RESUMO
Tuberculosis (TB) is a disease of global importance. There is an increasing recognition of the role of Toll like receptors, important pattern recognition receptors of host immune system, in determining the susceptibility or resistance to TB in various populations. In an attempt to examine the importance of Toll like receptors in immune response to Mycobacterium tuberculosis infection, we explored two variants each of TLR2 and TLR9 in a population residing in Uttar Pradesh, India. Genotyping was performed to detect -196 to -174 del polymorphism and G2258A SNP (Arg753Gln, rs5743708) in TLR2 gene and -T1237C (rs5743836) and G2848A (rs352140) SNP in TLR9 gene in patients with pulmonary TB and healthy controls. The A allele of G2848A SNP in TLR9 gene was found with a marginally higher frequency among TB patients as compared to healthy controls, suggesting that A allele at position 2848 of TLR9 gene may be associated with susceptibility to TB in North Indian population [p = 0.05, Mantel-Haenszel OR = 1.34, 95% CI (1.0-1.82)].
Assuntos
Receptor 2 Toll-Like/genética , Receptor Toll-Like 9/genética , Tuberculose Pulmonar/genética , Adulto , Idoso , Povo Asiático/genética , Estudos de Casos e Controles , Feminino , Frequência do Gene , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Polimorfismo de Nucleotídeo Único , Receptor 2 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologiaRESUMO
BACKGROUND: Mycobacterium tuberculosis (M. tuberculosis) modulates host immune response, mainly T cell responses for its own survival leading to disease or latent infection. The molecules and mechanisms utilized to accomplish immune subversion by M. tuberculosis are not fully understood. Understanding the molecular mechanism of T cell response to M. tuberculosis is important for development of efficacious vaccine against TB. METHODS: Here, we investigated effect of M. tuberculosis antigens Ag85A and ESAT-6 on T cell signalling events in CD3/CD28 induced Peripheral blood mononuclear cells (PBMCs) of PPD+ve healthy individuals and pulmonary TB patients. We studied CD3 induced intracellular calcium mobilization in PBMCs of healthy individuals and TB patients by spectrofluorimetry, CD3 and CD28 induced activation of mitogen activated protein kinases (MAPKs) in PBMCs of healthy individuals and TB patients by western blotting and binding of transcription factors NFAT and NFκB by Electrophorectic mobility shift assay (EMSA). RESULTS: We observed CD3 triggered modulations in free intracellular calcium concentrations in PPD+ve healthy individuals and pulmonary TB patients after the treatment of M. tuberculosis antigens. As regards the downstream signalling events, phosphorylation of MAPKs, Extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38 was curtailed by M. tuberculosis antigens in TB patients whereas, in PPD+ve healthy individuals only ERK1/2 phosphorylation was inhibited. Besides, the terminal signalling events like binding of transcription factors NFAT and NFκB was also altered by M. tuberculosis antigens. Altogether, our results suggest that M. tuberculosis antigens, specifically ESAT-6, interfere with TCR/CD28-induced upstream as well as downstream signalling events which might be responsible for defective IL-2 production which further contributed in T-cell unresponsiveness, implicated in the progression of disease. CONCLUSION: To the best of our knowledge, this is the first study to investigate effect of Ag85A and ESAT-6 on TCR- and TCR/CD28- induced upstream and downstream signalling events of T-cell activation in TB patients. This study showed the effect of secretory antigens of M. tuberculosis in the modulation of T cell signalling pathways. This inflection is accomplished by altering the proximal and distal events of signalling cascade which could be involved in T-cell dysfunctioning during the progression of the disease.