RESUMEN
Merkel Cell Carcinoma (MCC) is a rare neuroendocrine skin cancer. In our previous work, we decoded genes specifically deregulated by MCPyV early genes as opposed to other polyomaviruses and established functional importance of NDRG1 in inhibiting cellular proliferation and migration in MCC. In the present work, we found the SET protein, (I2PP2A, intrinsic inhibitor of PP2A) upstream of NDRG1 which was modulated by MCPyV early genes, both in hTERT-HK-MCPyV and MCPyV-positive (+) MCC cell lines. Additionally, MCC dermal tumour nodule tissues showed strong SET expression. Inhibition of the SET-PP2A interaction in hTERT-HK-MCPyV using the small molecule inhibitor, FTY720, increased NDRG1 expression and inhibited cell cycle regulators, cyclinD1 and CDK2. SET inhibition by shRNA and FTY720 also decreased cell proliferation and colony formation in MCPyV(+) MCC cells. Overall, these results pave a path for use of drugs targeting SET protein for the treatment of MCC.
Asunto(s)
Carcinoma de Células de Merkel , Movimiento Celular , Proliferación Celular , Poliomavirus de Células de Merkel , Proteína Fosfatasa 2 , Humanos , Poliomavirus de Células de Merkel/fisiología , Poliomavirus de Células de Merkel/genética , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/genética , Carcinoma de Células de Merkel/virología , Carcinoma de Células de Merkel/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Clorhidrato de Fingolimod/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Línea Celular Tumoral , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Infecciones por Polyomavirus/virología , Neoplasias Cutáneas/virología , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/genéticaRESUMEN
Since the outbreak of COVID-19, researchers have been working tirelessly to discover effective ways to combat coronavirus infection. The use of computational drug repurposing methods and molecular docking has been instrumental in identifying compounds that have the potential to disrupt the binding between the spike glycoprotein of SARS-CoV-2 and human ACE2 (hACE2). Moreover, the pseudovirus approach has emerged as a robust technique for investigating the mechanism of virus attachment to cellular receptors and for screening targeted small molecule drugs. Pseudoviruses are viral particles containing envelope proteins, which mediate the virus's entry with the same efficiency as that of live viruses but lacking pathogenic genes. Therefore, they represent a safe alternative to screen potential drugs inhibiting viral entry, especially for highly pathogenic enveloped viruses. In this review, we have compiled a list of antiviral plant extracts and natural products that have been extensively studied against enveloped emerging and re-emerging viruses by pseudovirus technology. The review is organized into three parts: (1) construction of pseudoviruses based on different packaging systems and applications; (2) knowledge of emerging and re-emerging viruses; (3) natural products active against pseudovirus-mediated entry. One of the most crucial stages in the life cycle of a virus is its penetration into host cells. Therefore, the discovery of viral entry inhibitors represents a promising therapeutic option in fighting against emerging viruses.
Asunto(s)
Antivirales , Productos Biológicos , SARS-CoV-2 , Internalización del Virus , Antivirales/farmacología , Antivirales/química , Humanos , Internalización del Virus/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Productos Biológicos/farmacología , Productos Biológicos/química , Productos Biológicos/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Extractos Vegetales/farmacología , Extractos Vegetales/química , Reposicionamiento de Medicamentos/métodos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/química , Evaluación Preclínica de Medicamentos/métodosRESUMEN
The continuous emergence of SARS-CoV-2 variants caused the persistence of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. The viral proteases are the most attractive targets for developing antiviral drugs. In this scenario, our study explores the use of HIV-1 protease inhibitors against SARS-CoV-2. An in silico screening of a library of HIV-1 proteases identified four anti-HIV compounds able to interact with the 3CLpro of SARS-CoV-2. Thus, in vitro studies were designed to evaluate their potential antiviral effectiveness against SARS-CoV-2. We employed pseudovirus technology to simulate, in a highly safe manner, the adsorption of the alpha (α-SARS-CoV-2) and omicron (ο-SARS-CoV-2) variants of SARS-CoV-2 and study the inhibitory mechanism of the selected compounds for cell-virus interaction. The results reported a mild activity against the viral proteases 3CLpro and PLpro, but efficient inhibitory effects on the internalization of both variants mediated by cathepsin B/L. Our findings provide insights into the feasibility of using drugs exhibiting antiviral effects for other viruses against the viral and host SARS-CoV-2 proteases required for entry.
