RESUMO
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
A previous sequence analysis of a US5 gene fragment of infectious laryngotracheitis virus (ILTV) performed in an Argentinian epidemiological study allowed to differentiate between wild and vaccine strains. This analysis also defined five ILTV haplotypes with specific variations at positions 461, 484, 832, 878 and 894 of the US5 gene. This characterization of viral strains may also be accomplished using the High-Resolution Melting Analysis (HRMA), which has been described as an effective, fast and sensitive method to detect mutations in PCR products. In the present study, an HRM protocol was developed with the aim of characterizing the circulating ILTV strains in Argentina. The specificity of this tool was confirmed in different DNA diluents, without interference from heterologous DNA or other cellular metabolites. Additionally, the salt concentration in the elution buffer used for DNA extraction did not alter the curve profiles. Higher concentrations of DNA (Ctâ 26.0) displayed well-defined curve profiles, whereas lower concentrations (Ctâ 32.5) exhibited more heterogeneous curves. The HRMA showed 97.49% concordance with the reference technique, i.e., sequencing. The HRM protocol has the capability to perform DNA amplification prior to its characterization. Thus, eventually this technique may be used simultaneously as a diagnostic tool. This advantage implies a significant reduction in the time and effort involved in sample processing.
Assuntos
Herpesvirus Galináceo 1 , Reação em Cadeia da Polimerase , DNA Viral/genética , Herpesvirus Galináceo 1/genéticaRESUMO
BACKGROUND: The arenavirus Junin virus (JUNV), causative agent of the argentine hemorrhagic fever, is able to modulate several signaling pathways involved in cell survival and multiplication. OBJECTIVES: We aimed to characterize the infection of rat osteoblasts (OBCs) with JUNV and its consequence on the modulation of osteogenic genes expression, thus studying the ability of this virus to induce cell differentiation. In addition, we evaluated the effect of purinergic agonists on viral replication. METHOD: Quantification of infectivity by plaque forming unit (PFU) assay, synthesis of viral proteins by western blot and immunofluorescence, and expression of osteogenic differentiation markers (ODM) by quantitative real-time polymerase chain reaction were employed. RESULTS: Infection of OBCs with JUNV (MOI 0.01 PFU/cell) showed a peak of infectivity, reaching 1.5 × 105 PFU/mL at the second day post-infection (p.i.). A marked restriction in multiplication was detected at day 7 p.i. that did not impair the establishment of a persistent stage of infection in OBCs. Analysis of mRNAs corresponding to ODM such as alkaline phosphatase, bone sialo-protein, and bone morphogenetic proteins (BMPs) 4 and 6 revealed that only the levels of BMP-6 were significantly higher in infected cells. Treatment with the purinergic agonists ATPγS, UTP, ADP, or UDP diminished viral titer and reduced the expression of the viral nucleoprotein. Also, treatment with 10 µM ATPγS reduced the stimulation of BMP-6 expression induced by the infection. CONCLUSIONS: These data demonstrate that JUNV is capable of infecting OBCs and point out BMP-6 as a key factor during this process.