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Ativação Linfocitária/imunologia , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Aciltransferases/imunologia , Aciltransferases/metabolismo , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Antígenos CD28/metabolismo , Cálcio/metabolismo , Humanos , Espaço Intracelular/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is one of the essential latent antigens for primary B-cell transformation. Previous studies established that EBNA3C facilitates degradation of several vital cell cycle regulators, including the retinoblastoma (pRb) and p27(KIP) proteins, by recruitment of the SCF(Skp2) E3 ubiquitin ligase complex. EBNA3C was also shown to be ubiquitinated at its N-terminal residues. Furthermore, EBNA3C can bind to and be degraded in vitro by purified 20S proteasomes. Surprisingly, in lymphoblastoid cell lines, EBNA3C is extremely stable, and the mechanism for this stability is unknown. In this report we show that EBNA3C can function as a deubiquitination enzyme capable of deubiquitinating itself in vitro as well as in vivo. Functional mapping using deletion and point mutational analysis showed that both the N- and C-terminal domains of EBNA3C contribute to the deubiquitination activity. We also show that EBNA3C efficiently deubiquitinates Mdm2, an important cellular proto-oncogene, which is known to be overexpressed in several human cancers. The data presented here further demonstrate that the N-terminal domain of EBNA3C can bind to the acidic domain of Mdm2. Additionally, the N-terminal domain of EBNA3C strongly stabilizes Mdm2. Importantly, EBNA3C simultaneously binds to both Mdm2 and p53 and can form a stable ternary complex; however, in the presence of p53 the binding affinity of Mdm2 toward EBNA3C was significantly reduced, suggesting that p53 and Mdm2 might share a common overlapping domain of EBNA3C. We also showed that EBNA3C enhances the intrinsic ubiquitin ligase activity of Mdm2 toward p53, which in turn facilitated p53 ubiquitination and degradation. Thus, manipulation of the oncoprotein Mdm2 by EBNA3C potentially provides a favorable environment for transformation and proliferation of EBV-infected cells.
Assuntos
Antígenos Virais/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antígenos Virais/genética , Linhagem Celular , Sistema Livre de Células , Antígenos Nucleares do Vírus Epstein-Barr , Herpesvirus Humano 4/genética , Humanos , Ligação Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , UbiquitinaçãoRESUMO
The C-C motif chemokine ligand-2 (CCL2) was evidenced to be associated with tuberculosis susceptibility in some ethnic groups. In the present study, effort was made to find out the association of CCL2-2518 A>G and -362 G>C variants with susceptibility to TB in a population from North India. The genotyping was carried out in 373 participants with pulmonary TB (PTB) and 248 healthy controls (HCs) for CCL2-2518 A>G and -362 G>C polymorphisms by PCR-RFLP and by melting curve analysis using fluorescence-labeled hybridization fluorescent resonance energy transfer (FRET) probes, respectively, followed by DNA sequencing in a few representative samples. Genotype and allele frequencies were compared by the chi-squared test and crude and Mantel-Haenszel (M-H) odds ratio (OR). OR was calculated using STATA/MP16.1 software. Further, CCL2, IL-12p70, IFN-γ, TNF-α, and TGF-ß levels were measured in serum samples of these participants using commercially available kits. Our analysis indicated that the homozygous mutant in both -2518 GG (OR = 2.07, p = 0.02) and -362 CC (OR = 1.92, p = 0.03) genotypes was associated with susceptibility to pulmonary TB. Further, heterozygous genotypes -2518AG (OR = 0.60, p = 0.003) and -362GC (OR = 0.64, p = 0.013) provide resistance from PTB disease. Haplotype analysis revealed AC haplotype (p = 0.006) to be a risk factor associated with PTB susceptibility. The serum CCL2 level was significantly elevated among participants with -2518 AA genotype compared to -2518 GG genotype. CCL2 level was observed to be positively correlated with IL12p70, IFN-γ and TNF-α, thus suggesting the immunological regulatory role of CCL2 against pulmonary tuberculosis. CCL2-2518 GG and -362 CC genotypes were found to be associated with susceptibility to pulmonary tuberculosis and CCL2-2518AG and CCL2-362GC with resistance from PTB. AC haplotype was found to be a risk factor for PTB in the present study. It may be hypothesized from the findings that -2518G allele could be responsible for lower production of CCL2 which leads to defective Th1 response and makes a host susceptible for pulmonary tuberculosis.
