RESUMEN
Human immunodeficiency virus type 1 (HIV-1) Tat, an important regulator of viral transcription, interacts with diverse cellular proteins and promotes or inhibits cell proliferation. Here, we show that ribosomal protein S3 (RPS3) plays an important role in mitosis through an interaction with α-tubulin and that Tat binds to and inhibits the localization of RPS3 in the mitotic spindle during mitosis. RPS3 colocalized with α-tubulin around chromosomes in the mitotic spindle. Depletion of RPS3 promoted α-tubulin assembly, while overexpression of RPS3 impaired α-tubulin assembly. Depletion of RPS3 resulted in aberrant mitotic spindle formation, segregation failure, and defective abscission. Moreover, ectopic expression of RPS3 rescued the cell proliferation defect in RPS3-knockdown cells. HIV-1 Tat interacted with RPS3 through its basic domain and increased the level of RPS3 in the nucleus. Expression of Tat caused defects in mitotic spindle formation and chromosome assembly in mitosis. Moreover, the localization of RPS3 in the mitotic spindle was disrupted when HIV-1 Tat was expressed in HeLa and Jurkat cells. These results suggest that Tat inhibits cell proliferation via an interaction with RPS3 and thereby disrupts mitotic spindle formation during HIV-1 infection. These results might provide insight into the mechanism underlying lymphocyte pathogenesis during HIV-1 infection.
Asunto(s)
Infecciones por VIH/metabolismo , VIH-1/fisiología , Proteínas Ribosómicas/metabolismo , Huso Acromático/virología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Proliferación Celular , Células HEK293 , Infecciones por VIH/patología , Infecciones por VIH/virología , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Células Jurkat , Huso Acromático/metabolismo , Huso Acromático/patología , Tubulina (Proteína)/metabolismoRESUMEN
The Human Papillomavirus (HPV) E2 protein, which inhibits the E6 and E7 viral oncogenes, is believed to have anti-oncogenic properties. Here, we challenge this view and show that HPV-18 E2 over-activates the Spindle Assembly Checkpoint (SAC) and induces DNA breaks in mitosis followed by aneuploidy. This phenotype is associated with interaction of E2 with the Mitotic Checkpoint Complex (MCC) proteins Cdc20, MAD2 and BUBR1. While BUBR1 silencing rescues the mitotic phenotype induced by E2, p53 silencing or presence of E6/E7 (inactivating p53 and increasing BUBR1 levels respectively) both amplify it. This work pinpoints E2 as a key protein in the initiation of HPV-induced cervical cancer and identifies the SAC as a target for oncogenic pathogens. Moreover, our results suggest a role of p53 in regulating the mitotic process itself and highlight SAC over-activation in a p53-negative context as a highly pathogenic event.
Asunto(s)
Aneuploidia , Papillomavirus Humano 18/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación de la Expresión Génica , Células HeLa , Interacciones Huésped-Patógeno , Papillomavirus Humano 18/genética , Humanos , Queratinocitos/metabolismo , Queratinocitos/virología , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/virología , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Transducción de Señal , Huso Acromático/genética , Huso Acromático/virología , Factores de Tiempo , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virologíaRESUMEN
Tat, the transactivation factor of human immunodeficiency virus type 1 (HIV-1), represents one of the major players mediating the loss of CD4-positive T-lymphocytes in HIV-1-infected patients, primarily due to the ability of Tat to trigger apoptosis. However, the molecular events underlying this process remain elusive. In this study, we provide evidence that Tat interacts with Eg5, a microtubule-associated motor protein, and allosterically modulates the ATPase activity of Eg5 by affecting ADP release from the enzyme's active centre. This action of Tat impairs the formation of the mitotic spindle and activates the spindle checkpoint, thereby blocking cell cycle progression at mitosis and leading to apoptosis. Further studies reveal that lysine 85 in the carboxyl terminus of Tat is critical for its interaction with Eg5 and hence its effects on Eg5 activity, mitotic progression, and apoptosis. These findings identify Tat as a viral regulator of Eg5 and provide novel insights into the mechanisms of action of Tat in mediating the reduction of CD4-positive T-lymphocytes.
Asunto(s)
Apoptosis , Linfocitos T CD4-Positivos/enzimología , VIH-1/metabolismo , Cinesinas/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Huso Acromático/enzimología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , VIH-1/genética , Humanos , Hidrólisis , Células Jurkat , Cinesinas/genética , Lisina , Mitosis , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Huso Acromático/virología , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Cells expressing human papillomavirus type 16 (HPV-16) E6 and E7 proteins exhibit deregulation of G2/M genes, allowing bypass of DNA damage arrest signals. Normally, cells with DNA damage that override the G2 damage checkpoint would precociously enter mitosis and ultimately face mitotic catastrophe and apoptotic cell death. However, E6/E7-expressing cells (E6/E7 cells) have the ability to enter and exit mitosis in the presence of DNA damage and continue with the next round of the cell cycle. Little is known about the mechanism that allows these cells to gain entry into and exit from mitosis. Here, we show that in the presence of DNA damage, E6/E7 cells have elevated levels of cyclin B, which would allow entry into mitosis. Also, as required for exit from mitosis, cyclin B is degraded in these cells, permitting initiation of the next round of DNA synthesis and cell cycle progression. Proteasomal degradation of cyclin B by anaphase-promoting complex/cyclosome (APC/C) is, in part, due to elevated levels of the E2-conjugating enzyme, Ubch10, and the substrate recognition protein, Cdc20, of APC/C. Also, in E6/E7 cells with DNA damage, while Cdc20 is complexed with BubR1, indicating an active checkpoint, it is also present in complexes free of BubR1, presumably allowing APC/C activity and slippage through the checkpoint.
