RESUMO
The New World (NW) mammarenavirus Junín (JUNV) is the etiological agent of Argentine hemorrhagic fever, a human endemic disease of Argentina. Promyelocytic leukemia protein (PML) has been reported as a restriction factor for several viruses although the mechanism/s behind PML-mediated antiviral effect may be diverse and are a matter of debate. Previous studies have reported a nuclear to cytoplasm translocation of PML during the murine Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) infection. This translocation was found to be mediated by the viral Z protein. Here, we show that PML restricts JUNV infection in human A549 cells. However, in contrast to LCVM, JUNV infection enhances PML expression and PML is not translocated to the cytoplasm neither it colocalizes with JUNV Z protein. Our study demonstrates that a NW mammarenavirus as JUNV interacts differently with the antiviral protein PML than LCMV.
Assuntos
Febre Hemorrágica Americana , Vírus Junin , Proteína da Leucemia Promielocítica , Células A549 , Febre Hemorrágica Americana/metabolismo , Humanos , Proteína da Leucemia Promielocítica/genética , Proteínas Virais , Replicação ViralRESUMO
Junin virus (JUNV), a highly pathogenic New World arenavirus, is the causative agent of Argentine hemorrhagic fever (AHF). The live-attenuated Candid #1 (Can) strain currently serves as a vaccine for at-risk populations. We have previously shown that the Can glycoprotein (GPC) gene is the primary gene responsible for attenuation in a guinea pig model of AHF. However, the mechanisms through which the GPC contributes to the attenuation of the Can strain remain unknown. A more complete understanding of the mechanisms underlying the attenuation and immunogenicity of the Can strain will potentially allow for the rational design of additional safe and novel vaccines. Here, we provide a detailed comparison of both RNA and protein expression profiles between both inter- and intra-segment chimeric JUNV recombinant clones expressing combinations of genes from the Can strain and the pathogenic Romero (Rom) strain. The recombinant viruses that express Can GPC, which were shown to be attenuated in guinea pigs, displayed different RNA levels and GPC processing patterns as determined by Northern and Western blot analyses, respectively. Analysis of recombinant viruses containing amino acid substitutions selected at different mouse brain passages during the generation of Can revealed that altered Can GPC processing was primarily due to the T168A substitution within G1, which eliminates an N-linked glycosylation motif. Incorporation of the T168A substitution in the Rom GPC resulted in a Can-like processing pattern of Rom GPC. In addition, JUNV GPCs containing T168A substitution were retained within the endoplasmic reticulum (ER) and displayed significantly lower cell surface expression than wild-type Rom GPC. Interestingly, the reversion A168T in Can GPC significantly increased GPC expression at the cell surface. Our results demonstrate that recombinant JUNV (rJUNV) expressing Can GPC display markedly different protein expression and elevated genomic RNA expression when compared to viruses expressing Rom GPC. Additionally, our findings indicate that the N-linked glycosylation motif at amino acid positions 166-168 is important for trafficking of JUNV GPC to the cell surface, and the elimination of this motif interferes with the GPC release from the ER.
Assuntos
Motivos de Aminoácidos , Arenavirus do Novo Mundo/imunologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Febre Hemorrágica Americana , Vacinas Virais , Animais , Arenavirus do Novo Mundo/genética , Linhagem Celular , Células Cultivadas , Cricetinae , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Expressão Gênica , Regulação Viral da Expressão Gênica , Glicoproteínas/química , Glicoproteínas/imunologia , Glicosilação , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/metabolismo , Febre Hemorrágica Americana/prevenção & controle , Febre Hemorrágica Americana/virologia , Humanos , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transcrição Gênica , Vacinas Virais/genética , Vacinas Virais/imunologia , VirulênciaRESUMO
Stress granules (SGs) are ephemeral cytoplasmic aggregates containing stalled translation preinitiation complexes involved in mRNA storage and triage during the cellular stress response. SG formation is triggered by the phosphorylation of the alpha subunit of eIF2 (eIF2α), which provokes a dramatic blockage of protein translation. Our results demonstrate that acute infection of Vero cells with the arenavirus Junín (JUNV), aetiological agent of Argentine haemorrhagic fever, does not induce the formation of SGs. Moreover, JUNV negatively modulates SG formation in infected cells stressed with arsenite, and this inhibition correlates with low levels of eIF2α phosphorylation. Transient expression of JUNV nucleoprotein (N) or the glycoprotein precursor (GPC), but not of the matrix protein (Z), inhibits SG formation in a similar manner, comparable to infectious virus. Expression of N and GPC also impaired eIF2α phosphorylation triggered by arsenite. A moderate inhibition of SG formation was also observed when DTT and thapsigargin were employed as stress inducers. In contrast, no inhibition was observed when infected cells were treated with hippuristanol, a translational inhibitor and inducer of SGs that bypasses the requirement for eIF2α phosphorylation. Finally, we analysed SG formation in persistently JUNV-infected cells, where N and GPC are virtually absent and truncated N products are expressed abundantly. We found that persistently infected cells show a quite normal response to arsenite, with SG formation comparable to that of uninfected cells. This suggests that the presence of GPC and/or N is crucial to control the stress response upon JUNV infection of Vero cells.