Assuntos
Proteína Morfogenética Óssea 6/genética , Vírus Junin/fisiologia , Osteoblastos/virologia , Osteogênese/genética , Animais , Diferenciação Celular , Células Cultivadas , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Agonistas Purinérgicos/farmacologia , Ratos , Transdução de Sinais , Replicação Viral/efeitos dos fármacosRESUMO
Mammarenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome that encodes the nucleocapsid protein (NP), the envelope glycoprotein precursor (GPC), the RNA polymerase (L), and a RING matrix protein (Z). Viral proteins are synthesized from subgenomic mRNAs bearing a capped 5' untranslated region (UTR) and lacking 3' poly(A) tail. We analyzed the translation strategy of Tacaribe virus (TCRV), a prototype of the New World mammarenaviruses. A virus-like transcript that carries a reporter gene in place of the NP open reading frame and transcripts bearing modified 5' and/or 3' UTR were evaluated in a cell-based translation assay. We found that the presence of the cap structure at the 5' end dramatically increases translation efficiency and that the viral 5' UTR comprises stimulatory signals while the 3' UTR,specifically the presence of a terminal C+G-rich sequence and/or a stem-loop structure, down-modulates translation. Additionally, translation was profoundly reduced in eukaryotic initiation factor (eIF) 4G-inactivated cells, whereas depletion of intracellular levels of eIF4E had less impact on virus-like mRNA translation than on a cell-like transcript. Translation efficiency was independent of NP expression or TCRV infection. Our results indicate that TCRV mRNAs are translated using a cap-dependent mechanism, whose efficiency relies on the interplay between stimulatory signals in the 5' UTR and a negative modulatory element in the 3' UTR. The low dependence on eIF4E suggests that viral mRNAs may engage yet-unknown noncanonical host factors for a cap-dependent initiation mechanism.IMPORTANCE Several members of the Arenaviridae family cause serious hemorrhagic fevers in humans. In the present report, we describe the mechanism by which Tacaribe virus, a prototypic nonpathogenic New World mammarenavirus, regulates viral mRNA translation. Our results highlight the impact of untranslated sequences and key host translation factors on this process. We propose a model that explains how viral mRNAs outcompete cellular mRNAs for the translation machinery. A better understanding of the mechanism of translation regulation of this virus can provide the bases for the rational design of new antiviral tools directed to pathogenic arenaviruses.
Assuntos
Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Arenavirus do Novo Mundo/genética , Regulação Viral da Expressão Gênica , Biossíntese de Proteínas , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Ribonucleico , Animais , Linhagem Celular , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Interações Hospedeiro-Patógeno , 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
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
Immune evasion strategies are important for the onset and the maintenance of viral infections. Many viruses have evolved mechanisms to counteract or suppress the host immune response. We have previously characterized two syncytial (syn) variants of Herpes simplex 1 (HSV-1) strain F, syn14-1 and syn17-2, obtained by selective pressure with a natural carrageenan. These variants showed a differential pathology in vaginal and respiratory mucosa infection in comparison with parental strain. In this paper, we evaluated the modulation of immune response in respiratory mucosa by these HSV-1 variants. We observed altered levels of Tumor Necrosis Factor-α and Interleukin-6 in lungs of animals infected with the syn14-1 and syn17-2 variants compared with the parental strain. Also, we detected differences in the recruitment of immune cells to the lung in syn variants infected mice. Both variants exhibit one point mutation in the sequence of the gene of glycoprotein D detected in the ectodomain of syn14-1 and the cytoplasmic tail of syn17-2. Results obtained in the present study contribute to the characterization of HSV-1 syn variants and the participation of the cellular inflammatory response in viral pathogenesis.
Assuntos
Citocinas/metabolismo , Herpesvirus Humano 1/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/patologia , Animais , Feminino , Herpesvirus Humano 1/genética , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/imunologia , Mucosa/patologia , Mutação Puntual , Infecções Respiratórias/virologia , Proteínas do Envelope Viral/genéticaRESUMO
Translation efficiency of viral mRNAs is a key factor defining both cytopathogenicity and virulence of viruses, which are entirely dependent on the cellular translation machinery to synthesize their proteins. This dependence has led them to develop different translational reprogramming strategies to ensure viral mRNAs can effectively compete with cellular mRNAs. Junin virus (JUNV) is a member of the family Arenaviridae, whose mRNAs are capped but not polyadenylated. In this work we evaluated the relevance to JUNV replication of the main components of the eIF4F complex: eIF4A, eIF4GI and eIF4E. We found the viral nucleoprotein (N) of JUNV colocalized with eIF4A and eIF4GI but not with eIF4E. Moreover, N could be immunoprecipitated in association with eIF4A and eIF4GI but not with eIF4E. Accordingly, functional impairment of eIF4A as well as eIF4GI reduced JUNV multiplication. By contrast, inhibition of eIF4E did not show a significant effect on JUNV protein synthesis. A similar situation was observed for another two members of arenaviruses: Tacaribe (TCRV) and Pichinde (PICV) viruses. Finally, the nucleoproteins of JUNV, TCRV and PICV were able to interact with 7 methyl-guanosine (cap), suggesting that the independence of JUNV multiplication on eIF4E, the cap-binding protein, may be due to the replacement of this factor by N protein.
