown of endogenous Mov10 by 80% resulted in a 2-fold reduction in virus production but no discernible impact on the infectivity of the viruses after normalization for the p24 input, suggesting that endogenous Mov10 was not required for viral infectivity. Overall, these results show that Mov10 can potently inhibit HIV-1 replication at multiple stages.
Assuntos
HIV-1/fisiologia , RNA Helicases/fisiologia , Replicação Viral/fisiologia , Desaminase APOBEC-3G , Linhagem Celular , Citidina Desaminase/fisiologia , Estruturas Citoplasmáticas/fisiologia , Estruturas Citoplasmáticas/virologia , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , HIV-1/genética , HIV-1/patogenicidade , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Processamento de Proteína Pós-Traducional , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Viral/genética , RNA Viral/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
HIV-1 Vpr, a nonstructural viral protein associated with virus particles, has a positive role in the efficient transport of PIC into the nucleus of non-dividing target cells and enhances virus replication in primary T cells. Vpr is a 96 amino acid protein and the structure by NMR shows three helical domains. Vpr has been shown to exist as dimers and higher order oligomers. Considering the multifunctional nature of Vpr, the contribution of distinct helical domains to the dimer/oligomer structure of Vpr and the relevance of this feature to its functions are not clear. To address this, we have utilized molecular modeling approaches to identify putative models of oligomerization. The predicted interface residues were subjected to site-directed mutagenesis and evaluated their role in intermolecular interaction and virion incorporation. The interaction between Vpr molecules was monitored by Bimolecular Fluorescence complementation (BiFC) method. The results show that Vpr forms oligomers in live cells and residues in helical domains play critical roles in oligomerization. Interestingly, Vpr molecules defective in oligomerization also fail to incorporate into the virus particles. Based on the data, we suggest that oligomerization of Vpr is essential for virion incorporation property and may also have a role in the events associated with virus infection.
Assuntos
HIV-1/metabolismo , Vírion/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/química , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Linhagem Celular , HIV-1/química , HIV-1/genética , Humanos , Conformação Molecular , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Vírion/química , Vírion/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genéticaRESUMO
Xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus, has been isolated from human prostate cancer tissue and from activated CD4(+) T cells and B cells of patients with chronic fatigue syndrome, suggesting an association between XMRV infection and these two diseases. Since APOBEC3G (A3G) and APOBEC3F (A3F), which are potent inhibitors of murine leukemia virus and Vif-deficient human immunodeficiency virus type 1 (HIV-1), are expressed in human CD4(+) T cells and B cells, we sought to determine how XMRV evades suppression of replication by APOBEC3 proteins. We found that expression of A3G, A3F, or murine A3 in virus-producing cells resulted in their virion incorporation, inhibition of XMRV replication, and G-to-A hypermutation of the viral DNA with all three APOBEC3 proteins. Quantitation of A3G and A3F mRNAs indicated that, compared to the human T-cell lines CEM and H9, prostate cell lines LNCaP and DU145 exhibited 50% lower A3F mRNA levels, whereas A3G expression in 22Rv1, LNCaP, and DU145 cells was nearly undetectable. XMRV proviral genomes in LNCaP and DU145 cells were hypermutated at low frequency with mutation patterns consistent with A3F activity. XMRV proviral genomes were extensively hypermutated upon replication in A3G/A3F-positive T cells (CEM and H9), but not in A3G/A3F-negative cells (CEM-SS). We also observed that XMRV replication was susceptible to the nucleoside reverse transcriptase (RT) inhibitors zidovudine (AZT) and tenofovir and the integrase inhibitor raltegravir. In summary, the establishment of XMRV infection in patients may be dependent on infection of A3G/A3F-deficient cells, and cells expressing low levels of A3G/A3F, such as prostate cancer cells, may be ideal producers of infectious XMRV. Furthermore, the anti-HIV-1 drugs AZT, tenofovir, and raltegravir may be useful for treatment of XMRV infection.
