RESUMO
Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.
Assuntos
HIV-1/imunologia , Evasão da Resposta Imune , Imunidade Inata/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Ciclofilinas/metabolismo , Ciclosporina/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Macrófagos/citologia , Macrófagos/patologia , Chaperonas Moleculares/metabolismo , Monócitos/citologia , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Receptores de Reconhecimento de Padrão , Internalização do Vírus , Replicação Viral/imunologia , Fatores de Poliadenilação e Clivagem de mRNA/deficiência , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Polycomb proteins maintain cell identity by repressing the expression of developmental regulators specific for other cell types. Polycomb repressive complex-2 (PRC2) catalyzes trimethylation of histone H3 lysine-27 (H3K27me3). Although repressed, PRC2 targets are generally associated with the transcriptional initiation marker H3K4me3, but the significance of this remains unclear. Here, we identify a class of short RNAs, approximately 50-200 nucleotides in length, transcribed from the 5' end of polycomb target genes in primary T cells and embryonic stem cells. Short RNA transcription is associated with RNA polymerase II and H3K4me3, occurs in the absence of mRNA transcription, and is independent of polycomb activity. Short RNAs form stem-loop structures resembling PRC2 binding sites in Xist, interact with PRC2 through SUZ12, cause gene repression in cis, and are depleted from polycomb target genes activated during cell differentiation. We propose that short RNAs play a role in the association of PRC2 with its target genes.
Assuntos
RNA/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/fisiologia , Conformação de Ácido Nucleico , Proteínas do Grupo Polycomb , Regiões Promotoras Genéticas , RNA/química , RNA/genética , Proteínas Repressoras/genética , Linfócitos T/citologia , Linfócitos T/fisiologiaRESUMO
BACKGROUND: Human Immunodeficiency Virus 1 (HIV-1) exhibits a wide range of interactions with the host cell but whether viral proteins interact with cellular RNA is not clear. A candidate interacting factor is the trans-activator of transcription (Tat) protein. Tat is required for expression of virus genes but activates transcription through an unusual mechanism; binding to an RNA stem-loop, the transactivation response element (TAR), with the host elongation factor P-TEFb. HIV-1 Tat has also been shown to alter the expression of host genes during infection, contributing to viral pathogenesis but, whether Tat also interacts with cellular RNAs is unknown. RESULTS: Using RNA immunoprecipitation coupled with microarray analysis, we have discovered that HIV-1 Tat is associated with a specific set of human mRNAs in T cells. mRNAs bound by Tat share a stem-loop structural element and encode proteins with common biological roles. In contrast, we do not find evidence that Tat associates with microRNAs or the RNA-induced silencing complex (RISC). The interaction of Tat with cellular RNA requires an intact RNA binding domain and Tat RNA binding is linked to an increase in RNA abundance in cell lines and during infection of primary CD4+ T cells by HIV. CONCLUSIONS: We conclude that Tat interacts with a specific set of human mRNAs in T cells, many of which show changes in abundance in response to Tat and HIV infection. This work uncovers a previously unrecognised interaction between HIV and its host that may contribute to viral alteration of the host cellular environment.
Assuntos
Infecções por HIV/virologia , HIV-1/genética , RNA Mensageiro/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Sequência de Bases , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Regulação Viral da Expressão Gênica , Células HEK293 , Humanos , MicroRNAs/genética , Dados de Sequência Molecular , Ligação Proteica/genética , Proteínas de Ligação a RNA/genética , Transcrição Gênica , Ativação Transcricional/genéticaRESUMO
CD8(+) T cells are major players in antiviral immunity against human immunodeficiency virus type 1 (HIV-1) through recognition of viral epitopes presented on the surface of infected cells. However, the early events involving HIV-1 epitope presentation to CD8(+) T cells remain poorly understood but are nonetheless crucial for the rapid clearance of virus-infected cells. Here, we comprehensively studied the kinetics of antigen presentation of two protective epitopes, KF11Gag and KK10Gag, restricted by HLA alleles B*57:01 and B*27:05, respectively, and compared these to KY9Pol and VL9Vpr epitopes in a single cycle of HIV-1 replication. We consistently demonstrate differences in epitope presentation kinetics, with very early presentation, within 3 h postinfection, for the protective KF11Gag, KK10Gag epitopes, and KY9Pol but only late presentation for VL9Vpr. We show that this early presentation relies on the antigen being presented from incoming viral particles and is correlated with rapid CD8(+) T cell activation and clearance of virus-infected cells. Additionally, our data indicate a dose-response dependency between the levels of CD8(+) T cell activation and the amount of virus inoculum. These data reflect a proof of principle emphasizing the importance of identifying early-presented viral epitopes for rapid elimination of HIV-1-infected cells.
Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Epitopos/imunologia , HIV-1/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Linhagem Celular , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Ativação Linfocitária , Replicação ViralRESUMO
Epstein-Barr virus (EBV) is present in all cases of endemic Burkitt lymphoma (BL) but in few European/North American sporadic BLs. Gene expression arrays of sporadic tumors have defined a consensus BL profile within which tumors are classifiable as "molecular BL" (mBL). Where endemic BLs fall relative to this profile remains unclear, since they not only carry EBV but also display one of two different forms of virus latency. Here, we use early-passage BL cell lines from different tumors, and BL subclones from a single tumor, to compare EBV-negative cells with EBV-positive cells displaying either classical latency I EBV infection (where EBNA1 is the only EBV antigen expressed from the wild-type EBV genome) or Wp-restricted latency (where an EBNA2 gene-deleted virus genome broadens antigen expression to include the EBNA3A, -3B, and -3C proteins and BHRF1). Expression arrays show that both types of endemic BL fall within the mBL classification. However, while EBV-negative and latency I BLs show overlapping profiles, Wp-restricted BLs form a distinct subgroup, characterized by a detectable downregulation of the germinal center (GC)-associated marker Bcl6 and upregulation of genes marking early plasmacytoid differentiation, notably IRF4 and BLIMP1. Importantly, these same changes can be induced in EBV-negative or latency I BL cells by infection with an EBNA2-knockout virus. Thus, we infer that the distinct gene profile of Wp-restricted BLs does not reflect differences in the identity of the tumor progenitor cell per se but differences imposed on a common progenitor by broadened EBV gene expression.
Assuntos
Linfoma de Burkitt/genética , Linfoma de Burkitt/virologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Transcriptoma , Latência Viral/genética , Antígenos Virais/biossíntese , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Antígenos Nucleares do Vírus Epstein-Barr/biossíntese , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/classificação , Humanos , Fatores Reguladores de Interferon/biossíntese , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Repressoras/biossíntese , Regulação para Cima , Proteínas Virais/biossínteseRESUMO
Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated non-dividing cells, but how they do this is unclear. The cytoplasmic NPC protein Nup358/RanBP2 was identified as an HIV-1 co-factor in previous studies. Here we report that HIV-1 capsid (CA) binds directly to the cyclophilin domain of Nup358/RanBP2. Fusion of the Nup358/RanBP2 cyclophilin (Cyp) domain to the tripartite motif of TRIM5 created a novel inhibitor of HIV-1 replication, consistent with an interaction in vivo. In contrast to CypA binding to HIV-1 CA, Nup358 binding is insensitive to inhibition with cyclosporine, allowing contributions from CypA and Nup358 to be distinguished. Inhibition of CypA reduced dependence on Nup358 and the nuclear basket protein Nup153, suggesting that CypA regulates the choice of the nuclear import machinery that is engaged by the virus. HIV-1 cyclophilin-binding mutants CA G89V and P90A favored integration in genomic regions with a higher density of transcription units and associated features than wild type virus. Integration preference of wild type virus in the presence of cyclosporine was similarly altered to regions of higher transcription density. In contrast, HIV-1 CA alterations in another patch on the capsid surface that render the virus less sensitive to Nup358 or TRN-SR2 depletion (CA N74D, N57A) resulted in integration in genomic regions sparse in transcription units. Both groups of CA mutants are impaired in replication in HeLa cells and human monocyte derived macrophages. Our findings link HIV-1 engagement of cyclophilins with both integration targeting and replication efficiency and provide insight into the conservation of viral cyclophilin recruitment.