Assuntos
Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Interações Hospedeiro-Patógeno , Vírus Junin/fisiologia , Proteínas do Nucleocapsídeo/metabolismo , Replicação Viral , Animais , Chlorocebus aethiops , Guanosina/análogos & derivados , Guanosina/metabolismo , Células VeroRESUMO
The modulation of purinergic receptors plays an important role in the regulation of bone formation by the osteoblast. On the other hand, bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß superfamily, regulate the differentiation of osteoprogenitor bone cells and stimulate bone formation. In this study, we investigate the effects of several nucleotides on osteoblast differentiation and function, and their relation with the gene expression of osteogenic proteins BMP-2, BMP-4 and BMP-5 as well as of differentiation markers alkaline phosphatase (ALP) and bone sialoprotein (BSP). Our results indicate that 100µM ATP, ATPγS and UTP, but not ADP, ADPßS or UDP, promote ALP activity in rat primary osteoblasts, showing a peak about day 7 of the treatment. ATP, ATPγS and UTP also increase the mRNA levels of ALP, BMP-2, BMP-4, BMP-5 and BSP. Both the ALP activity and ALP and BMP-4 mRNA increments induced by ATP and UTP are inhibited by Ly294002, a PI3K inhibitor, suggesting the involvement of PI3K/AKT signaling pathway in purinergic modulation of osteoblast differentiation. Furthermore, bone mineralization enhance 1 and 1.5 fold after culturing osteoblasts in the presence of 100µM ATP or UTP, respectively, but not of ADP or UDP for 22 days. This information suggests that P2Y2 receptors (responsive to ATP, ATPγS and UTP) enhance osteoblast differentiation involving PI3K/AKT signaling pathway activation and gene expression induction of ALP, BMP-2, BMP-4, BMP-5 and BSP. Our findings state a novel molecular mechanism that involves specific gene expression activation of osteoblast function by the purinoreceptors, which would be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.
Assuntos
Trifosfato de Adenosina/farmacologia , Proteínas Morfogenéticas Ósseas/genética , Calcificação Fisiológica/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Uridina Trifosfato/farmacologia , Animais , Animais Recém-Nascidos , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 5/genética , Proteína Morfogenética Óssea 5/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Calcificação Fisiológica/genética , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteína Oncogênica v-akt/metabolismo , Proteína Oncogênica v-akt/fisiologia , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Cultura Primária de Células , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
There is no specific chemotherapy approved for the treatment of pathogenic arenaviruses that cause severe hemorrhagic fever (HF) in the population of endemic regions in America and Africa. The present study reports the effects of the natural flavonoid quercetin (QUER) on the infection of A549 and Vero cells with Junín virus (JUNV), agent of the Argentine HF. By infectivity assays, a very effective dose-dependent reduction of JUNV multiplication was shown by cell pretreatment at 2-6 h prior to the infection at non-cytotoxic concentrations, with 50% effective concentration values in the range of 6.1-7.5 µg/mL. QUER was also active by post-infection treatment but with minor efficacy. Mechanistic studies indicated that QUER mainly affected the early steps of virus adsorption and internalization in the multiplication cycle of JUNV. Treatment with QUER blocked the phosphorylation of Akt without changes in the total protein expression, detected by Western blot, and the consequent perturbation of the PI3K/Akt pathway was also associated with the fluorescence redistribution from membrane to cytoplasm of TfR1, the cell receptor recognized by JUNV. Then, it appears that the cellular antiviral state, induced by QUER treatment, leads to the prevention of JUNV entry into the cell.
