RESUMEN
While combined antiretroviral therapy (cART) has had a great impact on the treatment of HIV-1 infection, the persistence of long-lived cells with an intact provirus precludes virus eradication and sterilizing cure. CRISPR/Cas9 genome editing has become an efficient tool to eradicate HIV-1 genome or prevent replication. Furthermore, regulation of Cas9 gene expression by HIV can induce mutations that could inactivate the proviral genome, making a gene therapy safe by preventing the induction of non-specific mutations, which could compromise the integrity of healthy cells. In this study, isolated HIV-1 LTR, INS and RRE sequences were used to regulate Cas9 expression in HEK293 cells, and guide RNAs (gRNAs) were designed to target mutations in HIV-1 conserved regions such as tat and rev regulatory genes. We demonstrate that Cas9 expression in our system is controlled by the HIV-1 Tat and Rev proteins, leading to self-regulation of gene edition, and showing a strong antiviral effect by inactivating HIV-1 replication. Sequencing analysis confirmed that viral genome was partially excised by multiplex editing (90% efficiency), and viral capsid protein (CA-p24) was undetectable. In conclusion, the self-regulated CRISPR/Cas9 system may be a reliable and accurate strategy for eliminating HIV-1 infection whose effect will be restricted to infected cells.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Inactivación de Virus , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Sistemas CRISPR-Cas , Edición Génica , Regulación Viral de la Expresión Génica , Células HEK293 , VIH-1/genética , Humanos , ARN Guía de Kinetoplastida/genética , Replicación Viral/genéticaRESUMEN
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
Translation initiation from the human immunodeficiency virus type-1 (HIV-1) mRNA can occur through a cap or an IRES dependent mechanism. Cap-dependent translation initiation of the HIV-1 mRNA can be inhibited by the instability element (INS)-1, a cis-acting regulatory element present within the gag open reading frame (ORF). In this study we evaluated the impact of the INS-1 on HIV-1 IRES-mediated translation initiation. Using heterologous bicistronic mRNAs, we show that the INS-1 negatively impact on HIV-1 IRES-driven translation in in vitro and in cell-based experiments. Additionally, our results show that the inhibitory effect of the INS-1 is not general to all IRESes since it does not hinder translation driven by the HCV IRES. The inhibition by the INS-1 was partially rescued in cells by the overexpression of the viral Rev protein or hnRNPA1.
Asunto(s)
Genes gag/genética , VIH-1/genética , Sistemas de Lectura Abierta/genética , Células HeLa , Humanos , Immunoblotting , Secuencias Reguladoras de Ácidos Nucleicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Human immunodeficiency virus (HIV)-1 encoded Rev is essential for export from the nucleus to the cytoplasm, of unspliced and singly spliced transcripts coding for structural and nonstructural viral proteins. This process is spatially and temporally coordinated resulting from the interactions between cellular and viral proteins. Here we examined the effects of the sub-cellular localization and dynamics of Rev on the efficiency of nucleocytoplasmic transport of HIV-1 Gag transcripts and virus particle production. Using confocal microscopy and fluorescence recovery after bleaching (FRAP), we report that NF90ctv, a cellular protein involved in Rev function, alters both the sub-cellular localization and dynamics of Rev in vivo, which drastically affects the accumulation of the viral protein p24. The CRM1-dependent nuclear export of Gag mRNA linked to the Rev Response Element (RRE) is dependent on specific domains of the NF90ctv protein. Taken together, our results demonstrate that the appropriate intracellular localization and dynamics of Rev could regulate Gag assembly and HIV-1 replication.