Assuntos
Proteínas do Capsídeo/metabolismo , Núcleo Celular/virologia , Ciclofilina A/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Replicação Viral , Transporte Ativo do Núcleo Celular/fisiologia , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Replicação Viral/fisiologiaRESUMO
TRIMCyps are anti-retroviral proteins that have arisen independently in New World and Old World primates. All TRIMCyps comprise a CypA domain fused to the tripartite domains of TRIM5alpha but they have distinct lentiviral specificities, conferring HIV-1 restriction in New World owl monkeys and HIV-2 restriction in Old World rhesus macaques. Here we provide evidence that Asian macaque TRIMCyps have acquired changes that switch restriction specificity between different lentiviral lineages, resulting in species-specific alleles that target different viruses. Structural, thermodynamic and viral restriction analysis suggests that a single mutation in the Cyp domain, R69H, occurred early in macaque TRIMCyp evolution, expanding restriction specificity to the lentiviral lineages found in African green monkeys, sooty mangabeys and chimpanzees. Subsequent mutations have enhanced restriction to particular viruses but at the cost of broad specificity. We reveal how specificity is altered by a scaffold mutation, E143K, that modifies surface electrostatics and propagates conformational changes into the active site. Our results suggest that lentiviruses may have been important pathogens in Asian macaques despite the fact that there are no reported lentiviral infections in current macaque populations.
Assuntos
Ciclofilina A/genética , Infecções por HIV/genética , Macaca/genética , Proteínas Mutantes Quiméricas/genética , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Ciclofilina A/metabolismo , Evolução Molecular , Infecções por Lentivirus/genética , Macaca/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes Quiméricas/metabolismo , Mutação , Filogenia , Estrutura Quaternária de Proteína , Proteínas/genética , Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína LigasesRESUMO
Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation.
Assuntos
Infecções por HIV , HIV-1 , Vírus da Imunodeficiência Símia , Animais , Humanos , Filogenia , Vírus da Imunodeficiência Símia/metabolismo , Capsídeo/metabolismo , HIV-1/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Infecções por HIV/epidemiologia , Infecções por HIV/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Targeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by 'T v T' fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in 'self-enrichment' yielding populations 99.6% TCR-/CD3-/CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic 'off-target' activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.
Assuntos
Antígenos CD7/genética , Complexo CD3/genética , Imunoterapia Adotiva/métodos , Leucemia de Células T/terapia , Linfócitos T/imunologia , Animais , Antígenos CD7/química , Antígenos CD7/metabolismo , Complexo CD3/antagonistas & inibidores , Complexo CD3/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Feminino , Edição de Genes , Humanos , Leucemia de Células T/imunologia , Leucemia de Células T/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Expression microarrays are increasingly used to obtain large scale transcriptomic information on a wide range of biological samples. Nevertheless, there is still much debate on the best ways to process data, to design experiments and analyse the output. Furthermore, many of the more sophisticated mathematical approaches to data analysis in the literature remain inaccessible to much of the biological research community. In this study we examine ways of extracting and analysing a large data set obtained using the Agilent long oligonucleotide transcriptomics platform, applied to a set of human macrophage and dendritic cell samples. RESULTS: We describe and validate a series of data extraction, transformation and normalisation steps which are implemented via a new R function. Analysis of replicate normalised reference data demonstrate that interarray variability is small (only around 2% of the mean log signal), while interarray variability from replicate array measurements has a standard deviation (SD) of around 0.5 log(2) units ( 6% of mean). The common practise of working with ratios of Cy5/Cy3 signal offers little further improvement in terms of reducing error. Comparison to expression data obtained using Arabidopsis samples demonstrates that the large number of genes in each sample showing a low level of transcription reflect the real complexity of the cellular transcriptome. Multidimensional scaling is used to show that the processed data identifies an underlying structure which reflect some of the key biological variables which define the data set. This structure is robust, allowing reliable comparison of samples collected over a number of years and collected by a variety of operators. CONCLUSIONS: This study outlines a robust and easily implemented pipeline for extracting, transforming normalising and visualising transcriptomic array data from Agilent expression platform. The analysis is used to obtain quantitative estimates of the SD arising from experimental (non biological) intra- and interarray variability, and for a lower threshold for determining whether an individual gene is expressed. The study provides a reliable basis for further more extensive studies of the systems biology of eukaryotic cells.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Arabidopsis/genética , DNA Complementar/genética , Regulação da Expressão Gênica , Genoma Humano , Humanos , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
HIV-1 must replicate in cells that are equipped to defend themselves from infection through intracellular innate immune systems. HIV-1 evades innate immune sensing through encapsidated DNA synthesis and encodes accessory genes that antagonize specific antiviral effectors. Here, we show that both particle associated, and expressed HIV-1 Vpr, antagonize the stimulatory effect of a variety of pathogen associated molecular patterns by inhibiting IRF3 and NF-κB nuclear transport. Phosphorylation of IRF3 at S396, but not S386, was also inhibited. We propose that, rather than promoting HIV-1 nuclear import, Vpr interacts with karyopherins to disturb their import of IRF3 and NF-κB to promote replication in macrophages. Concordantly, we demonstrate Vpr-dependent rescue of HIV-1 replication in human macrophages from inhibition by cGAMP, the product of activated cGAS. We propose a model that unifies Vpr manipulation of nuclear import and inhibition of innate immune activation to promote HIV-1 replication and transmission.
