RESUMO
Latently infected resting CD4(+) T cells are a major barrier to HIV cure. Understanding how latency is established, maintained and reversed is critical to identifying novel strategies to eliminate latently infected cells. We demonstrate here that co-culture of resting CD4(+) T cells and syngeneic myeloid dendritic cells (mDC) can dramatically increase the frequency of HIV DNA integration and latent HIV infection in non-proliferating memory, but not naïve, CD4(+) T cells. Latency was eliminated when cell-to-cell contact was prevented in the mDC-T cell co-cultures and reduced when clustering was minimised in the mDC-T cell co-cultures. Supernatants from infected mDC-T cell co-cultures did not facilitate the establishment of latency, consistent with cell-cell contact and not a soluble factor being critical for mediating latent infection of resting CD4(+) T cells. Gene expression in non-proliferating CD4(+) T cells, enriched for latent infection, showed significant changes in the expression of genes involved in cellular activation and interferon regulated pathways, including the down-regulation of genes controlling both NF-κB and cell cycle. We conclude that mDC play a key role in the establishment of HIV latency in resting memory CD4(+) T cells, which is predominantly mediated through signalling during DC-T cell contact.
Assuntos
Linfócitos T CD4-Positivos/virologia , Células Dendríticas/fisiologia , HIV-1/fisiologia , Células Mieloides/fisiologia , Latência Viral , Linfócitos T CD4-Positivos/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Proliferação de Células , Células Cultivadas , Redes Reguladoras de Genes , Células HEK293 , Humanos , Análise em Microsséries , Transcriptoma , Latência Viral/genética , Latência Viral/imunologiaRESUMO
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
Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1) structural proteins (matrix, capsid and nucleocapsid), enzymes (protease, reverse transcriptase, RNAse H and integrase) and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection.
Assuntos
Arsenicais/metabolismo , Cisteína , HIV-1/metabolismo , Coloração e Rotulagem/métodos , Proteínas Virais/química , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Células HEK293 , HIV-1/enzimologia , HIV-1/genética , HIV-1/patogenicidade , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Especificidade por SubstratoRESUMO
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.