RESUMO
Dengue virus (DENV), a member of the genus Flavivirus, family Flaviviridae, is the most widespread viral pathogen transmitted to humans by mosquitoes. Despite the increased incidence of DENV infection, there are no antiviral drugs available for treatment or prevention. Phenothiazines are heterocyclic compounds with various pharmacological properties that are very adaptable for drug repurposing. In the present report, we analyzed the antiviral activity against DENV and the related Zika virus (ZIKV) of trifluoperazine (TFP), a phenothiazine derivative in clinical use as an antipsychotic and antiemetic agent. TFP exhibited dose-dependent inhibitory activity against the four DENV serotypes and ZIKV in monkey Vero cells at non-cytotoxic concentrations with 50% effective concentration values in the range 1.6-6.4 µM. A similar level of antiviral efficacy was exhibited by TFP against flavivirus infection in the human cell lines A549 and HepG2. Mechanistic studies, performed using time-dependent infectivity assays, real-time RT-PCR, Western blot, and immunofluorescence techniques, indicated that uncoating of the virus during penetration into the cell was the main target for TFP in infected cells, but the compound also exerted a minor effect on a late stage of the virus multiplication cycle. This study demonstrates that TFP, a pharmacologically active phenothiazine, is a selective inhibitor of DENV multiplication in cell culture. Our findings open perspectives for the repositioning of phenothiazines like TFP with a wide spectrum of antiviral efficacy as potential agents for the control of pathogenic flaviviruses.
Assuntos
Antieméticos , Antipsicóticos , Vírus da Dengue , Dengue , Infecção por Zika virus , Zika virus , Animais , Antieméticos/farmacologia , Antieméticos/uso terapêutico , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Chlorocebus aethiops , Dengue/tratamento farmacológico , Humanos , Fenotiazinas/farmacologia , Fenotiazinas/uso terapêutico , Trifluoperazina/farmacologia , Trifluoperazina/uso terapêutico , Células Vero , Replicação ViralRESUMO
In the present study, we analyzed the modulation of p38 cell signaling by Junín virus (JUNV) and evaluated the antiviral activity of p38 inhibitors against JUNV. While JUNV induced a progressive activation of p38 throughout the infection in Vero cells, a partial downregulation of p38 phosphorylation was observed in HEK293 and HeLa cells. The compounds SB203580 and SB202190, which are selective inhibitors of p38, significantly reduced viral protein expression and viral yield in the cell lines examined, indicating that the p38 signaling pathway might be a promising antiviral target against JUNV infection.
Assuntos
Vírus Junin , Animais , Antivirais/farmacologia , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Vírus Junin/fisiologia , Transdução de Sinais , Células Vero , Replicação ViralRESUMO
We have previously shown that the infection of cell cultures with the arenaviruses Junín (JUNV), Tacaribe (TCRV), and Pichindé promotes the phosphorylation of mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases 1 and 2 (ERK1/2) and that this activation is required for the achievement of a productive infection. Here we examined the contribution of ERK1/2 in early steps of JUNV and TCRV multiplication. JUNV adsorption, internalization, and uncoating were not affected by treatment of cultured cells with U0126, an inhibitor of the ERK1/2 signaling pathway. In contrast, U0126 caused a marked reduction in viral protein expression and RNA synthesis, while JUNV RNA synthesis was significantly augmented in the presence of an activator of the ERK1/2 pathway. Moreover, U0126 impaired the expression of a reporter gene in a TCRV-based replicon system, confirming the ability of the compound to hinder arenavirus macromolecular synthesis. By using a cell-based assay, we determined that the inhibitor did not affect the translation of a synthetic TCRV-like mRNA. No changes in the phosphorylation pattern of the translation factor eIF2α were found in U0126-treated cells. Our results indicate that U0126 impairs viral RNA synthesis, thereby leading to a subsequent reduction in viral protein expression. Thus, we conclude that ERK1/2 signaling activation is required for an efficient arenavirus RNA synthesis.