Assuntos
Infecções por Arenaviridae , Arenavirus , Chlorocebus aethiops , Animais , Quercetina/farmacologia , Flavonoides , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Células VeroRESUMO
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
Heterogeneous nuclear ribonucleoproteins A and B (hnRNPs A/B), cellular RNA-binding proteins that participate in splicing, trafficking, translation and turnover of mRNAs, have been implicated in the life cycles of several cytoplasmic RNA viruses. Here, we demonstrate that silencing of hnRNPs A1 and A2 significantly reduces the replication of the arenavirus Junín virus (JUNV), the aetiological agent of Argentine haemorrhagic fever. While acute JUNV infection did not modify total levels of expression of hnRNPs A/B in comparison with uninfected cells, non-cytopathic persistent infection exhibited low levels of these cell proteins. Furthermore, acutely infected cells showed a cytoplasmic relocalization of overexpressed hnRNP A1, probably related to the involvement of this protein in virus replicative cycle. This cytoplasmic accumulation was also observed in cells expressing viral nucleoprotein (N), and co-immunoprecipitation studies revealed the interaction between hnRNP A1 and N protein. By contrast, a predominantly nuclear distribution of overexpressed hnRNP A1 was found during persistent infection, even in the presence of endogenous or overexpressed N protein, indicating a differential modulation of nucleo-cytoplasmic trafficking in acute and persistent JUNV infections.
Assuntos
Transporte Ativo do Núcleo Celular , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Interações Hospedeiro-Patógeno , Vírus Junin/patogenicidade , RNA Viral/metabolismo , Replicação Viral , Animais , Chlorocebus aethiops , Ribonucleoproteína Nuclear Heterogênea A1 , Imunoprecipitação , Ligação Proteica , Células Vero , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Antiviral therapy against herpes simplex virus based on sulfated polysaccharides, like carrageenans, represents a new alternative for genital herpes infections treatment and arises the concern about the appearance of resistant viral populations. METHODS: We characterized the F strain of herpes simplex virus-1 passaged in the presence of a natural carrageenan isolated from the red seaweed Gigartina skottbergii in view of the virulence for mice of isolated viral clones. RESULTS: Viral clones (syn14-1 and syn17-2) showed a syncytial phenotype and a mild resistance to carrageenan, heparin, acyclovir, and brivudine. Both clones were avirulent for BALB/c mice when inoculated intravaginally, whereas F strain produced high mortality. Attenuation correlated with low levels of TNF-[alpha], interleukin-6, and IFN-[gamma] in vaginal lavages although virus titers were similar to those obtained for F strain. On the contrary, they showed a marked virulence when inoculated intranasally leading to a generalized spreading of virus. CONCLUSIONS: Results confirm the hypothesis that selection of herpes simplex virus-1 with a carrageenan in vitro leads to the emergence of variants with a differential virulence when compared to the original virus. This finding should be addressed when an antiviral therapy against genital herpes infection employing a natural carrageenan is under consideration.