Assuntos
Quimiocina CCL2/genética , Predisposição Genética para Doença , Mycobacterium tuberculosis , Polimorfismo de Nucleotídeo Único , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/etiologia , Adolescente , Adulto , Alelos , Estudos de Casos e Controles , Citocinas/genética , Feminino , Frequência do Gene , Estudos de Associação Genética , Genótipo , Haplótipos , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Vigilância da População , Adulto JovemRESUMO
Epstein-Barr virus (EBV) was the first human DNA virus to be associated with cancer. Its oncogenic potential was further demonstrated by its ability to transform primary B lymphocytes in vitro. EBV nuclear antigen 3C (EBNA3C) is one of a small subset of latent antigens critical for the transformation of human primary B lymphocytes. Although EBNA3C has been shown to modulate several cellular functions, additional targets involved in cellular transformation remain to be explored. EBNA3C can recruit key components of the SCF(Skp2) ubiquitin ligase complex. In this report, we show that EBNA3C residues 130 to 190, previously shown to bind to the SCF(Skp2) complex, also can strongly associate with the c-Myc oncoprotein. Additionally, the interaction of EBNA3C with c-Myc was mapped to the region of c-Myc that includes the highly conserved Skp2 binding domain. Skp2 has been shown to regulate c-Myc stability and also has been shown to function as a coactivator of transcription for c-Myc target genes. We now show that the EBV latent oncoprotein EBNA3C can stabilize c-Myc and that the recruitment of both c-Myc and its cofactor Skp2 to c-Myc-dependent promoters can enhance c-Myc-dependent transcription. This same region of EBNA3C also recruits and modulates the activity of retinoblastoma and p27, both major regulators of the mammalian cell cycle. The inclusion of c-Myc in the group of cellular targets modulated by this domain further accentuates the importance of these critical residues of EBNA3C in bypassing the cell cycle checkpoints.
Assuntos
Antígenos Virais/metabolismo , Herpesvirus Humano 4/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sítios de Ligação , Western Blotting , Linhagem Celular , Antígenos Nucleares do Vírus Epstein-Barr , Humanos , Imunoprecipitação , Ligação Proteica , Mapeamento de Interação de Proteínas , RNA Mensageiro/biossíntese , Proteínas Quinases Associadas a Fase S/metabolismo , Transcrição GênicaRESUMO
Epstein-Barr virus (EBV) is a gammaherpesvirus of the Lymphocryptovirus genus, which infects greater than 90% of the world's population. Infection is nonsymptomatic in healthy individuals, but has been associated with a number of lymphoproliferative disorders when accompanied by immunosuppression. Like all herpesviruses, EBV has both latent and lytic replication programs, which allows it to evade immune clearance and persist for the lifetime of the host. Latent infection is characterized by replication of the viral genome as an integral part of the host cell chromosomes, and the absence of production of infectious virus. A further layer of complexity is added in that EBV can establish three distinct latency programs, in each of which a specific set of viral antigens is expressed. In most malignant disorders associated with EBV, the virus replicates using one of these three latency programs. In the most aggressive latency program, only 11 of the hitherto 85 identified open reading frames in the EBV genome are expressed. The other two latency programs express even smaller subsets of this repertoire of latent genes. The onset of the AIDS pandemic and the corresponding increase in individuals with acquired immunodeficiency resulted in a sharp increase in EBV-mediated AIDS-associated malignancies. This has sparked a renewed interest in EBV biology and pathogenesis.
Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Neoplasias/complicações , Neoplasias/virologia , Infecções por Vírus Epstein-Barr/patologia , Genes Virais , Humanos , Ativação ViralRESUMO
Electrospun carbon nanofibers (CNFs) decorated with copper oxide nanoparticles were successfully synthesized using a one step and modified hydroxyl ion assisted alcohol reduction method. The X-ray diffraction pattern reveals the presence of (-111), (111) and (-202) crystal planes of monoclinic copper oxide (CuO). The CuO coated CNF air annealed at 250 °C exhibited an electrical conductivity of 251 S m-1. The CuO coated CNFs were employed as a novel flexible matrix for a cholesterol biosensor. The biosensor exhibited a high sensitivity of 75 µA mM-1 cm-2 over the linear range (0.12 mM to 12.93 mM) of cholesterol. This coating method offers easy and inexpensive processing with a flexible and robust supporting structure for biosensing applications.
RESUMO
Human epidermal keratinocytes are an important target for gene therapy because they can be easily expanded in culture and used to generate skin substitutes for the treatment of wounds, genetic diseases of the skin, and for delivery of proteins to the systemic circulation. Although retroviral transduction results in permanent genetic modification, differentiation and loss of transduced cells from the epidermis results in temporary transgene expression. To ensure permanent genetic modification, epidermal stem cells must be transduced with high efficiency. We evaluated gene transfer on two different substrates and found that the efficiency of gene transfer is substantially higher on a substrate of recombinant fibronectin (FN), when compared to tissue culture plastic (TCP). The rate of retroviral transduction on FN is four times faster than transduction on tissue culture plates and is independent of polybrene (PB). The transduction efficiency correlates with the levels of expression of integrin subunits alpha5, alpha2, and beta1, which have been shown to correlate with stem cell phenotype. Notably, cells that adhere rapidly to FN are transduced more efficiently than slowly adherent cells. In addition, integrin-blocking antibodies decrease the efficiency of gene transfer in a dose-dependent manner. Our results suggest that FN may enhance retroviral gene transfer to the least differentiated cells, thereby increasing the potential of genetically modified keratinocytes to treat short- and long-term disease states.
Assuntos
Meios de Cultura/farmacologia , Fibronectinas/farmacologia , Vetores Genéticos/fisiologia , Integrina alfa2/fisiologia , Integrina alfa5/fisiologia , Integrina beta1/fisiologia , Queratinócitos/virologia , Retroviridae/fisiologia , Transdução Genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Adesão Celular , Técnicas de Cultura de Células/instrumentação , Células Cultivadas/metabolismo , Células Cultivadas/virologia , Vetores Genéticos/genética , Brometo de Hexadimetrina/farmacologia , Humanos , Integrina alfa2/biossíntese , Integrina alfa2/genética , Integrina alfa5/biossíntese , Integrina alfa5/genética , Integrina beta1/biossíntese , Integrina beta1/genética , Queratinócitos/metabolismo , Plásticos , Proteínas Recombinantes/farmacologia , Retroviridae/genéticaRESUMO
Infection with Mycobacteriumtuberculosis possibly depends on host genetic factors and is thought to be the major cause of differential susceptibility to the disease. In the present study, 205 pulmonary tuberculosis cases and 127 healthy controls were studied for the association of Toll-like Receptor (TLR) variants (TLR1 variants 743A>G and 1805T>G, and TLR6 variant 745 C>T) in north Indian population. The frequency of heterozygous genotypes (AG) in TB cases (0.47) and HCs (0.61), differed significantly (p value = 0.02). The association of AG genotypes in HCs was adjusted for gender as gender was observed to be a confounder and M-H OR was found to be 0.62 (p = 0.044). On categorizing the cases basing on AFB smear positivity, the heterozygous genotypes (AG) was found to be associated with low bacillary load (scanty and 1+) (P = 0.002). No association was observed for either TLR1 1805 T>G or TLR6 745 C>T polymorphism. Level of serum IL6 was found to be significantly higher among healthy controls with TLR1 GG genotype compared to healthy controls with AA (p = 0.035) and AG (p = 0.005) genotypes. Thus, it may be suggested that the heterozygous condition for TLR1 743 A>G provide resistance from the disease. However, in depth study is required to understand the mechanism for possible protective responses.
