RESUMEN
Translation initiation of the human immunodeficiency virus-type 1 (HIV-1) genomic mRNA (vRNA) is cap-dependent or mediated by an internal ribosome entry site (IRES). The HIV-1 IRES requires IRES-transacting factors (ITAFs) for function. In this study, we evaluated the role of the heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a potential ITAF for the HIV-1 IRES. In HIV-1-expressing cells, the depletion of hnRNPK reduced HIV-1 vRNA translation. Furthermore, both the depletion and overexpression of hnRNPK modulated HIV-1 IRES activity. Phosphorylations and protein arginine methyltransferase 1 (PRMT1)-induced asymmetrical dimethylation (aDMA) of hnRNPK strongly impacted the protein's ability to promote the activity of the HIV-1 IRES. We also show that hnRNPK acts as an ITAF for the human T cell lymphotropic virus-type 1 (HTLV-1) IRES, present in the 5'UTR of the viral sense mRNA, but not for the IRES present in the antisense spliced transcript encoding the HTLV-1 basic leucine zipper protein (sHBZ). This study provides evidence for a novel role of the host hnRNPK as an ITAF that stimulates IRES-mediated translation initiation for the retroviruses HIV-1 and HTLV-1.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo K , Retroviridae , Humanos , Regiones no Traducidas 5' , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Sitios Internos de Entrada al Ribosoma/genética , Fosforilación , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Retroviridae/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The B. safensis RGM 2450 and B. siamensis RGM 2529 strains were isolated from the rhizosphere of plants presenting resilience to abiotic and biotic stress conditions. To understand the implications of bacteria in resilience, a genomic and experimental analysis was carried out on their biostimulant and phytopathogenic antagonist properties. Genome analyses of both strains indicated that they have the potential to synthesize bioactive compounds such as the battery of non-ribosomal peptides, polyketides, extracellular enzymes and phytohormones. These results were consistent with the antagonistic activities of both strains against the phytopathogens Botrytis cinerea, Colletotrichum acutatum, Fusarium oxysporum and Phytophtora cinnamomi. They also showed the capacity to solubilize phosphorus, fix nitrogen and produce indole acetic acid. This was observed in tomato seedlings grown from seeds inoculated with the mixture of strains which presented significantly greater length as well as wet and dry weight in comparison with the treatments individually inoculated with each strain and the control. Accordingly, the combination of B. safensis RGM 2450 and B. siamensis RGM 2529 showed synergistic biostimulant activity. These findings contribute new knowledge of the genomic and metabolomic properties taking part in the symbiotic interactions between these strains and the plants and uphold the combined use of both strains as a biostimulant.
RESUMEN
During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that deposition of N6-methyladenosine (m6A) regulates full-length RNA packaging. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promoted the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and contributes to full-length RNA demethylation. We further identified two highly conserved adenosines within the 5'-UTR that have a crucial functional role in m6A methylation and packaging of the full-length RNA. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the HIV-1 full-length RNA molecules that will be used as viral genomes.
Asunto(s)
VIH-1 , Regiones no Traducidas 5' , Adenosina/genética , Adenosina/metabolismo , Productos del Gen gag/genética , VIH-1/metabolismo , Metilación , ARN Viral/genética , ARN Viral/metabolismo , Virión/metabolismoRESUMEN
Tellurium oxyanion, tellurite (TeO 3 -2), is a highly toxic compound for many organisms. Its presence in the environment has increased over the past years due to industrial manufacturing processes and has been associated with adverse effects on human health. Although tellurite induces the phosphorylation of eIF2α, DNA damage and oxidative stress, the molecular mechanisms related to the cellular responses to tellurite-induced stress are poorly understood. In this work, we evaluated the ability of tellurite to induce phosphorylation of eIF2α, stress granules (SGs) assembly and their relationship with DNA damage in U2OS cells. We demonstrate that tellurite promotes the assembly of bona fide cytoplasmic SGs. Unexpectedly, tellurite also induces the assembly of nuclear SGs. Interestingly, we observed that the presence of tellurite-induced nuclear SGs correlates with γH2AX foci. However, although H2O2 also induce DNA damage, no nuclear SGs were observed. Our data show that tellurite promotes the assembly of cytoplasmic and nuclear SGs in response to oxidative stress and DNA damage, revealing a new aspect of cellular stress response mediated by the assembly of nuclear stress granules.