Asunto(s)
COVID-19 , Proteasas de Cisteína , Humanos , SARS-CoV-2/genética , Inhibidores de Proteasas/farmacología , Antivirales/farmacología , Antivirales/uso terapéutico , Cisteína Endopeptidasas/genética , Proteasas Virales , Simulación del Acoplamiento MolecularRESUMEN
Epstein-Barr (EBV) is a human γ-herpesvirus that undergoes both a productive (lytic) cycle and a non-productive (latent) phase. The virus establishes enduring latent infection in B lymphocytes and productive infection in the oral mucosal epithelium. Like other herpesviruses, EBV expresses its genes in a coordinated pattern during acute infection. Unlike others, it replicates its DNA during latency to maintain the viral genome in an expanding pool of B lymphocytes, which are stimulated to divide upon infection. The reactivation from the latent state is associated with a productive gene expression pattern mediated by virus-encoded transcriptional activators BZLF-1 and BRLF-1. EBV is a highly transforming virus that contributes to the development of human lymphomas. Though viral vectors and mRNA platforms have been used to develop an EBV prophylactic vaccine, currently, there are no vaccines or antiviral drugs for the prophylaxis or treatment of EBV infection and EBV-associated cancers. Natural products and bioactive compounds are widely studied for their antiviral potential and capability to modulate intracellular signaling pathways. This review was intended to collect information on plant-derived products showing their antiviral activity against EBV and evaluate their feasibility as an alternative or adjuvant therapy against EBV infections and correlated oncogenesis in humans.
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Productos Biológicos , Infecciones por Virus de Epstein-Barr , Magnoliopsida , Humanos , Productos Biológicos/farmacología , Productos Biológicos/uso terapéutico , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Linfocitos B , Carcinogénesis , AntiviralesRESUMEN
IMPORTANCE: Here, we demonstrate that the direct binding of p53 on the IL-18 promoter region regulates its gene expression. However, the presence of E6 and E7 from human papillomavirus type 38 impairs this mechanism via a new inhibitory complex formed by DNA methyltransferase 1 (DNMT1)/PKR/ΔNp73α, which binds to the region formerly occupied by p53 in primary keratinocytes.
Asunto(s)
Citocinas , Proteína p53 Supresora de Tumor , Humanos , Citocinas/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Proteínas E7 de Papillomavirus/genética , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Tumor suppressor p53 and its related proteins, p63 and p73, can be synthesized as multiple isoforms lacking part of the N- or C-terminal regions. Specifically, high expression of the ΔNp73α isoform is notoriously associated with various human malignancies characterized by poor prognosis. This isoform is also accumulated by oncogenic viruses, such as Epstein-Barr virus (EBV), as well as genus beta human papillomaviruses (HPV) that appear to be involved in carcinogenesis. To gain additional insight into ΔNp73α mechanisms, we have performed proteomics analyses using human keratinocytes transformed by the E6 and E7 proteins of the beta-HPV type 38 virus as an experimental model (38HK). We find that ΔNp73α associates with the E2F4/p130 repressor complex through a direct interaction with E2F4. This interaction is favored by the N-terminal truncation of p73 characteristic of ΔNp73 isoforms. Moreover, it is independent of the C-terminal splicing status, suggesting that it could represent a general feature of ΔNp73 isoforms (α, ß, γ, δ, ε, ζ, θ, η, and η1). We show that the ΔNp73α-E2F4/p130 complex inhibits the expression of specific genes, including genes encoding for negative regulators of proliferation, both in 38HK and in HPV-negative cancer-derived cell lines. Such genes are not inhibited by E2F4/p130 in primary keratinocytes lacking ΔNp73α, indicating that the interaction with ΔNp73α rewires the E2F4 transcriptional program. In conclusion, we have identified and characterized a novel transcriptional regulatory complex with potential implications