RESUMO
A common method for cell-associated virus isolation involves disruption of infected cells by a combination of hypotonic burst, freeze-thaw cycles (F-T) and sonication. This protocol was also originally used for the preparation of cell-free extract containing the MX strain of lymphocytic choriomeningitis virus (LCMV), which is preferentially propagated by cell-to-cell contact and does not release distinct virions into the medium. In the present study, we compared different approaches to virus isolation. Based on virus yield, we show that deionized water lysis is the fastest and most effective method for releasing LCMV MX infectious viral particles from persistently infected cells. Moreover, we demonstrate that freeze-thaw cycles and sonication do not improve virus isolation. This simple protocol could be used for isolation of other viruses, the life cycle of which is strictly cell-associated and therefore are difficult to release in large amounts from host cells.
Assuntos
Vírus da Coriomeningite Linfocítica/isolamento & purificação , Nucleoproteínas/isolamento & purificação , Vírion/isolamento & purificação , Virologia/métodos , Células A549 , Animais , Anticorpos Antivirais , Soluções Tampão , Técnicas de Cultura de Células , Chlorocebus aethiops , Meios de Cultura/química , Congelamento , Células HeLa , Humanos , Coriomeningite Linfocítica/diagnóstico , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/crescimento & desenvolvimento , Camundongos , Células VeroRESUMO
Dexamethasone is a synthetic glucocorticoid frequently used to suppress side-effects of anticancer chemotherapy. In the present study, we showed that dexamethasone treatment leads to concentration-dependent downregulation of cancer-associated marker, carbonic anhydrase IX (CA IX), at the level of promoter activity, mRNA and protein expression in 2D and 3D cancer cell models. The effect of dexamethasone on CA IX expression under hypoxic conditions is predominantly mediated by impaired transcriptional activity and decreased protein level of the main hypoxic transcription factor HIF-1α. In addition, CA9 downregulation can be caused by protein-protein interactions between activated glucocorticoid receptors, major effectors of glucocorticoid action, and transcription factors that trigger CA9 transcription (e.g. AP-1). Moreover, we identified a potential NF-κB binding site in the CA9 promoter and propose the involvement of NF-κB in the dexamethasone-mediated inhibition of CA9 transcription. As high level of CA IX is often linked to aggressive tumor behavior, poor prognosis and chemo- and radiotherapy resistance, uncovering its reduction after dexamethasone treatment and implication of additional regulatory mechanisms can be relevant for the CA IX-related clinical applications.
Assuntos
Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX/metabolismo , Dexametasona/farmacologia , Regulação para Baixo , Glucocorticoides/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , NF-kappa B/metabolismo , Antígenos de Neoplasias/genética , Sítios de Ligação , Anidrase Carbônica IX/genética , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Células MCF-7 , Regiões Promotoras Genéticas , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologiaRESUMO
Lambda interferons inhibit replication of many viruses, but their role in the inhibition of lymphocytic choriomeningitis virus (LCMV) infection remains unclear. In this study, we examined the antiviral effects of interferon (IFN)-λ2 and IFN-λ3 against LCMV in A549 cells. We found that IFN-λ2 is a more potent inhibitor of LCMV strain MX compared with IFN-λ3, whereas both cytokines have similar antiviral effects against an immunosuppressive variant of LCMV, clone-13. We also demonstrated that the antiviral activity of IFN-λ2 is more effective if it is delivered early rather than after establishment of a long-term infection, suggesting that virus replication is only partially responsive to the cytokine. In agreement with this observation, we showed that LCMV infection significantly reduces IFNLR1 mRNA expression in infected cells. In addition, LCMV infection, to some extent, compromises the signal transduction pathway of IFN-λ2. This implies that IFN receptors as well as their downstream signaling components could be selectively targeted either directly by LCMV proteins or indirectly by cellular factor(s) that are induced or activated by LCMV infection.
Assuntos
Antivirais/farmacologia , Interferons/farmacologia , Vírus da Coriomeningite Linfocítica/efeitos dos fármacos , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Cinética , Coriomeningite Linfocítica/virologia , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interferon/metabolismo , Fator de Transcrição STAT1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Receptor de Interferon gamaRESUMO
The physiological context of virus-infected cells can markedly affect multiplication and spread of the virus progeny. During persistent infection, the virus exploits the host cell without disturbing its vital functions. However, microenvironmental hypoxia can uncouple this intimate relationship and escalate virus pathogenesis. Accumulating evidence suggests that hypoxia-inducible factor (HIF) modulates gene expression of the viruses that pass through a DNA stage, contain hypoxia-responsive promoter elements, and replicate in the nucleus. Here we show that hypoxia can influence the gene expression and transmission of the cytoplasmic RNA virus lymphocytic choriomeningitis virus (LCMV), which is a neglected human pathogen and teratogen. The MX strain of LCMV, which we used as a model, replicates in a persistent mode in human HeLa cells, fails to produce mature envelope glycoproteins, and spreads through cell-cell contacts in the absence of extracellular infectious virions. Both exposure of MX-infected HeLa cells to chronic hypoxia and gene transfer approaches led to increased virus RNA transcription and higher levels of the viral proteins via a HIF-dependent mechanism. Moreover, hypoxia enhanced the formation of infectious virions capable of transmitting LCMV by cell-free medium. This LCMV "reactivation" might have health-compromising consequences in hypoxia-associated situations, such as fetal development and ischemia-related pathologies.