Assuntos
Infecções por HIV/imunologia , Evasão da Resposta Imune/fisiologia , Imunidade Inata/imunologia , Replicação Viral/fisiologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/imunologia , Transporte Ativo do Núcleo Celular/fisiologia , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Carioferinas/imunologia , Carioferinas/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Most gene therapy lentiviral vector (LV) production platforms employ HEK293T cells expressing the oncogenic SV40 large T-antigen (TAg) that is thought to promote plasmid-mediated gene expression. Studies on other viral oncogenes suggest that TAg may also inhibit the intracellular autonomous innate immune system that triggers defensive antiviral responses upon detection of viral components by cytosolic sensors. Here we show that an innate response can be generated after HIV-1-derived LV transfection in HEK293T cells, particularly by the transgene, yet, remarkably, this had no effect on LV titer. Further, overexpression of DNA sensing pathway components led to expression of inflammatory cytokine and interferon (IFN) stimulated genes but did not result in detectable IFN or CXCL10 and had no impact on LV titer. Exogenous IFN-ß also did not affect LV production or transduction efficiency in primary T cells. Additionally, manipulation of TAg did not affect innate antiviral responses, but stable expression of TAg boosted vector production in HEK293 cells. Our findings demonstrate a measure of innate immune competence in HEK293T cells but, crucially, show that activation of inflammatory signaling is uncoupled from cytokine secretion in these cells. This provides new mechanistic insight into the unique suitability of HEK293T cells for LV manufacture.
RESUMO
Dendritic cells (DC) generated from MUTZ-3, an immortalized acute myeloid leukaemia-derived cell line, have potential application as a model for the study of human DC, and as a tool with which to stimulate immunotherapeutic responses to cancer. However, the relationship of MUTZ-3 DC to their non-transformed counterparts remains incompletely understood. Immunoselected CD14+ MUTZ-3 cells were used to generate a homogeneous population of DC (M3DC). These cells had a cell surface phentoype and morphology characteristic of conventional monocyte-derived DC (MDDC). Whole genome transcriptome comparison of M3DC and MDDC however, revealed extensive differences between these two cell types. Functional ontology-based data analysis revealed three enriched clusters of genes downregulated in M3DC, with functions in pathogen recognition, DC maturation and cytokine/chemokine signalling. Downregulation of protein expression was confirmed for several of these genes. The molecular differences were accompanied by a profoundly impaired phenotypic and functional response of M3DC to microbial stimulation. The immortalized phenotype of MUTZ-3 therefore reflects not only deregulated proliferative capacity, but substantial perturbation of normal antigen-presenting cell function. These results have important implications for studies using MUTZ-3 as a model of MDDC or for cancer immunotherapy.