in oncogenesis. IMPORTANCE The TP53 gene is mutated in about 50% of human cancers. In contrast, the TP63 and TP73 genes are rarely mutated but rather expressed as ΔNp63 and ΔNp73 isoforms in a wide range of malignancies, where they act as p53 antagonists. Accumulation of ΔNp63 and ΔNp73, which is associated with chemoresistance, can result from infection by oncogenic viruses such as EBV or HPV. Our study focuses on the highly carcinogenic ΔNp73α isoform and uses a viral model of cellular transformation. We unveil a physical interaction between ΔNp73α and the E2F4/p130 complex involved in cell cycle control, which rewires the E2F4/p130 transcriptional program. Our work shows that ΔNp73 isoforms can establish interactions with proteins that do not bind to the TAp73α tumor suppressor. This situation is analogous to the gain-of-function interactions of p53 mutants supporting cellular proliferation.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Infecciones por Papillomavirus , Humanos , Transformación Celular Neoplásica , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción E2F4/genética , Factor de Transcripción E2F4/metabolismo , Expresión Génica , Herpesvirus Humano 4/genética , Virus del Papiloma Humano , Queratinocitos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína Sustrato Asociada a CrK/metabolismo , Neoplasias/metabolismoRESUMEN
Several studies reported the presence of a recently discovered polyomavirus (PyV), Lyon IARC PyV (LIPyV), in human and domestic animal specimens. LIPyV has some structural similarities to well-established animal and human oncogenic PyVs, such as raccoon PyV and Merkel cell PyV (MCPyV), respectively. In this study, we demonstrate that LIPyV early proteins immortalize human foreskin keratinocytes. LIPyV LT binds pRb, accordingly cell cycle checkpoints are altered in primary human fibroblasts and keratinocytes expressing LIPyV early genes. Mutation of the pRb binding site in LT strongly affected the ability of LIPyV ER to induced HFK immortalization. LIPyV LT also binds p53 and alters p53 functions activated by cellular stresses. Finally, LIPyV early proteins activate telomerase reverse transcriptase (hTERT) gene expression, via accumulation of the Sp1 transcription factor. Sp1 recruitment to the hTERT promoter is controlled by its phosphorylation, which is mediated by ERK1 and CDK2. Together, these data highlight the transforming properties of LIPyV in in vitro experimental models, supporting its possible oncogenic nature. IMPORTANCE Lyon IARC PyV is a recently discovered polyomavirus that shows some structural similarities to well-established animal and human oncogenic PyVs, such as raccoon PyV and Merkel cell PyV, respectively. Here, we show the capability of LIPyV to efficiently promote cellular transformation of primary human cells, suggesting a possible oncogenic role of this virus in domestic animals and/or humans. Our study identified a novel virus-mediated mechanism of activation of telomerase reverse transcriptase gene expression, via accumulation of the Sp1 transcription factor. In addition, because the persistence of infection is a key event in virus-mediated carcinogenesis, it will be important to determine whether LIPyV can deregulate immune-related pathways, similarly to the well-established oncogenic viruses.
Asunto(s)
Infecciones por Polyomavirus , Poliomavirus , Animales , Carcinogénesis , Fibroblastos/virología , Humanos , Queratinocitos/virología , Poliomavirus de Células de Merkel/genética , Poliomavirus/genética , Poliomavirus/metabolismo , Infecciones por Polyomavirus/virología , Factor de Transcripción Sp1/metabolismo , Telomerasa/genética , Proteína p53 Supresora de Tumor/metabolismoAsunto(s)
ADN Viral/genética , Inflamasomas/farmacología , Neoplasias Experimentales , Papillomaviridae/genética , Proteínas E7 de Papillomavirus/genética , Neoplasias Cutáneas/genética , Rayos Ultravioleta/efectos adversos , Animales , Carcinogénesis , Ratones , Ratones Transgénicos , Proteínas E7 de Papillomavirus/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
