RESUMO
Human cytomegalovirus (HCMV) modulates numerous cellular pathways to facilitate infection. Iron is essential to many cellular processes and is often incorporated into proteins and enzymes involved in oxidative phosphorylation and DNA synthesis and repair, among others. Despite its prominent role in the cell, little is known about the regulation of iron metabolism during HCMV infection. Herein, we observe modulation of the transferrin receptor (TfR) during infection and a corresponding change in the cellular labile iron pool. TfR and the iron pool are increased early during infection and then return to mock levels at the late stages of infection. We identified the cellular ubiquitin ligase MARCH1 as an important regulator of TfR. MARCH1 plays a proviral role during infection, as its knockdown leads to a decrease in infectious titers. Knockdown of MARCH1 also leads to an increase in ROS, lipid peroxidation, and mitochondrial dysfunction. Inhibiting an early increase in TfR expression during infection also decreases virus production. These findings indicate the importance of tightly regulating iron metabolism during HCMV infection to facilitate efficient virus production. IMPORTANCE Iron is essential for cells, playing important roles in energy generation, DNA replication, and gene expression. During infection, HCMV alters many cellular processes to aid its replication. We found that iron levels are tightly regulated during infection and that dysregulation of iron levels alters the ability to produce infectious virions. We also found that HCMV inactivates many of the cellular safeguards put in place to deal with excess iron. Thus, infected cells become more susceptible to variations in iron levels, which could be exploited as a therapeutic strategy for dealing with HCMV infections.
Assuntos
Infecções por Citomegalovirus , Ferro , Ubiquitina-Proteína Ligases , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/fisiopatologia , Humanos , Ferro/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Both arginase (ARG2) and human cytomegalovirus (HCMV) have been implicated in tumorigenesis. However, the role of ARG2 in the pathogenesis of glioblastoma (GBM) and the HCMV effects on ARG2 are unknown. We hypothesize that HCMV may contribute to tumorigenesis by increasing ARG2 expression. RESULTS: ARG2 promotes tumorigenesis by increasing cellular proliferation, migration, invasion and vasculogenic mimicry in GBM cells, at least in part due to overexpression of MMP2/9. The nor-NOHA significantly reduced migration and tube formation of ARG2-overexpressing cells. HCMV immediate-early proteins (IE1/2) or its downstream pathways upregulated the expression of ARG2 in U-251 MG cells. Immunostaining of GBM tissue sections confirmed the overexpression of ARG2, consistent with data from subsets of Gene Expression Omnibus. Moreover, higher levels of ARG2 expression tended to be associated with poorer survival in GBM patient by analyzing data from TCGA. METHODS: The role of ARG2 in tumorigenesis was examined by proliferation-, migration-, invasion-, wound healing- and tube formation assays using an ARG2-overexpressing cell line and ARG inhibitor, N (omega)-hydroxy-nor-L-arginine (nor-NOHA) and siRNA against ARG2 coupled with functional assays measuring MMP2/9 activity, VEGF levels and nitric oxide synthase activity. Association between HCMV and ARG2 were examined in vitro with 3 different GBM cell lines, and ex vivo with immunostaining on GBM tissue sections. The viral mechanism mediating ARG2 induction was examined by siRNA approach. Correlation between ARG2 expression and patient survival was extrapolated from bioinformatics analysis on data from The Cancer Genome Atlas (TCGA). CONCLUSIONS: ARG2 promotes tumorigenesis, and HCMV may contribute to GBM pathogenesis by upregulating ARG2.
Assuntos
Arginase/biossíntese , Citomegalovirus/fisiologia , Glioblastoma/virologia , Arginase/genética , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Citomegalovirus/genética , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/virologia , Progressão da Doença , Glioblastoma/irrigação sanguínea , Glioblastoma/enzimologia , Glioblastoma/patologia , Humanos , Imuno-Histoquímica , Neovascularização Patológica/enzimologia , Neovascularização Patológica/patologia , Neovascularização Patológica/virologia , Transfecção , Regulação para CimaRESUMO
Human cytomegalovirus (HCMV) was reported to downregulate the Wnt/ß-catenin pathway. Induction of Axin1, the negative regulator of the Wnt pathway, has been reported as an important mechanism for inhibition of ß-catenin. Since Tankyrase (TNKS) negatively regulates Axin1, we investigated the effect of HCMV on TNKS expression and poly-ADP ribose polymerase (PARsylation) activity, during virus replication. Starting at 24 h post infection, HCMV stabilized the expression of TNKS and reduced its PARsylation activity, resulting in accumulation of Axin1 and reduction in its PARsylation as well. General PARsylation was not changed in HCMV-infected cells, suggesting specific inhibition of TNKS PARsylation. Similarly, treatment with XAV939, a chemical inhibitor of TNKS' activity, resulted in the accumulation of TNKS in both non-infected and HCMV-infected cell lines. Reduction of TNKS activity or knockdown of TNKS was beneficial for HCMV, evidenced by its improved growth in fibroblasts. Our results suggest that HCMV modulates the activity of TNKS to induce Axin1, resulting in inhibition of the ß-catenin pathway. Since HCMV replication is facilitated by TNKS knockdown or inhibition of its activity, TNKS may serve as an important virus target for control of a variety of cellular processes.