Assuntos
Antivirais/farmacologia , Citidina Desaminase/imunologia , Gammaretrovirus/imunologia , Vírus da Leucemia Murina/imunologia , Neoplasias da Próstata/virologia , Infecções por Retroviridae/imunologia , Infecções Tumorais por Vírus/imunologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Linhagem Celular Tumoral , Citidina Desaminase/genética , Humanos , Masculino , Camundongos , Mutação , Organofosfonatos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Pirrolidinonas/farmacologia , Raltegravir Potássico , Infecções por Retroviridae/tratamento farmacológico , Tenofovir , Infecções Tumorais por Vírus/tratamento farmacológico , Zidovudina/farmacologiaRESUMO
Dendritic cells (DC) play a pivotal role in transmission and dissemination of HIV-1. Earlier studies reported that DC present at the site of infection trap virus particles via DC-SIGN and transfer the virus to the interacting naïve T cells. This prompted us to ask the question whether DC could acquire virus from infected T cells during DC-T cell interaction. To address this, we investigated the likely transfer of virus from HIV-1 infected T cells to DC and the underlying mechanisms involved. Results indicate that DC acquire virus from infected T cells via antigen uptake mechanism and this results in infection of DC with expression of proteins directed by viral DNA. Further studies with HIV-1 lacking the Env protein also resulted in infection of DC. The use of antibodies against DC-SIGN and DC-SIGN-R ruled out a role for receptor in the infection of DC. Additional data show that DC infection is directly correlated with the ability of DC to take up antigen from infected T cells. Overall, these studies provide evidence to suggest that HIV-1, besides infecting immune cells, also utilizes immunological mechanism(s) to acquire and disseminate virus.
Assuntos
Células Dendríticas/virologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/metabolismo , Linfócitos T/virologia , Linfócitos T CD4-Positivos/virologia , Moléculas de Adesão Celular/metabolismo , Proteínas de Fluorescência Verde/química , Humanos , Células Jurkat , Lectinas Tipo C/metabolismo , Leucócitos Mononucleares/citologia , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Microscopia de Fluorescência/métodos , Fenótipo , Plasmídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Linfócitos T/metabolismoRESUMO
HIV-1 exploits the cellular machinery to replicate in the host cells by targeting a wide range of host factors at different stages of its life cycle. HIV-1 also induces detrimental effects in the infected and uninfected bystander cells resulting in dysregulation including interference in immune effector functions. The latter is specifically linked to the immune evasion strategies of the virus. In addition to the essential roles of structural proteins (Gag, Pol & Env), HIV-1 encoded auxiliary proteins such as Nef, Vif, Vpu, and Vpr through their interaction with the host cellular partners facilitate viral replication and dissemination. HIV-1 Vpr, a virion-associated molecule, has been implicated to play a role in the early events in virus life cycle. Vpr is a pleiotropic protein that exerts a range of effects including inhibition of cell proliferation, induction of apoptosis and modulation of a number of immune molecules. These functions could be in part responsible for Vpr induced immune evasion and virus replication. Appreciating this view is the genetic variation in vpr gene reflected in the form of polymorphisms at the amino acid level that may contribute to the potential CTL escape of the virus. It is likely that Vpr mediated dysregulation of host immune response contributes, in part, to the progression of disease. This review focuses on the recent advances regarding HIV-1 Vpr mediated immunopathogenesis and the mechanistic insight from in vitro and in vivo studies.
Assuntos
HIV-1/patogenicidade , Fatores de Virulência/fisiologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/fisiologia , HIV-1/imunologia , Humanos , Tolerância ImunológicaRESUMO
Programmed Death-1 (PD-1), a member of T cell costimulatory molecules is expressed in high levels on antigen specific T cells during chronic viral infection, whereas PD-1 expression in the context of HIV-1 infected CD4+ T cells is not known. Here we report that productively infected CD4+ T cells lose PD-1, whereas bystander cells were unaffected. Additionally, p24+/PD-1 negative cells are less susceptible to apoptosis compared to bystander cells in the same infected milieu. Similar results were observed in vivo, as infected T cells isolated from HIV-1+ individuals have significantly low level of PD-1 and the observed loss of PD-1 in vivo is independent of viral load, CD4 count, and/or antiviral treatment. Together these results indicate that productively infected cells are resistant to early apoptosis by downregulating PD-1, whereas PD-1 enhances the susceptibility of effector T cells to apoptosis suggesting a dual role for PD-1 during HIV-1 infection.
Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Apoptose , Linfócitos T CD4-Positivos/virologia , HIV-1/imunologia , Antirretrovirais/uso terapêutico , Antígenos CD , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/química , Proteína do Núcleo p24 do HIV/análise , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Humanos , Receptor de Morte Celular Programada 1 , Carga ViralRESUMO
Human immunodeficiency virus type 1 (HIV-1) infection has been implicated in impairing various aspects of NK cell function in viremic condition, and several viral factors contribute to these defects. Here, we evaluated the effect of HIV-1 Vpr on NK cell cytolytic function and cytokine (gamma interferon [IFN-gamma]) production in the context of infection and exposure. Our data indicate that NK cells derived from a peripheral blood mononuclear cell culture infected in vitro with HIV-1 vpr(+) virus or exposed to recombinant Vpr protein exhibited reduced target cell killing in conjunction with diminished expression of CD107a and reduced IFN-gamma production compared to their Vpr-negative counterparts. This Vpr-induced NK cell defect is in part through differential regulation of interleukin-12 and transforming growth factor beta production by the infected target cells and concomitant activation of Smad3 signaling pathway. Collectively, these results illustrate the ability of Vpr to impair NK cell-mediated innate immune functions indirectly by dysregulating multiple cytokines in the infected target cells, thus increasing disease severity and affecting the final outcome in HIV-1 infection.
Assuntos
Citocinas/biossíntese , HIV-1/imunologia , Células Matadoras Naturais/imunologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/imunologia , Células Cultivadas , Citocinas/imunologia , Testes Imunológicos de Citotoxicidade , Humanos , Células Matadoras Naturais/virologia , Leucócitos Mononucleares/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/biossínteseRESUMO
Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) plays a crucial role in viral replication and pathogenesis by inducing cell cycle arrest, apoptosis, translocation of preintegration complex, potentiation of glucocorticoid action, impairment of dendritic cell (DC) maturation, and T-cell activation. Recent studies involving the direct effects of Vpr on DCs and T cells indicated that HIV-1 containing Vpr selectively impairs phenotypic maturation, cytokine network, and antigen presentation in DCs and dysregulates costimulatory molecules and cytokine production in T cells. Here, we have further investigated the indirect effect of HIV-1 Vpr(+) virus-infected DCs on the bystander CD8(+) T-cell population. Our results indicate that HIV-1 Vpr(+) virus-infected DCs dysregulate CD8(+) T-cell proliferation and induce apoptosis. Vpr-containing virus-infected DC-mediated CD8(+) T-cell killing occurred in part through enhanced tumor necrosis factor alpha production by infected DCs and subsequent induction of death receptor signaling and activation of the caspase 8-dependent pathway in CD8(+) T cells. Collectively, these results provide evidence that Vpr could be one of the important contributors to the host immune escape by HIV-1 through its ability to dysregulate both directly and indirectly the DC biology and T-cell functions.
Assuntos
Apoptose/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , HIV-1/fisiologia , Linfócitos T/citologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , Células Cultivadas , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Human immunodeficiency virus type 1 (HIV-1) viral proteins disrupt the normal host cellular immune pathways thus exploiting the cellular machinery for replication, survival and to escape host immune attack. Here we evaluated the direct effects of HIV-1 Vpr-mediated immune modulation of infected T cells. Vpr specifically downregulated the expression of CD28 and increased the expression of CTLA-4, whereas no significant difference in the expression of CD25 and HLA-DR was observed. Interferon gamma (IFN-gamma) production in T cells was evaluated as a measure of the downstream effector functions. Results indicate that Vpr significantly inhibited IFN-gamma production and this may, in part, due to Vpr's ability to inhibit the nuclear translocation of NF-kappaB, and its transcriptional regulation. Together these results support that HIV-1 Vpr selectively dysregulates the immune functions at multiple levels and exerts its inhibitory effects in the presence of other viral proteins.