Tumor suppressors can exert pro-proliferation functions in specific contexts. In the beta human papillomavirus type 38 (HPV38) experimental model, the viral proteins E6 and E7 promote accumulation of a wild-type (WT) p53 form in human keratinocytes (HKs), promoting cellular proliferation. Inactivation of p53 by different means strongly decreases the proliferation of HPV38 E6/E7 HKs. This p53 form is phosphorylated at S392 by the double-stranded RNA-dependent protein kinase PKR, which is highly activated by HPV38. PKR-mediated S392 p53 phosphorylation promotes the formation of a p53/DNMT1 complex, which inhibits expression of integrin alpha 1 (ITGA1), a repressor of epidermal growth factor receptor (EGFR) signaling. Ectopic expression of ITGA1 in HPV38 E6/E7 HKs promotes EGFR degradation, inhibition of cellular proliferation, and cellular death. Itga1 expression was also inhibited in the skin of HPV38 transgenic mice that have an elevated susceptibility to UV-induced skin carcinogenesis. In summary, these findings reveal the existence of a specific WT p53 form that displays pro-proliferation properties.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Proliferación Celular , Queratinocitos/patología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/complicaciones , Proteínas Represoras/metabolismo , Neoplasias Cutáneas/etiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo , Humanos , Queratinocitos/inmunología , Queratinocitos/virología , Ratones , Ratones Transgénicos , Proteínas Oncogénicas Virales/genética , Papillomaviridae/aislamiento & purificación , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/virología , Proteínas Represoras/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The beta human papillomaviruses (HPVs) are subdivided into 5 species (beta-1 to beta-5), and they were first identified in the skin. However, the beta-3 species appears to be more highly represented in the mucosal epithelia than in the skin. Functional studies have also highlighted that beta-3 HPV49 shares some functional similarities with mucosal high-risk (HR) HPV16. Here, we describe the characterization of the in vitro transforming properties of the entire beta-3 species, which includes three additional HPV types: HPV75, HPV76, and HPV115. HPV49, HPV75, and HPV76 E6 and E7 (E6/E7), but not HPV115 E6 and E7, efficiently inactivate the p53 and pRb pathways and immortalize or extend the life span of human foreskin keratinocytes (HFKs). As observed for HR HPV16, cell cycle deregulation mediated by beta-3 HPV E6/E7 expression leads to p16INK4a accumulation, whereas no p16INK4a was detected in beta-2 HPV38 E6/E7 HFKs. As shown for HPV49 E6, HPV75 and HPV76 E6s degrade p53 by an E6AP/proteasome-mediated mechanism. Comparative analysis of cellular gene expression patterns of HFKs containing E6 and E7 from HR HPV16, beta-3 HPV types, and beta-2 HPV38 further highlights the functional similarities of HR HPV16 and beta-3 HPV49, HPV75, and HPV76. The expression profiles of these four HPV HFKs show some similarities and diverge substantially from those of beta-3 HPV115 E6/E7 and beta-2 HPV38 E6/E7 HFKs. In summary, our data show that beta-3 HPV types share some mechanisms with HR HPV types and pave the way for additional studies aiming to evaluate their potential role in human pathologies.IMPORTANCE Human papillomaviruses are currently classified in different genera. Mucosal HPVs belonging to the alpha genus have been clearly associated with carcinogenesis of the mucosal epithelium at different sites. Beta HPV types have been classified as cutaneous. Although findings indicate that some beta HPVs from species 1 and 2 play a role, together with UV irradiation, in skin cancer, very little is known about the transforming properties of most of the beta HPVs. This report shows the transforming activity of E6 and E7 from beta-3 HPV types. Moreover, it highlights that beta-3 HPVs share some biological properties more extensively with mucosal high-risk HPV16 than with beta-2 HPV38. This report provides new paradigms for a better understanding of the biology of the different HPV types and their possible association with lesions at mucosal and/or cutaneous epithelia.