Assuntos
Polimorfismo Genético , Receptor 1 Toll-Like/genética , Receptor 6 Toll-Like/genética , Tuberculose Pulmonar/genética , Adolescente , Adulto , Idoso , Alelos , Carga Bacteriana , Estudos de Casos e Controles , Criança , Feminino , Frequência do Gene , Predisposição Genética para Doença , Haplótipos , Heterozigoto , Humanos , Índia , Interleucina-6/genética , Interleucina-6/imunologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Receptor 1 Toll-Like/imunologia , Receptor 6 Toll-Like/imunologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/patologiaRESUMO
Elimination of tuberculosis (TB) largely depends upon definitive rapid diagnosis and treatment. Widely used diagnostic tests do not qualify for use in a developing country due to lack of either desired accuracy or their cost. In the present study an enzyme-linked immunosorbent assay was used to evaluate the diagnostic potential of an immuno-dominant 30/32-kDa mycolyl transferase complex (Ag85 complex) and Mycobacterium tuberculosis-specific proteins (ESAT-6 and CFP-10) of the RD1 region. Higher sensitivity (84.1%) with Ag85 complex was observed compared with ESAT-6 (64.9%) and CFP-10 (66%), with almost similar specificity (Ag85: 85.2%, ESAT-6: 88.9%, CFP-10: 85.2%), whereas the individual components of Ag85 complex, i.e. Ag85A, Ag85B, and Ag85C, showed sensitivities of 44.6, 34, and 80.9% and specificities of 55.6, 74.1, and 40.7% respectively. A cocktail of Ag85 complex, ESAT-6, CFP-10, Ag85A, Ag85B, and Ag85C antigens also could not help in increasing either sensitivity (51.1%) or specificity (85.2%). Furthermore, immunoblot analysis using clinical isolates as well as a standard strain (H37Rv) of M. tuberculosis also showed strong reactivity of sera from TB patients to Ag85 complex and, to a lesser extent, also to ESAT-6. To conclude, use of Ag85 complex along with ESAT-6 and CFP-10 seems to be promising in minimizing the heterogeneous sero-responses of adult TB cases.
Assuntos
Aciltransferases/análise , Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Mycobacterium tuberculosis/imunologia , Tuberculose/diagnóstico , Aciltransferases/imunologia , Adolescente , Adulto , Idoso , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Sensibilidade e EspecificidadeRESUMO
Primary effusion lymphoma (PEL) is a common cancer in AIDS patients closely associated with Kaposi's sarcoma-associated herpesvirus (KSHV). Previously, we showed that KSHV latency associated nuclear antigen (LANA) stabilizes intracellular activated Notch1 (ICN) involved in maintenance of the malignant phenotype of KSHV infected PEL cells in vitro. The gamma-secretase inhibitor (GSI) which specifically blocks the production of ICN slows down the proliferation of the KSHV infected PEL cell lines BCBL1, BC3 as well as JSC1 in vitro. In this study, we extended these studies to explore the possibility that manipulation of the Notch signaling by GSI would prevent the growth of the PEL tumors in vivo. We observed that the onset of tumorigenesis of KSHV infected PELs was significantly delayed in GSI treated SCID mice harboring the PEL cell lines. We also found that GSI treatment resulted in necrosis as well as apoptosis in tumors generated by the xenotransplanted KSHV positive PEL cell lines. In contrast, GSI had no effect on mice harboring BJAB cells, a KSHV negative Burkitt's lymphoma cell line where ICN levels were negligible. Our study provides further evidence to suggest that targeted downregulation of abnormal Notch signaling has therapeutic potential for KSHV related primary effusion lymphomas.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Dipeptídeos/uso terapêutico , Infecções por Herpesviridae , Herpesvirus Humano 8/patogenicidade , Linfoma de Efusão Primária/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Receptor Notch1/antagonistas & inibidores , Infecções Tumorais por Vírus , Secretases da Proteína Precursora do Amiloide/fisiologia , Animais , Antígenos Virais/fisiologia , Apoptose , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral/transplante , Herpesvirus Humano 4/isolamento & purificação , Herpesvirus Humano 4/patogenicidade , Herpesvirus Humano 8/isolamento & purificação , Linfoma de Efusão Primária/patologia , Linfoma de Efusão Primária/virologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Necrose , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/fisiologia , Distribuição Aleatória , Receptor Notch1/fisiologia , Transdução de Sinais/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this appendix, several basic methods are described for preparation of primary B lymphocyte.