Assuntos
Infecções por Citomegalovirus/enzimologia , Citomegalovirus/fisiologia , Tanquirases/metabolismo , Via de Sinalização Wnt , Linhagem Celular Tumoral , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Interações Hospedeiro-Patógeno , Humanos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Tanquirases/genética , Replicação ViralRESUMO
Proteolytic shedding of ligands for the NK group 2D (NKG2D) receptor is a strategy used by tumors to modulate immune recognition by NK cells and cytotoxic T cells. A number of metalloproteases, especially those of the A disintegrin and metalloprotease (ADAM) family, can mediate NKG2D ligand cleavage and this process can be modulated by expression of the thiol isomerase ERp5. In this article, we describe that an increased shedding of the NKG2D ligand MICA is observed postinfection with several strains of human CMV due to an enhanced activity of ADAM17 (TNF-α converting enzyme) and matrix metalloprotease 14 caused by a reduction in the expression of the endogenous inhibitor of metalloproteases tissue inhibitors of metalloproteinase 3 (TIMP3). This decrease in TIMP3 expression correlates with increased expression of a cellular miRNA known to target TIMP3, and we also identify a human CMV-encoded microRNA able to modulate TIMP3 expression. These observations characterize a novel viral strategy to influence the shedding of cell-surface molecules involved in immune response modulation. They also provide an explanation for previous reports of increased levels of various ADAM17 substrates in the serum from patients with CMV disease. Consistent with this hypothesis, we detected soluble MICA in serum of transplant recipients with CMV disease. Finally, these data suggest that it might be worthwhile to prospectively study ADAM17 activity in a larger group of patients to assay whether this might be a useful biomarker to identify patients at risk for development of CMV disease.
Assuntos
Infecções por Citomegalovirus/imunologia , Regulação para Baixo/imunologia , Regulação Viral da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , MicroRNAs/genética , Inibidor Tecidual de Metaloproteinase-3/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Linhagem Celular Tumoral , Citomegalovirus/genética , Citomegalovirus/imunologia , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/genética , Regulação para Baixo/genética , Antígenos de Histocompatibilidade Classe I/sangue , Humanos , Metaloproteinase 14 da Matriz/sangue , Metaloproteinase 14 da Matriz/metabolismo , MicroRNAs/biossíntese , Cultura Primária de Células , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia , Inibidor Tecidual de Metaloproteinase-3/sangue , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
NK cells represent an important component of the innate immune response against infection and tumors. Age-associated changes in NK cell phenotype have been previously reported that can be responsible of functional NK cell deficiency. The aim of this work was to analyze the effect CMV seropositivity and aging on the distribution of NK cell subsets with a focus on the expression of cytotoxicity-related molecules and on the expression of CD94/NKG2 heterodimers and CD57 on these NK cell subsets. Our results show that CMV seropositivity in young individuals does not significantly affect peripheral blood NK cell percentage and NK cell subsets defined by the use of CD56 and CD16 markers. In contrast a significant increase in the percentage of NK cells is observed in elderly donors, all of them are CMV seropositive, when compared with young CMV seropositive subjects. A decrease in the percentage of CD56bright NK cells, either fully immature CD16 negative or CD16+ and an increase in the CD56-CD16+ subset are also found in the elderly. CMV seropositivity either in healthy young or elderly individuals is associated to the expression of CD94/NKG2C dimers and high expression of CD57on the CD56dimCD16+ NK cell subset. CD56-CD16+ NK cells, which are expanded in the elderly, show a decreased expression of granzymes A and B and an increased expression of CD94/NKG2C and CD57 in CMV seropositive young donors when compared with CMV seronegative young individuals. These results indicate that CMV and age have a different effect on NK cell phenotype and emphasize the relevance of including the determination of CMV serostatus in those studies addressed to analyze the immune response in the elderly.