Assuntos
Arenavirus do Novo Mundo/fisiologia , Interações Hospedeiro-Patógeno , Sistema de Sinalização das MAP Quinases , Replicação Viral , Animais , Butadienos/metabolismo , Linhagem Celular , Inibidores Enzimáticos/metabolismo , Nitrilas/metabolismo , RNA Viral/biossíntese , Proteínas Virais/biossínteseRESUMO
Dengue virus (DENV) is the most prevalent mosquito borne viral pathogen worldwide. In this work we first evaluated the antiviral activity of natural and synthetic ß-carbolines against DENV-2 multiplication in cell cultures. We determined that the natural ß-carboline harmol and a synthetic harmine derivative, 9N-methylharmine, exhibit inhibitory effect on DENV-2 production without virucidal activity. The active compounds were inhibitory of all DENV serotypes, being DENV-2 the more susceptible to their antiviral action. The mode of action of 9N-methylharmine against DENV-2 was further explored. We determined that the derivative neither affects viral adsorption-internalization events nor viral RNA synthesis. The quantification of intracellular and extracellular viral genomes and infectious virus particles indicated that 9N-methylharmine would impair the maturation and release of virus particles to the extracellular medium affecting the spreading of the infection. Furthermore, we also determined that 9N-methylharmine antiviral activity is not related to the ability of the compound to downregulate p38 MAPK phosphorylation.
Assuntos
Antivirais/farmacologia , Carbolinas/química , Carbolinas/farmacologia , Vírus da Dengue/efeitos dos fármacos , Animais , Carbolinas/síntese química , Chlorocebus aethiops , Vírus da Dengue/genética , Descoberta de Drogas , Genoma Viral/efeitos dos fármacos , Harmina/análogos & derivados , Harmina/química , Harmina/farmacologia , Humanos , Fosforilação/efeitos dos fármacos , RNA Viral/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacosRESUMO
The endocytic uptake and intracellular trafficking for penetration of DENV-3 strain H-87 into Vero cells was analyzed by using several biochemical inhibitors and dominant negative mutants of cellular proteins. The results presented show that the infective entry of DENV-3 into Vero cells occurs through a non-classical endocytosis pathway dependent on low pH and dynamin, but non-mediated by clathrin. After uptake, DENV-3 transits through early endosomes to reach Rab 7-regulated late endosomes, and according with the half-time for ammonium chloride resistance viral nucleocapsid is released into the cytosol approximately at 12 min post-infection. Furthermore, the influence of the clathrin pathway in DENV-3 infective entry in other mammalian cell lines of human origin, such as A549, HepG2 and U937 cells, was evaluated demonstrating that variable entry pathways are employed depending on the host cell. Results show for the first time the simultaneous coexistence of infective and non -infective routes for DENV entry into the host cell, depending on the usage of clathrin-mediated endocytosis.
Assuntos
Clatrina/metabolismo , Vírus da Dengue/fisiologia , Dengue/virologia , Endocitose/fisiologia , Internalização do Vírus , Aedes , Animais , Células Cultivadas , Chlorocebus aethiops , Dengue/patologia , Células Hep G2 , Humanos , Testes de Sensibilidade Microbiana , Prognóstico , Células U937 , Células VeroRESUMO
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are cellular factors involved in the replication of several viruses. In this study we analyzed the expression and intracellular localization of hnRNP A2 and hnRNP K in cell cultures infected with two viruses that cause human hemorrhagic fevers: dengue virus type 2 (DENV-2) and Junín virus (JUNV). We determined that DENV-2 promoted the cytoplasmic translocation of hnRNP K and to a lesser extent of hnRNP A2, meanwhile, JUNV infection induced an increase in hnRNP K cytoplasmic localization whereas hnRNP A2 remained mainly in the nucleus of infected cells. Both hnRNP K and hnRNP A2 were localized predominantly in the nucleus of JUNV persistently-infected cells even after superinfection with JUNV indicating that persistent infection does not alter nucleo-cytoplasmic transport of these hnRNPs. Total levels of hnRNP K expression were unaffected by DENV-2 or JUNV infection. In addition we determined, using small interfering RNAs, that hnRNP K knockout inhibits DENV-2 and JUNV multiplication. Our results indicate that DENV-2 and JUNV induce hnRNP K cytoplasmic translocation to favor viral multiplication.