Assuntos
Centrifugação com Gradiente de Concentração/métodos , Separação Imunomagnética/métodos , Leucócitos Mononucleares/citologia , Linfócitos B/citologia , HumanosRESUMO
Epstein-Barr virus (EBV) was first reported as the etiological agent of Burkitt's lymphoma in 1964. Since then, EBV has also been associated with nasopharyngeal carcinoma, which is highly prevalent in Southeast Asia, as well as infectious mononucleosis, complications of AIDS, and transplant-related B cell lymphomas. This virus has further been linked with T cell lymphomas and Hodgkin's disease, establishing the concept of a wide spectrum of EBV-associated malignant disorders. So far, there are a number of EBV-infected cell lines established that can be induced for production of infectious viral progeny and that facilitate the study of the mechanism of EBV-related infection, transformation, and oncogenesis. This unit describes procedures for the preparation of EBV virion particles and in vitro infection of cells with EBV. In addition, procedures for quantitation and storage of the virus are provided.
Assuntos
Linfócitos B/virologia , Transformação Celular Viral , Células Epiteliais/virologia , Herpesvirus Humano 4 , Virologia/métodos , Animais , Linhagem Celular , Células Cultivadas , Meios de Cultura , Herpesvirus Humano 4/crescimento & desenvolvimento , Herpesvirus Humano 4/isolamento & purificação , Herpesvirus Humano 4/patogenicidade , Humanos , Camundongos , Manejo de Espécimes/métodosRESUMO
Deregulation of the evolutionarily conserved Notch signaling is highly correlated with oncogenesis. Intracellular activated Notch (ICN) is a protooncogene linked to the transcription activation of a number of cellular genes involved in cell cycle regulation, differentiation, and proliferation. Stability of ICN is tightly regulated by the Sel10-mediated ubiquitin-proteasome pathway. Sel10 can function as a negative regulator of Notch and exhibits activities of a tumor-suppressor protein. This article shows that the Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) directly interacts with Sel10 and forms a complex in KSHV-infected cells. This results in suppression of ICN ubiquitination and degradation. The carboxyl terminus of LANA interacts with the F-box and WD40 domains of Sel10 and competes with ICN for binding to Sel10. This elevated level of ICN is also critical for maintaining the enhanced proliferation of KSHV-infected tumor cells. These findings describe a mechanism by which the KSHV-encoded LANA protein regulates ubiquitination of ICN mediated by the F-box component of the E3 ligase Sel10, leading to proliferation of the virus-infected cells.
Assuntos
Antígenos Virais/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Herpesvirus Humano 8/metabolismo , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Antígenos Virais/genética , Sítios de Ligação , Proteínas de Ciclo Celular/química , Linhagem Celular , Proliferação de Células , Transformação Celular Viral , Proteínas F-Box/química , Proteína 7 com Repetições F-Box-WD , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/patogenicidade , Humanos , Modelos Biológicos , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Proto-Oncogenes , Receptores Notch/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
Epstein-Barr virus (EBV) infects and transforms resting B lymphocytes in vitro. The virus can also cause B cell lymphomas in immunosuppressed humans. Indeed, EBV-mediated post-transplant lymphoproliferative disease causes significant complications in transplant recipients, including loss of the transplanted organ and even death. The limited treatment options include, nonspecific targeting of B cell surface antigens with monoclonal antibodies or withdrawal of immunosuppression. These therapies fail in approximately 50% of patients. Clearly, treatments that specifically target EBV-infected cells are desirable. The EBV antigen EBNA3C regulates cell cycle by targeting critical cellular complexes such as cyclin A/cdk2, SCF(Skp2), and Rb. Here, we use a 20-amino-acid EBNA3C-derived peptide, fused to an HIV TAT tag for efficient delivery, to disrupt cell cycle regulation by EBNA3C. The peptide inhibited hyperproliferation of EBV-infected B cell lines and reduced in vitro immortalization of primary B lymphocytes by EBV. Importantly, the peptide inhibited lymphoblastoid outgrowth from the blood of an EBV-positive transplant patient in vitro.