Assuntos
Produtos do Gene vpr/imunologia , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1 , Linfócitos T/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Antígenos CD28/metabolismo , Antígeno CTLA-4 , Núcleo Celular/metabolismo , Células Cultivadas , Humanos , Interferon gama/biossíntese , Leucócitos Mononucleares , Ativação Linfocitária , NF-kappa B/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
The HIV-1 viral protein R, Vpr, increases virus replication in T cells and is necessary for the optimal infection of primary monocytes/macrophages and other non-dividing cells. Vpr interacts with the cellular glucocorticoid receptor (GR) and transactivates the HIV-1 LTR through glucocorticoid response element (GRE), an event that can be blocked by the GR antagonist, mifepristone. Results demonstrated that Vpr-induced transactivation of the HIV-1 LTR was inhibited by mifepristone in a dose-dependent manner by >60% at a 10 microM concentration. Infectivity assays using X4 and R5 viruses demonstrated antiviral effects on a dose-dependent regimen of mifepristone. The effects of mifepristone were also tested in latently infected cells that could be activated with extracellular Vpr protein and results indicated specific inhibition of virus reactivation in the presence of this antagonist.
Assuntos
Fármacos Anti-HIV/farmacologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Produtos do Gene vpr/metabolismo , HIV-1/efeitos dos fármacos , Mifepristona/farmacologia , Receptores de Glucocorticoides/metabolismo , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Proteína do Núcleo p24 do HIV/biossíntese , Repetição Terminal Longa de HIV/genética , Repetição Terminal Longa de HIV/fisiologia , HIV-1/fisiologia , Células HeLa , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Regiões Promotoras Genéticas , Ativação Transcricional , Ativação Viral , Latência Viral , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Induction of the 17-kDa ubiquitin-like protein ISG15/UCRP and its subsequent conjugation to cellular targets is the earliest response to type I interferons. The polypeptide is synthesized as a precursor containing a carboxyl-terminal extension whose correct processing is required for subsequent ligation of the exposed mature carboxyl terminus. Recombinant pro-ISG15 is processed in extracts of human lung fibroblasts by a constitutive 100-kDa enzyme whose activity is unaffected by type I interferon stimulation. The processing enzyme has been purified to apparent homogeneity by a combination of ion exchange and hydrophobic chromatography and found to be stimulated 12-fold by micromolar concentrations of ubiquitin. Analysis of the products of pro-ISG15 processing enzyme demonstrates specific cleavage exclusively at the Gly(157)-Gly(158) peptide bond to generate a mature ISG15 carboxyl terminus. Irreversible inhibition of pro-ISG15 processing activity by thiol-specific alkylating agents and a pH rate dependence conforming to titration of a single group of pK(a) 8.1 indicate the 100-kDa enzyme is a thiol protease. Partial sequencing of a trypsin-derived peptide indicates the enzyme is either the human ortholog of yeast Ubp1 or a Ubp1-related protein. As yeast do not contain ISG15, these results suggest that a ubiquitin-specific enzyme was recruited for pro-ISG15/UCRP processing by adaptive divergence.
Assuntos
Cisteína Endopeptidases/química , Citocinas , Interferon beta/farmacologia , Precursores de Proteínas/metabolismo , Ubiquitinas/análogos & derivados , Endopeptidases/química , Inibidores Enzimáticos/farmacologia , Fibroblastos , Humanos , Concentração de Íons de Hidrogênio , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Análise de Sequência , Especificidade por Substrato , Células Tumorais Cultivadas , Ubiquitinas/metabolismoRESUMO
Herpes simplex virus-type 1 (HSV-1) encodes both the small (UL40) and large (UL39) subunits of the enzyme, ribonucleotide reductase. Treatment of HSV-1-infected cells with interferon-alpha (IFN-alpha) reduced the levels of both enzyme subunits. Reduced steady state levels of the large subunit were demonstrated by immunoblot using polyclonal antibody specific for the viral enzyme. Reduction in the amount of small subunit was shown by a reduction in the electron spin resonance signal derived from the iron-containing tyrosyl free-radical present in this subunit. Treatment of cells with 100 IU/ml of IFN-alpha decreased levels of both subunits resulting in a reduction in enzyme activity as measured by conversion of CDP to dCDP. The decrease in the amount of the large subunit was not due to a reduction in the level of its mRNA. The combination of IFN-alpha and ACV treatment of human cornea stromal cells did not result in a further reduction in amounts of ribonucleotide reductase relative to that detected with IFN-alpha alone. The IFN-alpha-induced reduction in ribonucleotide reductase activity is the likely cause of decreased levels of dGTP which we have previously demonstrated in IFN-alpha-treated, infected cells.