Assuntos
Antivirais/farmacologia , Carragenina/farmacologia , Variação Genética , Células Gigantes/fisiologia , Herpesvirus Humano 1/patogenicidade , Seleção Genética , Animais , Chlorocebus aethiops , Feminino , Herpes Genital/patologia , Herpes Genital/virologia , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/classificação , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Rodófitas/química , Alga Marinha/química , Células Vero , VirulênciaRESUMO
Our previous studies reported the inhibitory action against arenaviruses of antiretroviral zinc finger-reactive compounds provided by the National Cancer Institute (USA). These compounds were able to inactivate virions as well as to reduce virus yields from infected cells. Here, the inactivation of the arenavirus Junín (JUNV), agent of Argentine hemorrhagic fever, by the aromatic disulfide NSC20625 was analyzed. The treatment of purified JUNV with this compound eliminated infectivity apparently through irreversible modifications in the matrix Z protein detected by: (a) alterations in the electrophoretic migration profile of Z under non-reducing conditions; (b) an electrodense labeling in the internal layer beneath the envelope and around the matrix Z protein, in negatively stained preparations; (c) changes in the subcellular localization of Z in cells transfected with a recombinant fusion protein JUNVZ-eGFP. The infection of Vero cells with JUNV inactivated particles was blocked at the uncoating of viral nucleocapsid from endosomes, providing new evidence for a functional role of Z in this stage of arenavirus cycle. Furthermore, the inactivated JUNV particles retained the immunoreactivity of the surface glycoprotein GP1 suggesting that this disulfide may be useful in the pursuit of an inactivating agent to obtain a vaccine antigen or diagnostic tool.
Assuntos
Infecções por Arenaviridae/tratamento farmacológico , Compostos Azo/farmacologia , Dissulfetos/farmacologia , Guanidinas/farmacologia , Vírus Junin/efeitos dos fármacos , Vírion/efeitos dos fármacos , Dedos de Zinco , Animais , Fármacos Anti-HIV/farmacologia , Infecções por Arenaviridae/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Chlorocebus aethiops , Proteínas de Fluorescência Verde , Vírus Junin/genética , Microscopia Eletrônica de Transmissão , Proteínas do Nucleocapsídeo/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Células Vero , Vírion/ultraestrutura , Inativação de VírusRESUMO
The syndrome viral haemorrhagic fever (VHF) designates a broad range of diseases that are caused by different viruses including members of the family Arenaviridae. Prophylaxis for Argentine Haemorrhagic Fever (AHF), caused by the arenavirus Junín (JUNV), has been achieved by the use of a live attenuated vaccine, named Candid#1. The standard treatment of AHF is transfusion of convalescent human plasma. Our aim was to develop an alternative and safer treatment for AHF based on the use of virus-neutralizing single domain antibodies (VHHs). We describe the first reported VHHs directed against an arenavirus. These VHHs could neutralize Candid#1 by altering virion binding/fusion. Surprisingly, the neutralizing VHHs appeared to be specific for the viral nucleoprotein (N) that is not known to be involved in arenavirus entry. Candid#1 VHH-escape viruses had acquired a predicted N-glycosylation site in the surface glycoprotein GP1 that is present in highly pathogenic JUNV strains. Accordingly, the Candid#1-neutralizing VHHs could not neutralize pathogenic JUNV strains, but they could still bind to cells infected with a pathogenic strain or the escape mutant viruses. These results show that the attenuated strains of JUNV can be potently neutralized by nucleoprotein-specific VHHs.
Assuntos
Anticorpos Neutralizantes/imunologia , Vírus Junin/imunologia , Nucleoproteínas/imunologia , Anticorpos de Domínio Único/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/imunologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Mutação Puntual/genética , Anticorpos de Domínio Único/isolamento & purificação , Proteínas Virais/química , Vacinas Virais/imunologiaRESUMO
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
Our perspective on nature has changed throughout history and at the same time has affected directly or indirectly our perception of biological processes. In that sense, the "fluxus" of information in a viral population arises a result of a much more complex process than the encoding of a protein by a gene, but as the consequence of the interaction between all the components of the genome and its products: DNA, RNA, and proteins and its modulation by the environment. Even modest "agents of life" like viruses display an intricate way to express their information. This conclusion can be withdrawn from the huge quantity of data furnished by new and potent technologies available now to analyze viral populations. Based on this premise, evolutive processes for viruses are now interpreted as a simultaneous and coordinated phenomenon that leads to global (i.e., not gradual or 'random') remodeling of the population. Our system of study involves the modulation of herpes simplex virus populations through the selective pressure exerted by carrageenans, natural compounds that interfere with virion attachment to cells. On this line, we demonstrated that the passaging of virus in the presence of carrageenans leads to the appearance of progeny virus phenotipically different from the parental seed, particularly, the emergence of syncytial (syn) variants. This event precedes the emergence of mutations in the population which can be readily detected five passages after from the moment of the appearance of syn virus. This observation can be explained taking into consideration that the onset of phenotypic changes may be triggered by "environmental-sensitive" glycoproteins. These "environmental-sensitive" glycoproteins may act by themselves or may transmit the stimulus to "adapter" proteins, particularly, proteins of the tegument, which eventually modulate the expression of genomic products in the "virocell." The modulation of the RNA network is a common strategy of the virocell to respond to environmental changes. This "fast" adaptive mechanism is followed eventually by the appearance of mutations in the viral genome. In this paper, we interpret these findings from a philosophical and scientific point of view interconnecting epigenetic action, exerted by carragenans from early RNA network-DNA interaction to late DNA mutation. The complexity of HSV virion structure is an adequate platform to envision new studies on this topic that may be complemented in a near future through the analysis of the genetic dynamics of HSV populations.