Assuntos
Infecções por Citomegalovirus/imunologia , Células Matadoras Naturais/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígeno CD56/metabolismo , Antígenos CD57/metabolismo , Infecções por Citomegalovirus/enzimologia , Feminino , Granzimas/metabolismo , Humanos , Células Matadoras Naturais/enzimologia , Subpopulações de Linfócitos/enzimologia , Subpopulações de Linfócitos/virologia , Masculino , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Perforina/metabolismo , Adulto JovemRESUMO
Human cytomegalovirus infection can lead to life-threatening clinical manifestations particularly in the immunocompromised host. Current therapy options face severe limitations leading to a continued search for alternative drug candidates. Viral replication is dependent on a balanced interaction between viral and cellular proteins. Especially protein kinases are important regulators of virus-host interaction indicated by remarkable kinome alterations induced upon HCMV infection. Here we report a novel approach of kinome profiling with an outcome that suggests an important role of specific cellular protein kinases, such as AMPK, ABL2 and Aurora A. Inhibition of AMPK and ABL kinases showed a significant reduction, whereas inhibition of Aurora A kinase led to a slight activation of HCMV replication, as measured in a GFP reporter-based replication assay. Furthermore, analysis of the mode of antiviral action suggested a substantial benefit for the efficiency of viral replication at the immediate early (AMPK) or early-late (ABL) phases of HCMV gene expression. In contrast, inhibition of Aurora A kinase promoted an enhancement of viral early-late gene expression, suggesting a putative role of Aurora A signaling in host defense. Thus, the combined data provide new information on host cell kinases involved in viral replication and uncovered potential targets for future antiviral strategies.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aurora Quinase A/metabolismo , Infecções por Citomegalovirus/enzimologia , Citomegalovirus/fisiologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Antivirais/farmacologia , Antivirais/uso terapêutico , Aurora Quinase A/antagonistas & inibidores , Benzamidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Peptídeos e Proteínas de Sinalização Intracelular , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Ácido Nucleico/uso terapêutico , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Pirimidinas/farmacologia , Serina-Treonina Quinase 3 , Replicação Viral/efeitos dos fármacosRESUMO
Members of the cytomegalovirus family each encode two or more genes with significant homology to G-protein coupled receptors (GPCRs). In rodent models of pathogenesis, these viral encoded GPCRs play functionally significant roles, as their deletion results in crippled viruses that cannot traffic properly and/or replicate in virally important target cells. Of the four HCMV encoded GPCRs, US28 has garnered the most attention due to the fact that it exhibits both agonist-independent and agonist-dependent signaling activity and has been demonstrated to promote cellular migration and proliferation. Thus, it appears that the CMV GPCRs play important roles in viral replication in vivo as well as promote the development of virus-associated pathology. In the current study we have utilized a series of HCMV/US28 recombinants to investigate the expression profile and signaling activities of US28 in a number of cell types relevant to HCMV infection including smooth muscle cells, endothelial cells and cells derived from glioblastoma multiforme (GBM) tumors. The results indicate that US28 is expressed and exhibits constitutive agonist-independent signaling activity through PLC-ß in all cell types tested. Moreover, while CCL5/RANTES and CX3CL1/Fractalkine both promote US28-dependent Ca(++) release in smooth muscle cells, this agonist-dependent effect appears to be cell-specific as we fail to detect US28 driven Ca(++) release in the GBM cells. We have also investigated the effects of US28 on signaling via endogenous GPCRs including those in the LPA receptor family. Our data indicate that US28 can enhance signaling via endogenous LPA receptors. Taken together, our results indicate that US28 induces a variety of signaling events in all cell types tested suggesting that US28 signaling likely plays a significant role during HCMV infection and dissemination in vivo.
Assuntos
Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/metabolismo , Receptores de Quimiocinas/metabolismo , Fosfolipases Tipo C/metabolismo , Proteínas Virais/metabolismo , Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Quimiocina CCL5/farmacologia , Quimiocina CX3CL1/farmacologia , Citomegalovirus/enzimologia , Citomegalovirus/patogenicidade , Humanos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismoRESUMO
Human cytomegalovirus (HCMV) starts its lytic replication cycle only in the G(0)/G(1) phase of the cell division cycle. S/G(2) cells can be infected but block the onset of immediate-early (IE) gene expression. This block can be overcome by inhibition of cyclin-dependent kinases (CDKs), suggesting that cyclin A2, the only cyclin with an S/G(2)-specific activity profile, may act as a negative regulator of viral gene expression. To directly test this hypothesis, we generated derivatives of an HCMV-permissive glioblastoma cell line that express cyclin A2 in a constitutive, cell cycle-independent manner. We demonstrate that even moderate cyclin A2 overexpression in G(1) was sufficient to severely compromise the HCMV replicative cycle after high-multiplicity infection. This negative effect was composed of a strong but transient inhibition of IE gene transcription and a more sustained alteration of IE mRNA processing, resulting in reduced levels of UL37 and IE2, an essential transactivator of viral early gene expression. Consistently, cyclin A2-overexpressing cells showed a strong delay of viral early and late gene expression, as well as virus reproduction. All effects were dependent on CDK activity, as a cyclin A2 mutant deficient in CDK binding was unable to interfere with the HCMV infectious cycle. Interestingly, murine CMV, whose IE gene expression is known to be cell cycle independent, is not affected by cyclin A2. Instead, it upregulates cyclin A2-associated kinase activity upon infection. Understanding the mechanisms behind the HCMV-specific action of cyclin A2-CDK might reveal new targets for antiviral strategies.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina A2/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Infecções por Citomegalovirus/enzimologia , Citomegalovirus/genética , Regulação para Baixo , Proteínas Imediatamente Precoces/metabolismo , Transativadores/metabolismo , Animais , Proteína Quinase CDC2/genética , Ciclo Celular , Linhagem Celular Tumoral , Ciclina A2/genética , Quinase 2 Dependente de Ciclina/genética , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Regulação Viral da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , Camundongos , Transativadores/genéticaRESUMO