Assuntos
Antígenos Virais , Linfócitos B/citologia , Linfócitos B/virologia , Proliferação de Células/efeitos dos fármacos , Herpesvirus Humano 4/patogenicidade , Fragmentos de Peptídeos/farmacologia , Antígenos Virais/genética , Antígenos Virais/metabolismo , Transformação Celular Viral , Células Cultivadas , Antígenos Nucleares do Vírus Epstein-Barr , Feminino , Produtos do Gene tat/genética , Produtos do Gene tat/farmacologia , Inibidores do Crescimento/farmacologia , Humanos , Transplante de Rim , Linfoma/patologia , Linfoma/virologia , Fragmentos de Peptídeos/farmacocinética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Latency-associated nuclear antigen (LANA) of KSHV is expressed in all forms of Kaposi's sarcoma-associated herpesvirus (KSHV)-mediated tumors and is important for TR-mediated replication and persistence of the virus. LANA does not exhibit any enzymatic activity by itself but is critical for replication and maintenance of the viral genome. To identify LANA binding proteins, we used a LANA binding sequence 1 DNA affinity column and determined the identities of a number of proteins associated with LANA. One of the identified proteins was uracil DNA glycosylase 2 (UNG2). UNG2 is important for removing uracil residues yielded after either misincorporation of dUTP during replication or deamination of cytosine. The specificity of the 'LANA-UNG2 interaction was confirmed by using a scrambled DNA sequence affinity column. Interaction of LANA and UNG2 was further confirmed by in vitro binding and coimmunoprecipitation assays. Colocalization of these proteins was also detected in primary effusion lymphoma (PEL) cells, as well as in a cotransfected KSHV-negative cell line. UNG2 binds to the carboxyl terminus of LANA and retains its enzymatic activity in the complex. However, no major effect on TR-mediated DNA replication was observed when a UNG2-deficient (UNG(-/-)) cell line was used. Infection of UNG(-/-) and wild-type mouse embryonic fibroblasts with KSHV did not reveal any difference; however, UNG(-/-) cells produced a significantly reduced number of virion particles after induction. Interestingly, depletion of UNG2 in PEL cells with short hairpin RNA reduced the number of viral genome copies and produced infection-deficient virus.
Assuntos
Antígenos Virais/metabolismo , DNA Glicosilases/metabolismo , Herpesvirus Humano 8/fisiologia , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Sequências Repetidas Terminais , Latência Viral/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Humanos , Imunoprecipitação , Linfócitos/química , Linfoma/virologia , Camundongos , Modelos Biológicos , Ligação ProteicaRESUMO
Pim kinases are proto-oncogenes that are upregulated in a number of B cell cancers, including Epstein-Barr Virus (EBV) associated Burkitt's lymphoma. They have also been shown to be upregulated in Kaposi sarcoma-associated herpes virus (KSHV) infected primary B cells. Most cells in KSHV-associated tumors are latently infected and express only a small subset of viral genes, with KSHV latency associated nuclear antigen (LANA) being constitutively expressed. LANA regulates the transcription of a large number of cellular and viral genes. Here, we show that LANA upregulates transcription from the Pim-1 promoter (pPim-1) and map this activation to a region in the promoter located within the sequence (-681 to +37). We show that LANA expressing cells can proliferate faster and are better protected from drug induced apoptosis. Since transition through cell cycle check points and anti-apoptosis are functions associated with Pim-1, it is likely that higher Pim-1 expression in cells expressing LANA is responsible, at least in part, for this effect. A Pim-1 phosphorylation site was also identified within the amino-terminal domain of LANA. Using in vitro kinase assays, we confirmed that LANA was indeed a Pim-1 substrate, and the failure of Pim-1 to phosphorylate LANA mutated at SS205/6RR identified this site as the specific serine residues phosphorylated by Pim-1. This report provides valuable insight into yet another cellular signaling pathway subverted by KSHV LANA and suggests a contribution to KSHV related oncogenesis.