Assuntos
Vírus da Dengue/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Interações Hospedeiro-Patógeno , Vírus Junin/fisiologia , Replicação Viral , Animais , Linhagem Celular , Núcleo Celular/química , Citoplasma/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , HumanosRESUMO
In previous work, we demonstrated that the arenavirus Junín virus (JUNV) is able to activate Akt by means of the phosphatidylinositol-3-kinase (PI3K) survival pathway during virus entry. This work extends our study, emphasizing the relevance of this pathway in the establishment and maintenance of persistent infection in vitro. During the course of infection, JUNV-infected Vero cells showed a typical cytopathic effect that may be ascribed to apoptotic cell death. Treatment of infected cultures with Ly294002, an inhibitor of the PI3K/Akt pathway, produced an apoptotic response similar to that observed for uninfected cells treated with the drug. This result suggests that virus-induced activation of the PI3K/Akt pathway does not deliver a strong enough anti-apoptotic signal to explain the low proportion of apoptotic cells observed during infection. Also, inhibition of the PI3K/Akt pathway during the acute stage of infection did not prevent the establishment of persistence. Furthermore, treatment of persistently JUNV-infected cells with Ly294002 did not alter viral protein expression. These findings indicate that despite the positive modulation of the PI3/Akt pathway during Junín virus entry, this would not play a critical role in the establishment and maintenance of JUNV persistence in Vero cells.
Assuntos
Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Febre Hemorrágica Americana/virologia , Vírus Junin/efeitos dos fármacos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Apoptose , Linhagem Celular , Chlorocebus aethiops , Febre Hemorrágica Americana/tratamento farmacológico , Vírus Junin/crescimento & desenvolvimento , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Células Vero , Proteínas Virais/biossínteseRESUMO
The aim of the present study was to analyze the influence of virus origin, mammalian or mosquito cell-derived, on antiviral susceptibility of DENV-2 to entry inhibitors and the association of this effect with any alteration in the mode of entry into the cell. To this end, ten serial passages of DENV-2 were performed in mosquito C6/36 cells or monkey Vero cells and the antiviral susceptibility of each virus passage to sulfated polysaccharides (SPs), like heparin and carrageenans, was evaluated by a virus plaque reduction assay. After serial passaging in Vero cells, DENV-2 became increasingly resistant to SP inhibition whereas the antiviral susceptibility was not altered in virus propagated in C6/36 cells. The change in antiviral susceptibility was associated to a differential mode of entry into the host cell. The route of endocytic entry for productive Vero cell infection was altered from a non-classical clathrin independent pathway for C6/36-grown virus to a clathrin-mediated endocytosis when the virus was serially propagated in Vero cells. Our results show the impact of the cellular system used for successive propagation of DENV on the initial interaction between the host cell and the virion in the next round of infection and the relevant consequences it might have during the in vitro evaluation of entry inhibitors.
Assuntos
Adaptação Biológica , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/crescimento & desenvolvimento , Farmacorresistência Viral , Internalização do Vírus/efeitos dos fármacos , Animais , Carragenina/farmacologia , Linhagem Celular , Chlorocebus aethiops , Culicidae , Análise Mutacional de DNA , Endocitose/efeitos dos fármacos , Heparina/farmacologia , Dados de Sequência Molecular , RNA Viral/genética , Análise de Sequência de DNA , Inoculações Seriadas , Ensaio de Placa ViralRESUMO
In the present work we investigated the importance of the Raf/MEK/ERK signalling pathway in the multiplication of the arenavirus Junín (JUNV) in monkey and human cell cultures. We established that JUNV induces a biphasic activation of ERK and we proved that a specific inhibitor of the ERK pathway, U0126, impairs viral replication. Furthermore, U0126 exerted inhibitory action against the arenaviruses Tacaribe and Pichinde. Moreover, treatment with known ERK activators such as phorbol 12-myristate 13-acetate and serum increased viral yields whereas ERK silencing by small interfering RNAs caused the inhibition of viral multiplication. Therefore, activation of the Raf/MEK/ERK signalling pathway is required to ensure efficient JUNV replication and may constitute a host target for the development of novel effective therapeutic strategies to deal with arenavirus infections.
Assuntos
Interações Hospedeiro-Patógeno , Vírus Junin/fisiologia , Sistema de Sinalização das MAP Quinases , Replicação Viral , Animais , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Haplorrinos , HumanosRESUMO
Among the members of the Arenaviridae family, Lassa virus and Junin virus generate periodic annual outbreaks of severe human hemorrhagic fever (HF) in endemic areas of West Africa and Argentina, respectively. Given the human health threat that arenaviruses represent and the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. Since diverse host cell pathways and enzymes are used by RNA viruses to fulfill their replicative cycle, the targeting of a host process has turned an attractive antiviral approach in the last years for many unrelated virus types. This strategy has the additional benefit to reduce the serious challenge for therapy of RNA viruses to escape from drug effects through selection of resistant variants triggered by their high mutation rate. This article focuses on novel strategies to identify inhibitors for arenavirus therapy, analyzing the potential for antiviral developments of diverse host factors essential for virus infection.