RESUMO
Rad50-interacting protein 1 (Rint1) associates with the DNA damage response protein Rad50 during the transition from the S phase to the G2/M phase and functions in radiation-induced G2 checkpoint control. It has also been demonstrated that Rint1 is essential in vesicle trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) through an interaction with Zeste-White 10 (ZW10). We have isolated a novel interaction between Rint1 and the human papillomavirus 16 (HPV16) transcription and replication factor E2. E2 binds to Rint1 within its ZW10 interaction domain, and we show that in the absence of E2, Rint1 is localized to the ER and associates with ZW10. E2 expression results in a disruption of the Rint1-ZW10 interaction and an accumulation of nuclear Rint1, coincident with a significant reduction in vesicle movement from the ER to the Golgi apparatus. Interestingly, nuclear Rint1 and members of the Mre11/Rad50/Nbs1 (MRN) complex were found in distinct E2 nuclear foci, which peaked during mid-S phase, indicating that the recruitment of Rint1 to E2 foci within the nucleus may also result in the recruitment of this DNA damage-sensing protein complex. We show that exogenous Rint1 expression enhances E2-dependent virus replication. Conversely, the overexpression of a truncated Rint1 protein that retains the E2 binding domain but not the Rad50 binding domain acts as a dominant negative inhibitor of E2-dependent HPV replication. Put together, these experiments demonstrate that the interaction between Rint1 and E2 has an important function in HPV replication.IMPORTANCE HPV infections are an important driver of many epithelial cancers, including those within the anogenital and oropharyngeal tracts. The HPV life cycle is tightly regulated and intimately linked to the differentiation of the epithelial cells that it infects. HPV replication factories formed in the nucleus are locations where viral DNA is copied to support virus persistence and amplification of infection. The recruitment of specific cellular protein complexes to these factories aids efficient and controlled viral replication. We have identified a novel HPV-host interaction that functions in the cellular response to DNA damage and cell cycle control. We show that the HPV E2 protein targets Rad50-interacting protein 1 (Rint1) to facilitate virus genome replication. These findings add to our understanding of how HPV replicates and the host cell pathways that are targeted by HPV to support virus replication. Understanding these pathways will allow further research into novel inhibitors of HPV genome replication.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Papillomavirus Humano 16/fisiologia , Proteínas Oncogênicas Virais/metabolismo , Replicação Viral , Sequência de Aminoácidos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Viral/biossíntese , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Origem de Replicação , Pontos de Checagem da Fase S do Ciclo CelularRESUMO
The metaphase chromosome spread technique and subsequent analysis of sister chromatid cohesion is used for (clinical) diagnosis of genetic abnormalities that can cause aberrant sister chromatid cohesion. In addition, the technique can be used to assess the contribution of novel genes to the cohesion establishment and maintenance pathways. Cells are swelled in a hypotonic solution and fixed in Carnoy's solution. Samples are then dropped onto glass slides, and the spread chromosomes are stained and visualized by microscopy. Defects in sister chromatid cohesion can be easily assessed using this method, examples of which are given.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Metáfase , Cromátides/ultraestrutura , Humanos , Microscopia/métodos , Coloração e Rotulagem/métodos , CoesinasRESUMO
The development of clinically useful histone deacetylase inhibitors has expanded greatly. In a preclinical study, we showed that panobinostat (LBH589) inhibits cell cycle progression of human head and neck squamous cell carcinoma (HNSCC) cell lines at G2/M and an associated decrease in expression of particular genes required for passage through G2 and mitosis. In this study we sought to analyse the mechanistic underpinnings of panobinostat-induced growth arrest. HNSCC cell lines were synchronised and progression through mitosis monitored. We demonstrate that panobinostat causes a marked G2 delay and mitotic defects. A loss of G2-specific Plk1 and Cyclin B1 expression and co-incident increase in p21(Waf1/Cip1) expression is also shown. Furthermore, we show a significant loss of E2F1 recruitment to the promoters of these genes in response to panobinostat treatment. These data provide mechanistic evidence of panobinostat-induced cell cycle arrest and highlight its potential as a chemotherapeutic agent for HNSCC.
Assuntos
Proteínas de Ciclo Celular/genética , Ciclina B1/genética , Fase G2/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Mitose/efeitos dos fármacos , Mitose/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fator de Transcrição E2F1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Panobinostat , Regiões Promotoras Genéticas , Carcinoma de Células Escamosas de Cabeça e Pescoço , Quinase 1 Polo-LikeRESUMO
It has been shown that the genomes of episomally maintained DNA viruses are tethered to host cell chromosomes during cell division, facilitating maintenance in dividing cells. The papillomavirus E2 protein serves this mechanism of viral genome persistence by simultaneously associating with chromatin and the viral genome during mitosis. Several host cell proteins are reported to be necessary for the association of E2 with chromatin including the cohesion establishment factor ChlR1. Here we use fluorescence resonance energy transfer (FRET) technology to confirm the interaction between BPV-1 E2 and ChlR1. Furthermore, we use synchronised live cells to study the temporal nature of this dynamic protein interaction and show that ChlR1 and E2 interact during specific phases of the cell cycle. These data provide evidence that the association of E2 with ChlR1 contributes to a loading mechanism during DNA replication rather than direct tethering during mitotic division.
Assuntos
Papillomavirus Bovino 1/fisiologia , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Virais/metabolismo , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligação ProteicaRESUMO
Following DNA replication, chromatid pairs are held together by a proteinacious complex called cohesin until separation during the metaphase-to-anaphase transition. Accurate segregation is achieved by regulation of both sister chromatid cohesion establishment and removal, mediated by post-translational modification of cohesin and interaction with numerous accessory proteins. Recent evidence has led to the conclusion that cohesin is also vitally important in the repair of DNA lesions and control of gene expression. It is now clear that chromosome segregation is not the only important function of cohesin in the maintenance of genome integrity.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Regulação da Expressão Gênica , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Segregação de Cromossomos/fisiologia , Dano ao DNA/genética , Dano ao DNA/fisiologia , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Modelos Biológicos , Ligação Proteica/genética , Ligação Proteica/fisiologia , CoesinasRESUMO
Viruses that maintain their genomes as extrachromosomal circular DNA molecules and establish infection in actively dividing cells must ensure retention of their genomes within the nuclear envelope in order to prevent genome loss. The loss of nuclear membrane integrity during mitosis dictates that paired host cell chromosomes are captured and organized by the mitotic spindle apparatus before segregation to daughter cells. This prevents inaccurate chromosomal segregation and loss of genetic material. A similar mechanism may also exist for the nuclear retention of extrachromosomal viral genomes or episomes during mitosis, particularly for genomes maintained at a low copy number in latent infections. It has been heavily debated whether such a mechanism exists and to what extent this mechanism is conserved among diverse viruses. Research over the last two decades has provided a wealth of information regarding the mechanisms by which specific tumour viruses evade mitotic and DNA damage checkpoints. Here, we discuss the similarities and differences in how specific viruses tether episomal genomes to host cell chromosomes during mitosis to ensure long-term persistence.