Asunto(s)
Proteínas de Ciclo Celular/genética , Regulación de la Expresión Génica , Papillomavirus Humano 16/fisiología , Queratinocitos/virología , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Proteínas Represoras/genética , Huso Acromático/virología , Enzimas Ubiquitina-Conjugadoras/genética , Proteínas Cdc20 , Ciclo Celular , Ciclina B/metabolismo , Daño del ADN , Humanos , Queratinocitos/metabolismo , Mitosis , Huso Acromático/patologíaRESUMEN
The nuclear mitotic apparatus (NuMA) protein is a component of the nuclear matrix in interphase cells and an essential protein for the formation of mitotic spindle poles. We used herpes simplex virus (HSV), an enveloped DNA virus that replicates in the nucleus, to study the intra-nuclear dynamics of NuMA in infected cells. This study shows that NuMA is extensively modified following HSV infection, including phosphorylation of an unidentified site(s), and that it depends to an extent on viral DNA synthesis. Although NuMA is insoluble in uninfected interphase cells, HSV infection induced solubilisation and dynamic relocalisation of NuMA, whereupon the protein became excluded from viral replication compartments -- sites of virus transcription and replication. Live cell, confocal imaging showed that NuMA localisation dramatically changed from the early stages (diffusely nuclear, excluding nucleoli) to late stages of infection (central diminuition, but remaining near the inner nuclear peripheries). In addition, NuMA knockdown using siRNA suggested that NuMA is important for efficient viral growth. In summary, we suggest that NuMA is required for efficient HSV infection, and identify further areas of research that address how the virus challenges host cell barriers.
Asunto(s)
ADN Viral/biosíntesis , Herpes Simple/genética , Herpesvirus Humano 1 , Interfase/fisiología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Huso Acromático/metabolismo , Animales , Antígenos Nucleares/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Chlorocebus aethiops , ADN Viral/ultraestructura , ADN Polimerasa Dirigida por ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Exodesoxirribonucleasas/metabolismo , Herpes Simple/patología , Humanos , Proteínas Asociadas a Matriz Nuclear/antagonistas & inhibidores , Proteínas Asociadas a Matriz Nuclear/genética , Ácido Fosfonoacético/farmacología , Fosforilación , Fosfotransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , ARN Interferente Pequeño , Huso Acromático/ultraestructura , Huso Acromático/virología , Transfección , Células Vero , Proteínas Virales/metabolismoRESUMEN
Incoming adenovirus type 2 (Ad2) and Ad5 shuttle bidirectionally along microtubules, biased to the microtubule-organizing center by the dynein/dynactin motor complex. It is unknown how the particles reach the nuclear pore complex, where capsids disassemble and viral DNA enters the nucleus. Here, we identified a novel link between nuclear export and microtubule-mediated transport. Two distinct inhibitors of the nuclear export factor CRM1, leptomycin B (LMB) and ratjadone A (RJA) or CRM1-siRNAs blocked adenovirus infection, arrested cytoplasmic transport of viral particles at the microtubule-organizing center or in the cytoplasm and prevented capsid disassembly and nuclear import of the viral genome. In mitotic cells where CRM1 is in the cytoplasm, adenovirus particles were not associated with microtubules but upon LMB treatment, they enriched at the spindle poles implying that CRM1 inhibited microtubule association of adenovirus. We propose that CRM1, a nuclear factor exported by CRM1 or a protein complex containing CRM1 is part of a sensor mechanism triggering the unloading of the incoming adenovirus particles from microtubules proximal to the nucleus of interphase cells.