Assuntos
Antígenos Virais/metabolismo , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Regulação para Cima , Antígenos Virais/genética , Apoptose , Linfócitos B/metabolismo , Linhagem Celular , Proliferação de Células , Células Epiteliais/metabolismo , Humanos , Proteínas Nucleares/genética , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-pim-1/genética , Transdução de Sinais , Especificidade por Substrato , Transcrição GênicaRESUMO
The epidermis is an attractive target for gene therapy because it is easily accessible and has great potential as an ectopic site for protein delivery in vivo. Genetically modified keratinocytes can be expanded in culture and used to generate three-dimensional skin equivalents, which can deliver therapeutic proteins either locally or systemically for the treatment of wounds or systemic diseases. Here we present an optimum protocol that yields consistently high retroviral gene transfer on a substrate of recombinant fibronectin (rFN). Gene transfer on rFN depends strongly on virus concentration and the density of target cells. Interestingly, the kinetics of gene transfer varies depending upon the origin--mouse or human--of virus-producer cells. Most notably, long-term growth and clonogenic assays show that transduction on rFN promotes gene transfer to epidermal stem cells and prevents loss of clonogenic potential due to exposure of cells to retroviral supernatant. In contrast, the traditional protocol transduces mostly differentiated keratinocytes. We also show that skin equivalents prepared from genetically modified keratinocytes display high levels of transgene expression, mainly in the suprabasal layers. Our results are important for cutaneous gene therapy and for biological studies that require efficient and permanent genetic modification.
Assuntos
Células Epidérmicas , Fibronectinas/química , Queratinócitos/metabolismo , Retroviridae/genética , Células-Tronco/metabolismo , Transdução Genética/métodos , Animais , Vetores Genéticos/genética , Humanos , Queratinócitos/química , Camundongos , Retroviridae/química , Células-Tronco/química , Engenharia Tecidual/métodos , Transgenes/genéticaRESUMO
Recombinant retroviruses have been shown to bind to fibronectin (FN) and increase the efficiency of gene transfer to a variety of cell types. Despite recent work to optimize gene transfer on recombinant FN, the mechanism of retrovirus binding to FN and the interactions of target cells with the bound virus remain elusive. We investigated the roles of virus surface glycoprotein (gp70), cell-conditioned medium, and proteoglycans in mediating retrovirus binding to FN. We also examined the role of Polybrene (PB) in these interactions. We found that gp70 is not involved in retrovirus binding to FN. Immobilization of the virus, however, does not overcome its receptor requirement, and gp70 is still needed for successful gene transfer. Our results clearly show that retrovirus binds FN through virus-associated heparan sulfate (HS) and that binding is necessary for transduction without PB. Two distinct modes of gene transfer occur depending on PB: (i) in the presence of PB, retrovirus interacts directly with the target cells; and (ii) in the absence of PB, retrovirus binds to FN and target cells interact with the immobilized virus. PB may promote the former mode by interacting with the virus HS and reducing the negative charge of the viral particles. Interestingly, the latter mode is more efficient, leading to significantly enhanced gene transfer. A better understanding of these interactions may provide insight into virus-cell interactions and lead to a more rational design of transduction protocols.