Asunto(s)
Alphapapillomavirus/genética , Alphapapillomavirus/patogenicidad , Células Epiteliales/virología , Membrana Mucosa/virología , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Alphapapillomavirus/clasificación , Animales , Células Cultivadas , Papillomavirus Humano 16/genética , Humanos , Queratinocitos/virología , Masculino , Ratones , Membrana Mucosa/citología , Células 3T3 NIH , Piel/virologíaRESUMEN
Merkel cell polyomavirus (MCPyV) is the first human polyomavirus etiologically associated with Merkel cell carcinoma (MCC), a rare and aggressive form of skin cancer. Similar to other polyomaviruses, MCPyV encodes early T antigen genes, viral oncogenes required for MCC tumor growth. To identify the unique oncogenic properties of MCPyV, we analyzed the gene expression profiles in human spontaneously immortalized keratinocytes (NIKs) expressing the early genes from six distinct human polyomaviruses (PyVs), including MCPyV. A comparison of the gene expression profiles revealed 28 genes specifically deregulated by MCPyV. In particular, the MCPyV early gene downregulated the expression of the tumor suppressor gene N-myc downstream-regulated gene 1 (NDRG1) in MCPyV gene-expressing NIKs and hTERT-MCPyV gene-expressing human keratinocytes (HK) compared to their expression in the controls. In MCPyV-positive MCC cells, the expression of NDRG1 was downregulated by the MCPyV early gene, as T antigen knockdown rescued the level of NDRG1. In addition, NDRG1 overexpression in hTERT-MCPyV gene-expressing HK or MCC cells resulted in a decrease in the number of cells in S phase and cell proliferation inhibition. Moreover, a decrease in wound healing capacity in hTERT-MCPyV gene-expressing HK was observed. Further analysis revealed that NDRG1 exerts its biological effect in Merkel cell lines by regulating the expression of the cyclin-dependent kinase 2 (CDK2) and cyclin D1 proteins. Overall, NDRG1 plays an important role in MCPyV-induced cellular proliferation.IMPORTANCE Merkel cell carcinoma was first described in 1972 as a neuroendocrine tumor of skin, most cases of which were reported in 2008 to be caused by a PyV named Merkel cell polyomavirus (MCPyV), the first PyV linked to human cancer. Thereafter, numerous studies have been conducted to understand the etiology of this virus-induced carcinogenesis. However, it is still a new field, and much work is needed to understand the molecular pathogenesis of MCC. In the current work, we sought to identify the host genes specifically deregulated by MCPyV, as opposed to other PyVs, in order to better understand the relevance of the genes analyzed on the biological impact and progression of the disease. These findings open newer avenues for targeted drug therapies, thereby providing hope for the management of patients suffering from this highly aggressive cancer.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular/genética , Proliferación Celular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Poliomavirus de Células de Merkel/genética , Poliomavirus de Células de Merkel/fisiología , Antígenos Virales de Tumores/genética , Antígenos Virales de Tumores/metabolismo , Carcinogénesis/genética , Carcinoma de Células de Merkel/virología , Línea Celular , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Humanos , Queratinocitos/virología , Infecciones por Polyomavirus/virología , Piel/patología , Neoplasias Cutáneas/genética , Transcriptoma , Infecciones Tumorales por Virus/virologíaRESUMEN
Human papillomaviruses (HPVs) infect the epithelia of skin or mucosa, where they can induce hyperproliferative lesions. More than 220 different HPV types have been characterized and classified into five different genera. Mucosal high-risk HPVs are causative for cancers of the anogenital region and oropharynx. Clinical data from patients with the rare genetic disorder epidermodysplasia verruciformis (EV) indicate that genus beta-HPVs cooperate with ultraviolet (UV) radiation in the development of cutaneous squamous cell carcinoma. In addition, epidemiological and biological findings indicate that beta-HPV types play a role in UV-mediated skin carcinogenesis also in non-EV individuals. However, the mechanisms used by these cutaneous viruses to promote epithelial carcinogenesis differ significantly from those of mucosal HPVs. Recent studies point to a delicate cross-talk of beta-HPVs with the cell-autonomous immunity of the host keratinocytes and the local immune microenvironment that eventually determines the fate of cutaneous HPV infection and the penetrance of disease. This review gives an overview of the critical interactions of genus beta-HPVs with the local immune system that allow the virus to complete its life cycle, to escape from extrinsic immunity, and eventually to cause chronic inflammation contributing to skin carcinogenesis. This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'.
Asunto(s)
Betapapillomavirus/fisiología , Susceptibilidad a Enfermedades/inmunología , Sistema Inmunológico/virología , Infecciones por Papillomavirus/inmunología , Neoplasias Cutáneas/inmunología , Animales , Susceptibilidad a Enfermedades/virología , Humanos , Sistema Inmunológico/inmunología , Ratones , Infecciones por Papillomavirus/virología , Neoplasias Cutáneas/virologíaRESUMEN
The nuclear factor κB (NF-κB) pathway plays a key role in innate and adaptive immunity, cell proliferation and survival, inflammation and tumors development. MiR-146a is an immune system regulator that has anti-inflammatory function in multiple cell types and conditions. Here we demonstrate activation of canonical NF-κB pathway in monocytic cells upon HSV-1 replication. By constructing and using a recombinant HSV-1\EGFP virus, we monitored the capability of the virus to recruit NF-κB and we report that the phosphorylation of p65 protein correlates with an active virus replication at single-cell level. In addition, we found that upregulation of miR-146a during viral replication is strictly dependent on NF-κB activation and correlates with tight control of the interleukin-1 receptor-associate kinase 1 (IRAK1). Accordingly, THP-1 DN IκBα cells, expressing a dominant negative mIκBα, did not show upregulation of miR-146a upon HSV-1 infection. Our data suggest that the expression of miRNA-146a modulates NF-κB activation through targeting IRAK1 during HSV-1 replication in THP-1 cells.