BACKGROUND: Human cytomegalovirus (HCMV) is the most common pathogen in uterus during pregnancy, which may lead to some serious results such as miscarriage, stillbirth, cerebellar malformation, fetus developmental retardation, but its pathogenesis has not been fully explained. The hypofunction of extravillous cytotrophoblast (EVT) invasion is the essential pathologic base of some complications of pregnancy. c-erbB-2 is a kind of oncogene protein and closely linked with embryogenesis, tissue repair and regeneration. Matrix metalloproteinase (MMP) is one of the key enzymes which affect EVT migration and invasion function. The expression level changes of c-erbB-2, MMP-2 and MMP-9 can reflect the changes of EVT invasion function. RESULTS: To explore the influence of HCMV on the invasion function of EVT, we tested the protein expression level changes of c-erbB-2, MMP-2 and MMP-9 in villous explant cultured in vitro infected by HCMV, with the use of immunohistochemistry SP method and western blot. We confirmed that HCMV can reproduce and spread in early pregnancy villus; c-erbB-2 protein mainly expressed in normal early pregnancy villous syncytiotrophoblast (ST) remote plasma membrane and EVT, especially remote EVT cell membrane in villous stem cell column, little expressed in ST proximal end cell membrane and interstitial cells; MMP-2 protein primarily expressed in early pregnancy villous EVT endochylema and rarely in villous trophoblast (VT), ST and interstitial cells; MMP-9 protein largely expressed in early pregnancy villous mesenchyme, EVT and VT endochylema. Compared with control group, the three kinds of protein expression level in early pregnancy villus of virus group significantly decreased (P < 0.05). CONCLUSION: HCMV can infect villus in vitro and cause the decrease of early pregnancy villous EVT's invasion function.
Assuntos
Diferenciação Celular , Vilosidades Coriônicas/virologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Complicações na Gravidez/virologia , Trofoblastos/citologia , Trofoblastos/virologia , Vilosidades Coriônicas/enzimologia , Vilosidades Coriônicas/fisiopatologia , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/fisiopatologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Gravidez , Complicações na Gravidez/enzimologia , Complicações na Gravidez/genética , Complicações na Gravidez/fisiopatologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Trofoblastos/enzimologiaRESUMO
BACKGROUND: Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that typically causes asymptomatic infections in healthy individuals but may lead to serious complications in newborns and immunodeficient individuals. The emergence of drug-resistant strains of HCMV has posed a need for the development of new drugs and treatment strategies. Antisense molecules are promising gene-targeting agents for specific regulation of gene expression. External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. The UL49-deletion BAC of HCMV was significantly defective in growth in human foreskin fibroblasts. Therefore, UL49 gene may serve as a potential target for novel drug development to combat HCMV infection. In this study, DNA-based EGS molecules were synthesized to target the UL49 mRNA of human cytomegalovirus (HCMV). RESULTS: By cleavage activity assessing in vitro, the EGS aimed to the cleavage site 324 nt downstream from the translational initiation codon of UL49 mRNA (i.e. EGS324) was confirmed be efficient to direct human RNase P to cleave the target mRNA sequence. When EGS324 was exogenously administered into HCMV-infected human foreskin fibroblasts (HFFs), a significant reduction of ~76% in the mRNA and ~80% in the protein expression of UL49 gene, comparing with the cells transfected with control EGSs. Furthermore, a reduction of about 330-fold in HCMV growth were observed in HCMV-infected HFFs treated with the EGS. CONCLUSIONS: These results indicated that UL49 gene was essential for replication of HCMV. Moreover, our study provides evidence that exogenous administration of a DNA-based EGS can be used as a potential therapeutic approach for inhibiting gene expression and replication of a human virus.
Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Regulação para Baixo/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Oligonucleotídeos/farmacologia , Ribonuclease P/metabolismo , Proteínas Estruturais Virais/genética , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , Infecções por Citomegalovirus/enzimologia , Humanos , Oligonucleotídeos/síntese química , Oligonucleotídeos/genética , Proteínas Estruturais Virais/metabolismoRESUMO
BACKGROUND: The mechanism by which human cytomegalovirus (HCMV) stimulates oncogenesis is unclear. Because cellular immortalization and transformation require telomerase activation by expression of the telomerase reverse transcriptase (hTERT) gene, we examined the role of HCMV in telomerase activation. METHODS: Normal human diploid fibroblasts (HDFs) and human malignant glioma (MG) cell lines were infected with HCMV or transfected with expression vectors encoding HCMV immediate early (IE) antigen 72 or 86. hTERT expression and promoter activity and telomerase activity were evaluated using reverse transcription-polymerase chain reaction, a luciferase reporter assay, and a telomeric repeat amplification protocol, respectively. hTERT promoter occupancy by the transcription factor Sp1, IE antigens, and histone deacetylases (HDACs) was assessed by chromatin immunoprecipitation. hTERT and IE protein expression in human primary glioblastoma multiforme (GBM) was determined immunohistochemically. All statistical tests were two-sided. RESULTS: In telomerase and hTERT-negative HDFs, HCMV infection induced constitutive hTERT expression and telomerase activation. The hTERT promoter activity in HDFs and MG cell lines was statistically significantly enhanced by HCMV in a dose-dependent manner (mean luciferase activity [arbitrary units] in control HDFs and in HDFs infected with HCMV at multiplicities of infection [MOIs] of 0.1 = 6 and 521, respectively, difference = 515, 95% CI = 178 to 850; mean activity at MOI of 1 and 10 = 8828 and 59,923, respectively; P < .001 comparing control with HCMV-infected cells at all MOIs). Ectopic expression of HCMV IE-72 protein also stimulated hTERT promoter activity in HDFs. HCMV-mediated transactivation of the hTERT gene was dependent on the presence of Sp1-binding sites in the hTERT promoter and was accompanied by increases in Sp1 binding, acetylation of histone H3, and a reduction in HDAC binding at the core promoter. In specimens of GBM, HCMV IE and hTERT proteins were colocalized in malignant cells and their levels paralleled each other. CONCLUSIONS: HCMV activates telomerase in both HDFs and malignant cells. These findings begin to reveal a novel mechanism by which HCMV infection may be linked to or modulate oncogenesis through telomerase activation.
Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/metabolismo , Fibroblastos/metabolismo , Glioma/enzimologia , Telomerase/metabolismo , Telômero/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Infecções por Citomegalovirus/enzimologia , Ativação Enzimática , Fibroblastos/enzimologia , Imunofluorescência , Glioblastoma/metabolismo , Glioma/metabolismo , Glioma/virologia , Humanos , Immunoblotting , Imuno-Histoquímica , Luciferases/metabolismo , Vírus Oncogênicos/metabolismo , RNA Mensageiro/metabolismo , Projetos de Pesquisa , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/genética , Telômero/genéticaRESUMO
External Guide Sequences (EGSs) represents a novel nucleic acid based gene interference approach to modulate gene expression. They are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. DNA-based EGS1386 with a size of 12 nt was chemically synthesized to target the mRNA coding for the UL49 gene of human cytomegalovirus (HCMV). The DNA-based EGS1386 molecule efficiently directed human RNase P to cleave the target mRNA sequence in vitro. A reduction of more than 50% in the levels of UL49 expression was observed in human cells treated with the DNA-based EGS1386 targeted UL49 assayed by fluorescent quantization PCR and Western blotting. This results showed that the DNA-EGS1386 can effectively guide the RNase P cut the target mRNA. Therefore, DNA-EGS can develop into a new gene silencing technology and potential of the anti-viral reagents.
Assuntos
Citomegalovirus/genética , DNA Viral/genética , Oligodesoxirribonucleotídeos/farmacologia , Ribonuclease P/metabolismo , Sequência de Bases , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/virologia , Evolução Molecular Direcionada/métodos , Regulação Viral da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease P/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Pequeno RNA não TraduzidoRESUMO
Rejection episodes and infections are common problems after organ transplantations (TX). Rejection can be diagnosed in liver-transplant (LTX) patients when liver-specific enzymes in the serum are elevated. As endomyocardial biopsy (EMB) is the gold standard for detecting heart transplant (HTX) rejection, serum parameters would permit more selective use of this invasive procedure. Cytomegalovirus (CMV) infections can have serious consequences for TX patients and so should be diagnosed and treated timely. At present, there are no suitable diagnostic methods other than CMV antigen pp65 and CMV polymerase chain reaction (PCR). Our study aimed to test the sensitivity of myeloperoxidase (MPO), an enzyme of neutrophilic granulocytes, as a new serum parameter in addition to established serum parameters and EMB for diagnosis of infection and rejection episodes after LTX and HTX. MPO in plasma from 246 blood samples (103 used for statistical analysis) from 27 patients (18 LTX and 9 HTX) was determined using ELISA; C-reactive protein (CRP), gamma-glutamyl-transpeptidase (GGT), white blood count and CMV pp65 antigen were monitored routinely. EMBs were performed at defined intervals after HTX. Results were analyzed with descriptive statistics, T-test, Wilcoxon test and Cox regression analysis, whereby a p<0.05 was viewed as significant. MPO values in TX patients with an infection (7 LTX, 2 HTX) were significantly higher than in TX patients without complications (control group) (253.9 microg/l vs. 116.6 microg/l, p=0.0194). In TX patients with rejections (6 LTX, 6 HTX), there is also a significant increase in comparison to controls (429.7 microg/l vs. 116.6 microg/l, p=0.0001). Data from individual TX patients, however, indicate that MPO levels rise distinctly earlier with infection (CMV) than with rejection, enabling earlier detection of the complication and initiation of suitable treatment. Our findings suggest that a larger and prospective study should be designed to evaluate the usefulness of MPO levels in assessing organ transplant recipients.