Assuntos
Aciclovir/farmacologia , Antivirais/farmacologia , Herpesvirus Humano 1/enzimologia , Interferon-alfa/farmacologia , Ribonucleotídeo Redutases/metabolismo , Animais , Northern Blotting , Western Blotting , Células Cultivadas , Chlorocebus aethiops , Córnea , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/metabolismo , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Interferon alfa-2 , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas Recombinantes , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/genética , Células Estromais , Tirosina/análogos & derivados , Tirosina/metabolismo , Células VeroRESUMO
The biological effect of type 1 interferons is proposed to arise in part from the conjugation of ubiquitin cross-reactive protein (UCRP), the ISG15 gene product, to intracellular target proteins in a process analogous to that of its sequence homolog ubiquitin, a highly conserved 8.6-kDa polypeptide whose ligation marks proteins for degradation via the 26 S proteasome. Inclusion of CoCl2 during the purification of recombinant UCRP blocks the proteolytic inactivation of the polypeptide occurring by cleavage of the carboxyl-terminal glycine dipeptide required for activation and subsequent ligation. Intact UCRP supports a low rate of ubiquitin-activating enzyme (E1)-dependent ATP:PPi exchange but fails to form a stoichiometric E1-UCRP thiol ester or undergo transfer to ubiquitin carrier protein (E2). The binding affinity of E1 for UCRP is significantly diminished relative to that of ubiquitin. These results suggest that UCRP conjugation proceeds through an enzyme pathway distinct from that of ubiquitin, at least with respect to the step of activation. This was confirmed for an in vitro conjugation assay in which 125I-UCRP could be ligated in an ATP-dependent reaction to proteins present within an A549 human lung carcinoma cell extract and could be competitively inhibited by excess unlabeled UCRP but not ubiquitin. Other results demonstrate that 125I-UCRP conjugation is significantly increased in cell extracts after 24 h of incubation in the presence of interferon-beta, consistent with the late induction of UCRP conjugating activity. Thus, interferon-responsive cells contain a pathway for UCRP ligation that is parallel but distinct from that of ubiquitin.
Assuntos
Interferons/fisiologia , Ubiquitinas/metabolismo , Trifosfato de Adenosina/metabolismo , Catálise , Ésteres/metabolismo , Humanos , Hidrólise , Pirofosfatase Inorgânica , Cinética , Pirofosfatases/metabolismo , Compostos de Sulfidrila/metabolismo , Células Tumorais Cultivadas , Ubiquitinas/biossíntese , Ubiquitinas/genéticaRESUMO
Gallium, a pharmacologically important metal which resembles iron, was shown in previous studies to inhibit ribonucleotide reductase. To better understand its mechanism of action, we have examined the interaction of gallium with the iron-dependent M2 subunit of ribonucleotide reductase. In its active form, M2 contains an iron center and a tyrosyl free radical which is detectable by ESR spectroscopy. In the present study, cytoplasmic extracts prepared from murine leukemic L1210 cells after an 18-hr incubation with 960 microM gallium nitrate displayed a > 60% inhibition in their M2 tyrosyl radical ESR signal. However, this signal was restored within 15 min to levels greater than that of controls by the addition of increasing concentrations of ferrous ammonium sulfate. Gallium citrate added directly to cytoplasmic extracts from control cells also decreased the tyrosyl radical signal, an effect which could be reversed by iron. Immunoblot analysis revealed that incubation with gallium did not diminish the amount of M2 protein in cells, thus indicating that the decrease in the tyrosyl radical signal was not due to a decrease in cellular M2 content. In immunoprecipitation studies of 59Fe-labeled M2, gallium displaced 55-60% of the 59Fe incorporated into M2. Our studies suggest that gallium displaces iron from the M2 subunit of ribonucleotide reductase, resulting in a loss of the tyrosyl radical and an accumulation of inactive M2 within the cell.