Asunto(s)
Herpesvirus Humano 1/fisiología , MicroARNs/metabolismo , Monocitos/metabolismo , FN-kappa B/metabolismo , Animales , Chlorocebus aethiops , Proteínas Fluorescentes Verdes/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crecimiento & desarrollo , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , MicroARNs/genética , Mutación/genética , Fosforilación , Células THP-1 , Células Vero , Replicación Viral/fisiologíaRESUMEN
The exposure to CCR5 (CC chemokine receptor 5) specific natural antibodies in vitro produces a Class B ß-arrestin2-dependent CCR5 retention with the aid of ERK1, due to the formation of a CCR5 signalosome, which remains stable for at least 48 h. Considering that ß-arrestins and MAPKs are receptive to environmental signals, their signal complexes could be one of the key junction for GPCRs internalization related signal transduction. Here, we demonstrate that, in T cells, the phosphorylation status of either CCR5 receptor or ERK1 protein is necessary to drive the internalized receptor into the early endosomes, forming the CCR5 signalosome. In particular, our data show that ß-arrestin2/ERK1 complex is a relevant transducer in the CCR5 signaling pathway. Understanding the mechanism of CCR5 regulation is essential for many inflammatory disorders, tumorigenesis and viral infection such as HIV.
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Autoanticuerpos/metabolismo , Endocitosis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Receptores CCR5/agonistas , Receptores CCR5/metabolismo , Transducción de Señal/efectos de los fármacos , Estaurosporina/farmacología , Línea Celular , Endosomas/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Receptores CCR5/ultraestructura , beta-Arrestinas/antagonistas & inhibidores , beta-Arrestinas/genética , beta-Arrestinas/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
The CC chemokine receptor 5 (CCR5) is responsible for immune and inflammatory responses by mediation of chemotactic activity in leukocytes, although it is expressed on different cell types. It has been shown to act as co-receptor for the human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV). Natural reactive antibodies (Abs) recognizing first loop (ECL1) of CCR5 have been detected in several pools of immunoglobulins from healthy donors and from several cohorts of either HIV-exposed but uninfected subjects (ESN) or HIV-infected individuals who control disease progression (LTNP) as well. The reason of development of anti-CCR5 Abs in the absence of autoimmune disease is still unknown; however, the presence of these Abs specific for CCR5 or for other immune receptors and mediators probably is related to homeostasis maintenance. The majority of anti-CCR5 Abs is directed to HIV binding site (N-terminus and ECL2) of the receptor. Conversely, it is well known that ECL1 of CCR5 does not bind HIV; thus, the anti-CCR5 Abs directed to ECL1 elicit a long-lasting internalization of CCR5 but not interfere with HIV binding directly; these Abs block HIV infection in either epithelial cells or CD4+ T lymphocytes and the mechanism differs from those ones described for all other CCR5-specific ligands. The Ab-mediated CCR5 internalization allows the formation of a stable signalosome by interaction of CCR5, ß-arrestin2 and ERK1 proteins. The signalosome degradation and the subsequent de novo proteins synthesis determine the CCR5 reappearance on the cell membrane with a very long-lasting kinetics (8 days). The use of monoclonal Abs to CCR5 with particular characteristics and mode of action may represent a novel mode to fight viral infection in either vaccinal or therapeutic strategies.
RESUMEN
The herpes simplex virus 1 is able to readdress different cellular pathways including cell cycle to facilitate its replication and spread. During infection, the progression of the cell cycle from G1 to S phase makes the cellular replication machinery accessible to viral DNA replication. In this work we established that HSV-1, in asynchronized HEp-2 cells, strictly controls cell cycle progression increasing S-phase population from 9 hours post infection until the end of HSV-1 replication. The G1/S phases progression depends on two important proteins, cyclin E and CDK2. We demonstrate that their phosphorylated status and then their activity during the infection is strongly correlated to viral replication events. In addition, HSV-1 is able to recruit and distribute ERK1/2 proteins in a spatio-temporal fashion, highlighting its downstream regulatory effects on cellular processes. According with this data, using chemical inhibitor U0126 and ERK dominant negative cells we found that the lack of ERK1 activity affects cyclin E protein accumulation, viral gene transcription and percentage of the cells in S phase, during the viral replication. These data suggested a complex interaction between ERK, cell cycle progression and HSV-1 replication.