Assuntos
Infecções por Citomegalovirus/diagnóstico , Rejeição de Enxerto/diagnóstico , Transplante de Coração , Transplante de Fígado , Peroxidase/sangue , Adulto , Biomarcadores/sangue , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/enzimologia , Ensaio de Imunoadsorção Enzimática , Feminino , Rejeição de Enxerto/sangue , Rejeição de Enxerto/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , Padrões de Referência , Estudos RetrospectivosRESUMO
Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in the kynurenine pathway of tryptophan catabolism and has been implicated in neurotoxicity and suppression of the antiviral T-cell response in HIV encephalitis (HIVE). Here we show that the Toll-like receptor 3 (TLR3) ligand poly(I:C) (PIC) induces the expression of IDO in human astrocytes. PIC was less potent than gamma interferon (IFN-gamma) but more potent than IFN-beta in inducing IDO. PIC induction of IDO was mediated in part by IFN-beta but not IFN-gamma, and both NF-kappaB and interferon regulatory factor 3 (IRF3) were required. PIC also upregulated TLR3, thereby augmenting the primary (IFN-beta) and secondary (IDO and viperin) response genes upon subsequent stimulation with PIC. In HIVE, the transcripts for TLR3, IFN-beta, IDO, and viperin were increased and IDO immunoreactivity was detected in reactive astrocytes as well as macrophages and microglia. PIC caused suppression of intracellular replication of human immunodeficiency virus pseudotyped with vesicular stomatitis virus G protein and human cytomegalovirus in a manner dependent on IRF3 and IDO. The involvement of IDO was demonstrated by partial but significant reversal of the PIC-mediated antiviral effect by IDO RNA interference and/or tryptophan supplementation. Importantly, the cytokine interleukin-1 abolished IFN-gamma-induced IDO enzyme activity in a nitric oxide-dependent manner without suppressing protein expression. Our results demonstrate that IDO is an innate antiviral protein induced by double-stranded RNA and suggest a therapeutic utility for PIC in human viral infections. They also show that IDO activity can be dissociated from protein expression, indicating that the local central nervous system cytokine and nitric oxide environment determines IDO function.
Assuntos
Astrócitos/imunologia , Encefalite Viral/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Indutores de Interferon/farmacologia , Poli I-C/farmacologia , Receptor 3 Toll-Like/imunologia , Replicação Viral/imunologia , Astrócitos/enzimologia , Astrócitos/virologia , Células Cultivadas , Citocinas/imunologia , Citocinas/farmacologia , Citomegalovirus/genética , Citomegalovirus/imunologia , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Encefalite Viral/enzimologia , Encefalite Viral/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Infecções por HIV/enzimologia , Infecções por HIV/genética , HIV-1/genética , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/biossíntese , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Cinurenina/genética , Cinurenina/imunologia , Cinurenina/metabolismo , Ligantes , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/virologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Microglia/enzimologia , Microglia/imunologia , Microglia/virologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Óxido Nítrico/imunologia , Óxido Nítrico/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas/imunologia , Proteínas/metabolismo , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/metabolismo , Triptofano/imunologia , Triptofano/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismoRESUMO
Matrix metalloproteinases (MMPs) have been identified as mediators of brain injury in HIV-associated neurological diseases. The activity of the 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) was detected by zymography in the cerebrospinal fluid (CSF) of 138 HIV-infected patients (40 with AIDS dementia, 83 with brain opportunistic infections and 15 neurologically asymptomatic), 26 HIV-seronegative individuals with inflammatory neurological diseases (IND) and 12 HIV-seronegative subjects with noninflammatory neurological diseases (NIND). MMP-2 was present in all CSF samples from HIV-seropositive and HIV-seronegative individuals, including those of subjects with NIND. On the contrary, MMP-9 was absent in the CSF of NIND controls, whereas the activity of this MMP was found in the 77 - 100% of CSF samples from HIV-infected patients, including those with HIV dementia, central nervous system (CNS) opportunistic infections or neurologically asymptomatic subjects. The highest levels of MMP-9 were found in the CSF of patients with cryptococcosis, cytomegalovirus encephalitis and tuberculous meningitis and were comparable with those found in the CSF of HIV-negative patients with multiple sclerosis or meningitis. A significant correlation between CSF MMP-9 activity and CSF cell count was found only in patients with HIV dementia. The increased CSF activity of MMPs capable to degrade components of the extracellular matrix of blood-brain barrier may contribute to the transendothelial migration of virus-infected cells into the CNS and development of HIV-associated neurologic damage.
