RESUMEN
In our search for new partners of the HIV-1 envelope glycoprotein (Env), we found that the cytoplasmic domain of the TMgp41 (TMgp41 CD) subunit of HIV-1 Env interacted with Luman, a transcription factor of the CREB/ATF family. Luman is anchored in the endoplasmic reticulum membrane and subjected to activation by regulated intramembrane proteolysis (RIP). The RIP process permits the release of the activated amino-terminal fragment of Luman into the cytoplasm, and its import into the nucleus. Here, we demonstrate that interaction between the TMgp41 CD and Luman requires a region encompassing the b-Zip and TM domains of Luman and decreases the stability of this factor. Moreover, we found that overexpression of a constitutively active form of Luman in cells transfected with HXB2R HIV-1 provirus decreased the intracellular expression of Gag and Env and led to a decrease in virion release. This negative effect of activated Luman on HIV-1 production was correlated to the inhibition of Tat transactivation of the HIV-1 LTR, which might be related to an interaction of activated Luman with Tat. Altogether, these results show that Luman acts as a partner of two major HIV-1 proteins: the TMgp41 Env subunit and Tat. The interaction between the TMgp41 subunit of Env and Luman affects the stability of the full-length Luman protein, the precursor of the activated, nuclear form of Luman, which acts negatively on Tat-mediated HIV-1 transactivation.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Productos del Gen tat/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Duplicado del Terminal Largo de VIH/genética , VIH-1 , Transcripción Genética , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Regulación Viral de la Expresión Génica , Productos del Gen gag/metabolismo , Productos del Gen tat/genética , Genes gag , Proteína gp41 de Envoltorio del VIH/genética , Humanos , Inmunoprecipitación , Provirus , Saccharomyces cerevisiae , Activación Transcripcional , Técnicas del Sistema de Dos Híbridos , Replicación Viral , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Activation of the human immunodeficiency virus type-1 (HIV-1) promoter in infected cells requires the sequential recruitment of several cellular factors to facilitate the formation of a processive elongation complex. The nucleosomal reorganization of the HIV-1 long terminal repeat (LTR) observed upon Tat stimulation suggests that chromatin-remodeling complexes could play a role during this process. Here, we reported that Tat interacts directly with Brm, a DNA-dependent ATPase subunit of the SWI/SNF chromatin-remodeling complex, to activate the HIV-1 LTR. Inhibition of Brm via small interfering RNAs impaired Tat-mediated transactivation of an integrated HIV-1 promoter. Furthermore, Brm is recruited in vivo to the HIV-1 LTR in a Tat-dependent manner. Interestingly, we found that Tat/Brm interaction is regulated by Tat lysine 50 acetylation. These data show the requirement of Tat-mediated recruitment of SWI/SNF chromatin-remodeling complex to HIV-1 promoter in the activation of the LTR.
Asunto(s)
Productos del Gen tat/fisiología , VIH-1/genética , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Acetilación , Secuencias de Aminoácidos , Arginina/genética , Línea Celular , Productos del Gen tat/genética , Humanos , Lisina/metabolismo , Mutación , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/fisiología , Secuencias Repetidas Terminales , Factores de Transcripción/genética , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
The human immunodeficiency virus type 1 (HIV-1) encodes a potent transactivator, Tat, which functions through binding to a short leader RNA, called transactivation responsive element (TAR). Recent studies suggest that Tat activates the HIV-1 long terminal repeat (LTR), mainly by adapting co-activator complexes, such as p300, PCAF and the positive transcription elongation factor P-TEFb, to the promoter. Here, we show that the proto-oncoprotein Hdm2 interacts with Tat and mediates its ubiquitination in vitro and in vivo. In addition, Hdm2 is a positive regulator of Tat-mediated transactivation, indicating that the transcriptional properties of Tat are stimulated by ubiquitination. Fusion of ubiquitin to Tat bypasses the requirement of Hdm2 for efficient transactivation, supporting the notion that ubiquitin has a non-proteolytic function in Tat-mediated transactivation.