RESUMO
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
Junín virus (JUNV), the etiological agent of the Argentine hemorrhagic fever, has a single-stranded RNA genome with ambisense expression which encodes for five proteins. In previous works we have demonstrated that the Z arenavirus matrix protein represents an attractive target for antiviral therapy. With the aim of studying a new alternative therapeutic mechanism, four Z-specific siRNAs (Z1- to Z4-siRNAs) were tested showing variable efficacy. The most effective inhibitor was Z2-siRNA targeted at the region encompassed by nt 179-197 of Z gene. The efficacy of this Z2-siRNA against JUNV was also demonstrated in virus-infected cells, by testing infectious virus plaque formation (92.8% JUNV yield reduction), viral RNA level or antigen expression, as well as in cells transfected with Z-specific reporter plasmids (91% reduction in expression of Z-EGFP fusion protein). Furthermore, the lack of effect of this Z-siRNA on the expression of other JUNV proteins, such as N and GPC, confirmed the specificity of action exerted by Z2-siRNA on Z transcript. Thus, the present study represents the first report of virus inhibition mediated by RNA interference for a New World arenavirus.
Assuntos
Infecções por Arenaviridae/virologia , Regulação para Baixo , Vírus Junin/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Replicação Viral , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Vírus Junin/química , Vírus Junin/fisiologia , Dados de Sequência Molecular , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Células VeroRESUMO
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.