Asunto(s)
Adenoviridae/metabolismo , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transporte Activo de Núcleo Celular , Adenoviridae/ultraestructura , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Endotelio Vascular/citología , Ácidos Grasos Insaturados/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Carioferinas/antagonistas & inhibidores , Microscopía Confocal , Centro Organizador de los Microtúbulos/efectos de los fármacos , Centro Organizador de los Microtúbulos/metabolismo , Mitosis , Pironas/farmacología , ARN Interferente Pequeño/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Huso Acromático/virología , Transferrina/farmacocinética , Venas Umbilicales/citología , Proteína Exportina 1RESUMEN
AIMS: Human herpesvirus 8 (HHV-8) has been identified in multicentric Castleman's disease and in angioimmunoblastic lymphadenopathies. However, the presence of the virus does not necessarily indicate an aetiological role in these conditions. This study investigates the cell types infected by HHV-8 in Castleman's disease and examines the correlation between HHV-8 and Castleman's disease lymph node angiogenesis. METHODS: Sixteen formalin fixed, paraffin wax embedded samples from patients with Castleman's disease (six multicentric, 10 solitary) were examined for the presence of HHV-8 using the polymerase chain reaction (PCR), non-isotopic in situ hybridisation, PCR in situ hybridisation (PCR-ISH), and real time quantitative TaqMan PCR to HHV-8 open reading frame 26 (ORF-26), and viral (v)-cyclin encoding regions. Vascularity was assessed using CD34, CD31, and factor VIII immunocytochemistry, and lymph nodes were scored as "low" or "high". RESULTS: Five multicentric Castleman's disease and two solitary Castleman's disease biopsies were positive for HHV-8. HHV-8 was identified in approximately 10% of intranodal B lymphocytes, in endothelial cells, and in subcapsular spindle cell proliferations. The copy number of HHV-8 was low at 10-50 copies/1000 cells. The highest copy number was in subcapsular spindle cells. There was no correlation between vascularity score and HHV-8 status. CONCLUSION: The preferential localisation of HHV-8 in subcapsular spindle cell proliferations (where early intranodal Kaposi's sarcoma initiates) and endothelial cells in Castleman's disease might finally explain the link between intranodal Kaposi's sarcoma and Castleman's disease.
Asunto(s)
Enfermedad de Castleman/virología , Herpesvirus Humano 8/aislamiento & purificación , Ganglios Linfáticos/irrigación sanguínea , Adulto , Antígenos CD34/metabolismo , Linfocitos B/virología , Enfermedad de Castleman/metabolismo , Endotelio Linfático/virología , Factor VIII/metabolismo , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/virología , Masculino , Persona de Mediana Edad , Sistemas de Lectura Abierta , Adhesión en Parafina , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Reacción en Cadena de la Polimerasa , Huso Acromático/virologíaRESUMEN
Modified viruses are used as gene transfer vectors because of their ability to transfer genetic material efficiently to the nucleus of a target cell. To better understand intracellular translocation of adenovirus serotype 5 (Ad), fluorophores were covalently conjugated to Ad capsids, and movement of fluorescent Ad within the cytoplasm was observed during the first hour of infection of a human lung epithelial carcinoma cell line (A549). Ad translocation was characterized with respect to its ability to achieve nuclear envelope localization as well as directed movement in the cytoplasm. Whereas Ad achieved efficient nuclear localization 60 min after infection of A549 cells under control conditions, depolymerization of the microtubule cytoskeleton by addition of 25 microM nocodazole reversibly inhibited development of nuclear localization. In contrast, depolymerization of microfilaments by addition of 1 microM cytochalasin D had no effect on nuclear localization. Direct video observation of Ad motility showed that nocodazole, but not cytochalasin D, caused a reversible decrease in rapid linear translocations of Ad in the cytoplasm of A549 cells. Microinjection of function-blocking antibodies against the microtubule-dependent motor protein, cytoplasmic dynein, but not kinesin, blocked nuclear localization of Ad, consistent with net minus end-directed motility indicated by accumulation of Ad at mitotic spindles. Fluorescence ratio imaging revealed a neutral pH in the environment of translocating Ad, leading to a model in which the interaction of Ad with an intact microtubule cytoskeleton and functional cytoplasmic dynein occurs after escape from endosomes and is a necessary prerequisite to nuclear localization of adenovirus serotype 5.
Asunto(s)
Adenoviridae/genética , Dineínas/fisiología , Endosomas/metabolismo , Vectores Genéticos/metabolismo , Microtúbulos/fisiología , Anticuerpos/administración & dosificación , Núcleo Celular/virología , Dineínas/inmunología , Humanos , Concentración de Iones de Hidrógeno , Microinyecciones , Microtúbulos/inmunología , Huso Acromático/virología , Células Tumorales CultivadasRESUMEN
The organization of the mitotic apparatus was studied in human embryo lung fibroblasts (HEL) and Vero cells at 4 days postinfection with human cytomegalovirus (HCMV) strain AD 169. The bipolar spindle was detected by immunofluorescence in p72-positive mitotic cells exhibiting a regular or C-metaphase-like chromosome configuration. Electron-microscopic study of C-metaphase-like cells revealed alteration of the centrosome structure which is characterized by the following features: (1) breakdown of the diplosome, (2) separation of the fibrillar material from centrioles, and (3) disruption of the centriolar cylinder. The spindle pole in the aberrant mitotic cells consisted of one or several foci of microtubules converging on the fibrillar aggregates. There are not any signs of the nuclear envelope reconstruction found in mitotic cells with highly condensed scattered chromosomes. Unlike in HEL cells, viral particles were not detected in Vero cells. A question arises as to whether centrosome injury is an integral part of the events leading to cell death unrelated to the reproduction of HCMV.