Assuntos
Complexo AIDS Demência/líquido cefalorraquidiano , Infecções Oportunistas Relacionadas com a AIDS/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/análise , Infecções por HIV/líquido cefalorraquidiano , HIV-1 , Metaloproteinase 2 da Matriz/líquido cefalorraquidiano , Metaloproteinase 9 da Matriz/líquido cefalorraquidiano , Doenças do Sistema Nervoso/líquido cefalorraquidiano , Complexo AIDS Demência/enzimologia , Infecções Oportunistas Relacionadas com a AIDS/enzimologia , Adolescente , Adulto , Barreira Hematoencefálica , Contagem de Linfócito CD4 , Contagem de Células , Movimento Celular , Líquido Cefalorraquidiano/citologia , Criança , Pré-Escolar , Infecções por Citomegalovirus/líquido cefalorraquidiano , Infecções por Citomegalovirus/enzimologia , Progressão da Doença , Encefalite Viral/líquido cefalorraquidiano , Encefalite Viral/enzimologia , Feminino , Infecções por HIV/enzimologia , Soronegatividade para HIV , Humanos , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , Meningites Bacterianas/líquido cefalorraquidiano , Meningites Bacterianas/enzimologia , Meningite Criptocócica/líquido cefalorraquidiano , Meningite Criptocócica/enzimologia , Pessoa de Meia-Idade , Doença dos Neurônios Motores/líquido cefalorraquidiano , Doença dos Neurônios Motores/enzimologia , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/enzimologia , Doenças do Sistema Nervoso/enzimologia , Toxoplasmose Cerebral/líquido cefalorraquidiano , Toxoplasmose Cerebral/enzimologia , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Meníngea/enzimologiaRESUMO
Recent evidence indicates activated mitogen-activated protein kinase (MAPK) p38 has a critical function in human cytomegalovirus (HCMV) viral DNA replication in infected human fibroblasts. To elucidate the mechanism of HCMV-mediated p38 activation, we have performed a detailed analysis of p38 activation and the kinases associated with this activation at different times postinfection. We demonstrate that p38 kinase activity is strongly increased following viral infection. Inhibition of this activity significantly inhibited HCMV-induced hyperphosphorylation of pRb and phosphorylation of heat shock protein 27, suggesting that p38 activation is involved in virus-mediated changes in host cell metabolism throughout the course of infection. We then provide evidence that p38 activation is mediated by different mechanisms at early times versus later times of infection. At early times of infection (8 to 14 h postinfection [hpi]), when p38 activation is first observed, no significant activation of the three kinases which can directly phosphorylate p38 (namely, MKK3, MKK6, and MKK4) is detected. Using vectors which express dominant negative proteins, we demonstrate that basal MKK6 kinase activity is necessary for HCMV-mediated p38 activation at these early times of infection (12 hpi). Then, we use ATP depletion to show that at 12 hpi, HCMV inhibits dephosphorylation of activated p38. These two experiments suggest that HCMV activates p38 by inhibition of dephosphorylation of p38. In contrast to early times of infection, at later times of infection (48 to 72 hpi), increased MKK3/6, but not MKK4, activity is observed. These results indicate that at early times of HCMV infection, increased steady-state levels of activated p38 is mediated at least in part by inhibition of dephosphorylation of p38, while at later times of infection p38 activation is due to increased activity of the upstream kinases MKK3 and MKK6. These findings indicate that HCMV has developed multiple mechanisms to ensure activation of the MAPK p38, a kinase critical to viral infection.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Proteínas de Choque Térmico , MAP Quinase Quinase 4 , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Trifosfato de Adenosina/metabolismo , Western Blotting , Células Cultivadas , Infecções por Citomegalovirus/enzimologia , Ativação Enzimática , Proteínas de Choque Térmico HSP27 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Cinética , MAP Quinase Quinase 3 , MAP Quinase Quinase 6 , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Chaperonas Moleculares , Proteínas de Neoplasias/metabolismo , Fosforilação , Testes de Precipitina , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Orthotopic liver transplantation (OLT) is now the only available treatment for end-stage liver disease; the major postoperative complications of OLT are rejection and infection. Fractionation of alkaline phosphatase (ALP) isoforms in serum by isoelectric focusing can be used to identify patients with complications. Reference ranges for liver-function tests (LFT) and liver ALP isoforms were established for post-OLT patients with stable postoperative courses and compared with those of patients with complications. We found canalicular, hepatocyte, and high-molecular-mass ALP to be statistically higher in nearly all patients with complications as compared with patients who had a stable postoperative course; these tests may identify patients requiring a liver biopsy. When used in conjunction with LFT and other clinical findings, ALP isoforms could aid in the monitoring of complications and treatment and in the adjustment of immunosuppressive therapy in stable OLT cases.