Assuntos
Arsenitos/farmacologia , Fator de Iniciação 2 em Eucariotos/genética , Vírus Junin/genética , Vírus Junin/patogenicidade , Animais , Chlorocebus aethiops , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/metabolismo , Febre Hemorrágica Americana/genética , Febre Hemorrágica Americana/metabolismo , Vírus Junin/metabolismo , Fosforilação , Plasmídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção/métodos , Células VeroRESUMO
Argentine haemorrhagic fever (AHF) is a systemic febrile syndrome characterized by several haematological and neurological alterations caused by Junín virus (JUNV), a member of the Arenaviridae family. Newborn mice are highly susceptible to JUNV and the course of infection has been associated with the viral strain used. Galectin-3 (Gal-3) is an animal lectin that has been proposed to play an important role in some central nervous system (CNS) diseases. In this study, we analysed Gal-3 expression at the transcriptional and translational expression levels during JUNV-induced CNS disease. We found that Candid 1 strain induced, with relatively low mortality, a subacute/chronic CNS disease with significant glia activation and upregulation of Gal-3 in microglia cells as well as in reactive astrocytes that correlated with viral levels. Our results suggest an important role for Gal-3 in viral-induced CNS disease.
Assuntos
Infecções por Arenaviridae/metabolismo , Encefalite Viral/metabolismo , Galectina 3/biossíntese , Vírus Junin/patogenicidade , Neuroglia/metabolismo , Neuroglia/virologia , Regulação para Cima/fisiologia , Animais , Animais Recém-Nascidos , Infecções por Arenaviridae/patologia , Astrócitos/metabolismo , Astrócitos/patologia , Astrócitos/virologia , Modelos Animais de Doenças , Encefalite Viral/patologia , Febre Hemorrágica Americana/metabolismo , Febre Hemorrágica Americana/patologia , Febre Hemorrágica Americana/virologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , Microglia/virologia , Neuroglia/patologiaRESUMO
In this paper we demonstrate that infection of cell cultures with the arenavirus Junín (JUNV), agent of the argentine haemorrhagic fever, leads to the activation of PI3K/Akt signalling pathway. Phosphorylation of Akt occurs early during JUNV infection of Vero cells and is blocked by the PI3K inhibitor, Ly294002. Infection of cells with UV-irradiated JUNV redeemed the pattern of stimulation observed for infectious virus indicating that an early stage of multiplication cycle would be enough to trigger activation. Treatment of cells with chlorpromazine abrogated phosphorylation of Akt upon JUNV infection suggesting virus internalization as responsible for activation. Inhibition of Akt phosphorylation by Ly294002 impaired viral protein synthesis and expression leading to a reduced infectious virus yield without blocking the onset of persistent stage of infection. This impairment is linked to a reduced amount of virus bound to cells probably due to a blockage on the recycling of transferrin cell-receptor, employed by the virus to adsorb to the cell surface. Early Akt activation was also observed in BHK-21 and A549 JUNV infected cells suggesting an important role of PI3K/Akt signalling in JUNV multiplication in vitro.
Assuntos
Febre Hemorrágica Americana/metabolismo , Vírus Junin/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Replicação Viral/efeitos dos fármacos , Animais , Chlorocebus aethiops , Cromonas/farmacologia , Cricetinae , Inibidores Enzimáticos/farmacologia , Febre Hemorrágica Americana/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Vírus Junin/efeitos dos fármacos , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Células Vero , Internalização do VírusRESUMO
Cultured astrocytes derived from newborn rat brain were inoculated with Junin virus (JV) to characterize their response to infection by means of their glial fibrillary acidic protein (GFAP) immunochemical profile. Samples from 1 to 11 days post-inoculation (pi), as well as matched controls, were serially harvested for GFAP labeling by peroxidase-antiperoxidase (PAP) method. It was only at day 3 that significantly greater values of GFAP staining (P < 0.05) were disclosed by three complementary approaches: image analysis, ELISA and immunoblot densitometry. Since such increase was abolished by Triton X-100 treatment, soluble GFAP fraction appeared responsible for the early though transient enhancement of GFAP immunoreactivity that followed viral inoculation.