Assuntos
Células Dendríticas/imunologia , Leucemia Mieloide Aguda/imunologia , Apresentação de Antígeno/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Regulação para Baixo/imunologia , Expressão Gênica/imunologia , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/genética , Receptores de Lipopolissacarídeos/análise , Família Multigênica/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Gênica/imunologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Nuclear import of the HIV-1 reverse transcription complex (RTC) is critical for infection of non dividing cells, and importin 7 (imp7) has been implicated in this process. To further characterize the function of imp7 in HIV-1 replication we generated cell lines stably depleted for imp7 and used them in conjunction with infection, cellular fractionation and pull-down assays. RESULTS: Imp7 depletion impaired HIV-1 infection but did not significantly affect HIV-2, simian immunodeficiency virus (SIVmac), or equine infectious anemia virus (EIAV). The lentiviral dependence on imp7 closely correlated with binding of the respective integrase proteins to imp7. HIV-1 RTC associated with nuclei of infected cells with remarkable speed and knock down of imp7 reduced HIV-1 DNA nuclear accumulation, delaying infection. Using an HIV-1 mutant deficient for reverse transcription, we found that viral RNA accumulated within nuclei of infected cells, indicating that reverse transcription is not absolutely required for nuclear import. Depletion of imp7 impacted on HIV-1 DNA but not RNA nuclear import and also inhibited DNA transfection efficiency. CONCLUSION: Although imp7 may not be essential for HIV-1 infection, our results suggest that imp7 facilitates nuclear trafficking of DNA and that HIV-1 exploits imp7 to maximize nuclear import of its DNA genome. Lentiviruses other than HIV-1 may have evolved to use alternative nuclear import receptors to the same end.
Assuntos
Transporte Ativo do Núcleo Celular , DNA Viral/metabolismo , HIV-1/fisiologia , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Linhagem Celular , Núcleo Celular/virologia , Endodesoxirribonucleases/metabolismo , Integrase de HIV/metabolismo , HIV-2/fisiologia , Humanos , Vírus da Anemia Infecciosa Equina/fisiologia , Ligação Proteica , Vírus da Imunodeficiência Símia/fisiologia , Replicação ViralRESUMO
The lytic gene expression of several members of the human herpesvirus family has been profiled by using gene-expression microarrays; however, the lytic cascade of roseoloviruses has not been studied in similar depth. Based on the complete DNA genome sequences of human herpesvirus 6 variant A (HHV-6A) and variant B (HHV-6B), we constructed a cDNA microarray containing DNA probes to their predicted open reading frames, plus 914 human genes. Gene-expression profiling of HHV-6B strain Z29 in SupT1 cells over a 60 h time-course post-infection, together with kinetic classification of the HHV-6B genes in the presence of either cycloheximide or phosphonoacetic acid, allowed the placement of HHV-6B genes into defined kinetic classes. Eighty-nine HHV-6B genes were divided into four different expression kinetic classes: eight immediate-early, 44 early, 33 late and four biphasic. Clustering of genes with similar expression profiles implied a shared function, thus revealing possible roles of previously uncharacterized HHV-6B genes.
Assuntos
Perfilação da Expressão Gênica , Herpesvirus Humano 6/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Replicação Viral , Antivirais/farmacologia , Linhagem Celular , Cicloeximida/farmacologia , Regulação Viral da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ácido Fosfonoacéticos/farmacologiaRESUMO
T cells engineered to express chimeric antigen receptors (CARs) against B cell antigens are being investigated as cellular immunotherapies. Similar approaches designed to target T cell malignancies have been hampered by the critical issue of T-on-T cytotoxicity, whereby fratricide or self-destruction of healthy T cells prohibits cell product manufacture. To date, there have been no reports of T cells engineered to target the definitive T cell marker, CD3 (3CAR). Recent improvements in gene editing now provide access to efficient disruption of such molecules on T cells, and this has provided a route to generation of 3CAR, CD3-specific CAR T cells. T cells were transduced with a lentiviral vector incorporating an anti-CD3ε CAR derived from OKT3, either before or after TALEN-mediated disruption of the endogenous TCRαß/CD3 complex. Only transduction after disrupting assembly of TCRαß/CD3 yielded viable 3CAR T cells, and these cultures were found to undergo self-enrichment for 3CAR+TCR-CD3- T cells without any further processing. Specific cytotoxicity against CD3ε was demonstrated against primary T cells and against childhood T cell acute lymphoblastic leukemia (T-ALL). 3CAR T cells mediated potent antileukemic effects in a human/murine chimeric model, supporting the application of cellular immunotherapy strategies against T cell malignancies. 3CAR provides a bridging strategy to achieve T cell eradication and leukemic remission ahead of conditioned allogeneic stem cell transplantation.