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Fase G1 , Herpes Simple/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fase S , Replicación Viral , Ciclo Celular/genética , Línea Celular , Células Cultivadas , Quinasa 2 Dependiente de la Ciclina/metabolismo , HumanosRESUMEN
Several lines of evidence indicate that cutaneous human papillomavirus (HPV) types belonging to the beta genus of the HPV phylogenetic tree synergize with UV radiation in the development of skin cancer. Accordingly, the E6 and E7 oncoproteins from some beta HPV types are able to deregulate pathways related to immune response and cellular transformation. Toll-like receptor 9 (TLR9), in addition to playing a role in innate immunity, has been shown to be involved in the cellular stress response. Using primary human keratinocytes as experimental models, we have shown that UV irradiation (and other cellular stresses) activates TLR9 expression. This event is closely linked to p53 activation. Silencing the expression of p53 or deleting its encoding gene affected the activation of TLR9 expression after UV irradiation. Using various strategies, we have also shown that the transcription factors p53 and c-Jun are recruited onto a specific region of the TLR9 promoter after UV irradiation. Importantly, the E6 and E7 oncoproteins from beta HPV38, by inducing the accumulation of the p53 antagonist ΔNp73α, prevent the UV-mediated recruitment of these transcription factors onto the TLR9 promoter, with subsequent impairment of TLR9 gene expression. This study provides new insight into the mechanism that mediates TLR9 upregulation in response to cellular stresses. In addition, we show that HPV38 E6 and E7 are able to interfere with this mechanism, providing another explanation for the possible cooperation of beta HPV types with UV radiation in skin carcinogenesis.IMPORTANCE Beta HPV types have been suggested to act as cofactors in UV-induced skin carcinogenesis by altering several cellular mechanisms activated by UV radiation. We show that the expression of TLR9, a sensor of damage-associated molecular patterns produced during cellular stress, is activated by UV radiation in primary human keratinocytes (PHKs). Two transcription factors known to be activated by UV radiation, p53 and c-Jun, play key roles in UV-activated TLR9 expression. The E6 and E7 oncoproteins from beta HPV38 strongly inhibit UV-activated TLR9 expression by preventing the recruitment of p53 and c-Jun to the TLR9 promoter. Our findings provide additional support for the role that beta HPV types play in skin carcinogenesis by preventing activation of specific pathways upon exposure of PHKs to UV radiation.
Asunto(s)
Transformación Celular Neoplásica/patología , Activación Enzimática/efectos de la radiación , Queratinocitos/metabolismo , Papillomaviridae/crecimiento & desarrollo , Proteínas E7 de Papillomavirus/metabolismo , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/efectos de la radiación , Proteínas Virales/metabolismo , Proliferación Celular/genética , Células Cultivadas , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Piel/parasitología , Piel/virología , Neoplasias Cutáneas/virología , Receptor Toll-Like 9/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos UltravioletaRESUMEN
CCR5 stimulation with natural ligands, such as RANTES, classically induces short-term internalization with transient activation of ß-arrestins and rapidly recycling on the cell surface. Here we discovered that, in T cells, natural CCR5 antibodies induce a CCR5-negative phenotype with the involvement of ß-arrestin2, which leads to the formation of a stable CCR5 signalosome with both ß-arrestin2 and ERK1. The activation of ß-arrestin2 is necessary to CCR5 signaling for the signalosome formation and stabilization. When all stimuli were washed out, ß-arrestin1 silencing favors the activity of ß-arrestin2 for the CCR5 signalosome retention. Interestingly, CCR5 turn from Class A trafficking pattern, normally used for its internalization with natural modulating molecules (i.e. RANTES), into a long lasting Class B type specifically induced by stimulation with natural anti-CCR5 antibodies. This new CCR5 pathway is relevant not only to study in depth the molecular basis of all pathologies where CCR5 is involved but also to generate new antidody-based therapeutics.