Assuntos
Fosfatase Alcalina/sangue , Isoenzimas/sangue , Transplante de Fígado , Adolescente , Adulto , Idoso , Doenças Biliares/enzimologia , Doenças Biliares/etiologia , Colangite/enzimologia , Colangite/etiologia , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/etiologia , Feminino , Rejeição de Enxerto/enzimologia , Humanos , Focalização Isoelétrica , Transplante de Fígado/efeitos adversos , Masculino , Pessoa de Meia-Idade , Valores de ReferênciaRESUMO
PURPOSE: To analyze the cortical gyral patterns and myelination patterns in a series of patients with congenital cytomegalovirus infections involving the central nervous system, to correlate them with known developmental events, and to develop a consistent theory regarding their embryogenesis. METHODS: The MR (11 patients) and CT (four patients) studies of 11 patients with congenital cytomegalovirus infections involving the brain were retrospectively reviewed. Analysis was made of myelination patterns, cortical gyral patterns, other areas of maldeveloped brain, and focal brain lesions. RESULTS: Lissencephaly was found in four patients. These patients had very thin cerebral cortices, extremely diminished volume of white matter, delayed myelination, small cerebella, and very enlarged lateral ventricles. Focal areas of dysplastic cortex, presumably polymicrogyria, were found in five patients. These patients had slightly thickened irregular cerebral cortices, slightly diminished volume of white matter, delayed myelination, variably small cerebella, and slightly enlarged lateral ventricles. Two patients had normal cerebral cortices, slightly diminished volume of white matter, delayed myelination, normal cerebella, and slightly enlarged lateral ventricles. Periventricular lesions, representing calcification, or perhaps blood, were seen in all groups. CONCLUSIONS: We postulate that the patients with lissencephaly suffer injury before 16 or 18 weeks gestational age, whereas those with regions of polymicrogyria are injured between approximately 18 and 24 weeks gestational age. Those with normal gyral patterns are probably injured during the third trimester and may have active infections at birth. Moreover, we propose that the finding of cerebellar hypoplasia and myelination delay in association with diffuse lissencephaly or cortical dysplasia should suggest the diagnosis of congenital cytomegalovirus infection.
Assuntos
Encefalopatias/congênito , Encefalopatias/microbiologia , Infecções por Citomegalovirus/congênito , Imageamento por Ressonância Magnética , Tomografia Computadorizada por Raios X , Encefalopatias/diagnóstico , Encefalopatias/diagnóstico por imagem , Encefalopatias/embriologia , Calcinose/diagnóstico , Calcinose/diagnóstico por imagem , Doenças Cerebelares/diagnóstico , Doenças Cerebelares/diagnóstico por imagem , Cerebelo/patologia , Córtex Cerebral/patologia , Hemorragia Cerebral/diagnóstico , Hemorragia Cerebral/diagnóstico por imagem , Ventrículos Cerebrais/patologia , Criança , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/diagnóstico por imagem , Infecções por Citomegalovirus/enzimologia , Doenças Desmielinizantes/diagnóstico , Doenças Desmielinizantes/diagnóstico por imagem , Hipocampo/diagnóstico por imagem , Hipocampo/patologia , Humanos , Processamento de Imagem Assistida por Computador , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Bainha de Mielina/fisiologia , Estudos RetrospectivosRESUMO
CMV has been reported to be associated with a DNA polymerase activity (DPA). In this communication its purification, characterization and potential diagnostic value were examined. CMV DNA polymerase was prepared from cell free supernatants of CMV (AD 169) infected cultures. Separation and purification of the enzyme was accomplished by column chromatography of the purified, lysed virus. CMV DPA was measured on an oligo (dT)-poly (dA) template primer. SDS-PAGE and western blot analysis under reducing conditions using an anti-CMV early antibody showed an 80 kDa protein band that was associated with the peak of polymerase activity. However, CMV isolates and CMV from urines from CMV retinitis patients immunoblotted by the same Ab revealed 140 kDa and 80 kDa bands under non-reducing and reducing conditions respectively, the latter was also associated with a 58 kDa band. The diagnostic value of the CMV associated DAP was tested using CMV positive urines. The latter demonstrated high PAA-sensitive DPA activity, compared to normal, HSV positive urines and urines from HBSAg positive patients. Taken collectively, these findings indicate the potential usefulness of CMV-associated DNA polymerase activity in the diagnosis and follow-up of patients with CMV-related illnesses.
Assuntos
Infecções por Citomegalovirus/microbiologia , Citomegalovirus/enzimologia , DNA Polimerase Dirigida por DNA/isolamento & purificação , DNA Polimerase Dirigida por DNA/metabolismo , Células Cultivadas , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/urina , Retinite por Citomegalovirus/enzimologia , Retinite por Citomegalovirus/microbiologia , Retinite por Citomegalovirus/urina , DNA Polimerase Dirigida por DNA/química , Fibroblastos , Humanos , ImmunoblottingRESUMO
Infection of human diploid embryonic lung (MRC5) cells by human cytomegalovirus (HCMV), strain AD169, increased the activity of a key enzyme in the synthesis of polyamines: S-adenosylmethionine decarboxylase (E.C. 4.1.1.50). The initial peak of S-adenosylmethionine decarboxylase activity occurred about 15 h postinfection. S-Adenosylmethionine decarboxylase was purified using a highly specific affinity chromatography step from HCMV-infected and control uninfected MRC5 cells. No difference was found between the two enzymes in their stability to heat or effect of pH on activity. Both enzymes were activated only by putrescine. The appKm for S-adenosylmethionine for the virus-induced enzyme was 1.7 times higher than the appKm for the control enzyme. The most dramatic difference observed was in the effect of high salt concentration on enzyme activity. S-Adenosylmethionine decarboxylase from HCMV-infected cells was unaffected by 0.8 M NaCl, whereas the enzyme from uninfected cells was inhibited by 50% at 0.45 M NaCl and was significantly inhibited at a concentration of 0.8 M NaCl. Thus, different forms of S-adenosylmethionine decarboxylase probably exist in infected and uninfected MRC5 cells.