Assuntos
Complexo CD3/imunologia , Imunoterapia , Leucemia/tratamento farmacológico , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Complexo CD3/genética , Engenharia Celular , Linhagem Celular Tumoral , Feminino , Terapia Genética , Humanos , Camundongos , Muromonab-CD3 , Receptores de Antígenos Quiméricos/genética , Transdução GenéticaRESUMO
This review summarizes current knowledge about the mixed lineage kinases (MLKs) and explores their potential role in inflammation and immunity. MLKs were identified initially as signalling molecules in the nervous system. They were also shown to play a role in the cell cycle. Further studies documented three groups of MLKs, and showed that they may be activated via the c-Jun NH(2) terminal kinase (JNK) pathway, and by Rho GTPases. The biochemistry of the MLKs has been investigated in considerable detail. Homodimerization and heterodimerization can occur, and both autophosphorylation and autoinhibition are seen. The interaction between MLKs and JNK interacting protein (JIP) scaffolds, and the resultant effects on mitogen activated protein kinases, have been identified. Clearly, there is some redundancy within the MLK pathway(s), since mice which lack the MLK3 molecule are not abnormal. However, using a combination of biochemical analysis and pharmacological inhibitors, several recent studies in vitro have suggested that MLKs are not only expressed in cells of the immune system (as well as in the nervous system), but also may be implicated selectively in the signalling pathway that follows on toll-like receptor ligation in innate sentinel cells, such as the dendritic cell.
Assuntos
Células Dendríticas/imunologia , MAP Quinase Quinase Quinases/fisiologia , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ciclo Celular , Ativação Enzimática , Humanos , Imunidade Inata , Inflamação , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores Toll-Like/metabolismoRESUMO
Dendritic cells (DCs) sense the presence of conserved microbial structures in their local microenvironment via specific pattern recognition receptors (PRRs). This leads to a programme of changes, which include migration and activation, and enables them to induce adaptive T cell immunity. Mitogen-activated protein kinases (MAPKs) are implicated in this response, but the pathways leading from PRR ligation to MAPK activation, and hence DC activation, are not fully understood. Recent studies in the nervous system have suggested that the mixed lineage kinase (MLK) family of MAPK kinase kinase proteins may be involved as an intermediary step between PRRs and MAPKs. Therefore, in this study, we have used a well-established DC model to explore the role of MLKs in these cells. Messenger RNA for MLKs 2, 3, 4 and DLK and protein for MLKs 2, 3 and DLK are found in DC. DC activation in response to model PRR ligands, such as LPS or poly (I:C), is accompanied by phosphorylation of MLK3. In contrast, another known PRR ligand, zymosan, induces little MLK3 phosphorylation. Inhibition of MLK activity using a pharmacological inhibitor, CEP11004, blocks p38 and Jun N-terminal kinase (JNK) MAPK activation in response to LPS and poly (I:C), but not zymosan. The inhibition is associated with a block in DC activation as measured by cell-surface marker expression and cytokine secretion. Thus, MLKs are expressed in DC, and are implicated in DC activation, and the involvement of MLKs appears to be selective, depending on the nature of the DC stimulus.
Assuntos
Células Dendríticas/enzimologia , MAP Quinase Quinase Quinases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antracenos/farmacologia , Apresentação de Antígeno , Carbazóis/farmacologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Lipopolissacarídeos/imunologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Zimosan/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Dendritic cells (DCs) are key antigen-presenting cells (APCs), which link innate and adaptive immunity, ultimately activating antigen-specific T cells. This review examines the relationship between the acute and chronic myeloid leukaemias and cells with DC properties. DCs are non-dividing terminally differentiated cells, and ex vivo leukaemic cells or cell lines show little similarity to DCs. However, many leukaemias differentiate further in response to defined stimuli, and retain a degree of lineage plasticity. Therefore, several studies have explored the response of leukaemic cells to the in vitro regimens used to differentiate ex vivo primary DCs. Recent data suggest that the most 'dendritic-like' cells can be derived from more undifferentiated myeloid leukaemias, such as the myelomonocytic Mutz-3 cell line. These findings have important implications for understanding the developmental origins of DCs, for harnessing the APC properties of this class of tumour to stimulate the therapeutic anti-tumour immunity, and for developing useful models for the study of human DC physiology and pathology. There is a strong rationale for further exploration of this class of tumour and its relationship to the normal DC.