RESUMO
Resumen En un estudio epidemiológico realizado previamente en Argentina, se analizó la secuencia de un fragmento del gen US5 del virus de la laringotraqueítis infecciosa (ILTV), lo que permitió diferenciar las cepas de campo de las vacunales. También esto permitió definir cinco haplotipos del ILTV, con variaciones específicas en las posiciones 461, 484, 832, 878 y 894 del gen US5. La caracterización de las cepas virales también puede lograrse mediante el análisis de la disociación de alta resolución o high-resolution melting analysis (HRMA), descripto como un método efectivo, rápido y sensible para detectar mutaciones en productos de PCR. En el presente estudio se desarrolló un protocolo de disociación de alta resolución con el objetivo de caracterizar cepas del ILTV circulantes en Argentina. Para ello,se confirmó la especificidad de esta herramienta en diferentes diluyentes del ADN de las muestras, sin observarse interferencias en presencia de ADN heterólogo u otros metabolitos celulares. Asimismo, la concentración de sales en el buffer de elución utilizado durante la extracción de ADN no alteró los perfiles de las curvas. Se obtuvieron perfiles bien definidos con concentraciones de ADN más elevadas (Ct = 26.0), mientras que concentraciones más bajas presentaron curvas heterogéneas (Ct = 32.5). El HRMA mostró una concordancia del 97.49% con la técnica de referencia, la secuenciación. El protocolo de disociación de alta resolución amplifica el ADN antes de su caracterización, por lo que esta técnica podría ser eventualmente utilizada para confirmar la presencia del ILTV y, al mismo tiempo, distinguir haplotipos, optimizando su valor como herramienta de diagnóstico. Esta característica implica una reducción significativa en el tiempo dedicado al procesamiento de muestras.
Assuntos
Reação em Cadeia da Polimerase , Herpesvirus Galináceo 1 , DNA Viral/genética , Herpesvirus Galináceo 1/genéticaRESUMO
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
This study presents the chemical composition and antiviral activity against herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) of sulfated galactan crude extracts and main fractions obtained from two red seaweeds collected in Brazil, Gymnogongrus griffithsiae and Cryptonemia crenulata. Most of the eighteen tested products, including homogeneous kappa/iota/nu carrageenan and DL-galactan hybrid, exhibited antiherpetic activity with inhibitory concentration 50% (IC50) values in the range 0.5-5.6 microg/ml, as determined in a virus plaque reduction assay in Vero cells. The galactans lacked cytotoxic effects and showed a broad spectrum of antiviral activity against HSV-1 and HSV-2. No direct virus inactivation was observed after virion treatment with the galactans. The mode of action of these compounds could be mainly ascribed to an inhibitory effect on virus adsorption. Most importantly, a significant protection against a murine vaginal infection with HSV-2 was afforded by topical treatment with the sulfated galactans.