RESUMEN
Translation initiation of the human immunodeficiency virus type-1 (HIV-1) full-length RNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the full-length RNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF inhibits HIV-1 and HIV-2 Gag synthesis from the full-length RNA. Our results indicate that CTIF associates with HIV-1 Rev through its N-terminal domain and is recruited onto the full-length RNA ribonucleoprotein complex in order to interfere with Gag synthesis. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that Rev interferes with the association of CTIF with CBP80 indicating that CTIF and Rev compete for the CBC subunit.
Asunto(s)
Factores Eucarióticos de Iniciación/fisiología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/biosíntesis , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Células Cultivadas , Regulación hacia Abajo , Células HEK293 , VIH-1/genética , VIH-1/metabolismo , Células HeLa , Humanos , Células Jurkat , Biosíntesis de Proteínas/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/fisiologíaRESUMEN
Gag synthesis from the full-length unspliced mRNA is critical for the production of the viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While most spliced mRNAs follow the canonical gene expression pathway in which the recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA relies on the viral protein Rev to reach the cytoplasm and recruit the host translational machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving nuclear export and translation of the unspliced mRNA. These functions of Rev are supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box RNA helicase eIF4AI, which translocates to the nucleus and cooperates with the viral protein to promote Gag synthesis. Finally, we show that the Rev/RRE axis is important for the assembly of a CBP80-eIF4AI complex onto the unspliced mRNA. Together, our results provide further evidence towards the understanding of the molecular mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 replication.
Asunto(s)
Factor 4A Eucariótico de Iniciación/genética , VIH-1/genética , Complejo Proteico Nuclear de Unión a la Caperuza/genética , ARN Mensajero/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Línea Celular , Factor 4A Eucariótico de Iniciación/metabolismo , VIH-1/metabolismo , Células HeLa , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Unión Proteica , Empalme del ARN , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Replicación Viral/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/biosíntesis , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The human immunodeficiency virus type-1 (HIV-1) unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs) containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1), Staufen double-stranded RNA binding protein 1/2 (STAU1/2), or components of miRNA-induced silencing complex (miRISC) and processing bodies (PBs). More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A), allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries.
Asunto(s)
VIH-1/genética , Interacciones Huésped-Patógeno , Estabilidad del ARN , ARN Viral/metabolismo , HumanosRESUMEN
DEAD-box RNA helicase DDX3 is a host factor essential for HIV-1 replication and thus, a potential target for novel therapies aimed to overcome viral resistance. Previous studies have shown that DDX3 promotes nuclear export and translation of the HIV-1 unspliced mRNA. Although the function of DDX3 during both processes requires its catalytic activity, it is unknown whether other domains surrounding the helicase core are involved. Here, we show the involvement of the N- and C-terminal domains of DDX3 in the regulation of HIV-1 unspliced mRNA translation. Our results suggest that the intrinsically disordered N-terminal domain of DDX3 regulates its functions in translation by acting prior to the recruitment of the 43S pre-initiation complex onto the viral 5'-UTR. Interestingly, this regulation was conserved in HIV-2 and was dependent on the CRM1-dependent nuclear export pathway suggesting a role of the RNA helicase in interconnecting nuclear export with ribosome recruitment of the viral unspliced mRNA. This specific function of DDX3 during HIV gene expression could be exploited as an alternative target for pharmaceutical intervention.
Asunto(s)
ARN Helicasas DEAD-box/genética , Infecciones por VIH/genética , VIH-1/genética , Carioferinas/genética , Receptores Citoplasmáticos y Nucleares/genética , Transporte Activo de Núcleo Celular/genética , Regulación Viral de la Expresión Génica , Infecciones por VIH/terapia , Infecciones por VIH/virología , VIH-1/patogenicidad , Interacciones Huésped-Patógeno/genética , Humanos , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Viral/genética , Replicación Viral/genética , Proteína Exportina 1RESUMEN
Post-transcriptional control in both HIV-1 and HIV-2 is a highly regulated process that commences in the nucleus of the host infected cell and finishes by the expression of viral proteins in the cytoplasm. Expression of the unspliced genomic RNA is particularly controlled at the level of RNA splicing, export, and translation. It appears increasingly obvious that all these steps are interconnected and they result in the building of a viral ribonucleoprotein complex (RNP) that must be efficiently translated in the cytosolic compartment. This review summarizes our knowledge about the genesis, localization, and expression of this viral RNP.