Abstract
BackgroundHuman immunodeficiency virus-1 (HIV-1) Tat protein plays an essential role in HIV gene transcription from the HIV-1 long terminal repeat (LTR) and replication. Transcriptional activity of Tat is modulated by several host factors, but the mechanism responsible for Tat regulation by host factors is not understood fully.ResultsUsing a yeast two-hybrid screening system, we identified Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) as a novel Tat-interacting partner. Here, we report its function as a positive regulator of Tat. In a coimmunoprecipitation assay, HIV-1 Tat interacted sufficiently with both endogenous and ectopically expressed NUCKS1. In a reporter assay, ectopic expression of NUCKS1 significantly increased Tat-mediated transcription of the HIV-1 LTR, whereas knockdown of NUCKS1 by small interfering RNA diminished Tat-mediated transcription of the HIV-1 LTR. We also investigated which mechanism contributes to NUCKS1-mediated Tat activation. In a chromatin immunoprecipitation assay (ChIP), knockdown of NUCKS1 interrupted the accumulation of Tat in the transactivation-responsive (TAR) region on the LTR, which then led to suppression of viral replication. However, NUCKS1 expression did not increase Tat nuclear localization and interaction with Cyclin T1. Interestingly, the NUCKS1 expression level was lower in latently HIV-1-infected cells than in uninfected parent cells. Besides, expression level of NUCKS1 was markedly induced, which then facilitated HIV-1 reactivation in latently infected cells.ConclusionTaken together, our data demonstrate clearly that NUCKS1 is a novel Tat coactivator that is required for Tat-mediated HIV-1 transcription and replication, and that it may contribute to HIV-1 reactivation in latently HIV-1 infected cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0067-y) contains supplementary material, which is available to authorized users.
Highlights
Human immunodeficiency virus-1 (HIV-1) Tat protein plays an essential role in HIV gene transcription from the HIV-1 long terminal repeat (LTR) and replication
From 44 positive clones resulting from nutritional selection, one clone was found to contain a cDNA of 732 bp (GenBank accession number NM_022731) encompassing the full-length open-reading frame of the NUCKS1 protein (Figure 1A)
To determine whether Tat interacts with cellular NUCKS1, Flag-tagged HIV-1 proteins (Flag–Tat, -Vpr and -Nef) and V5-tagged NUCKS1 (V5–NUCKS1) expression plasmids were introduced into HEK293 cells individually or in combination and analyzed by coimmunoprecipitation and a Western blot assay
Summary
Human immunodeficiency virus-1 (HIV-1) Tat protein plays an essential role in HIV gene transcription from the HIV-1 long terminal repeat (LTR) and replication. Human immunodeficiency virus type-1 (HIV-1) encodes a transcriptional activator (transactivator) protein, Tat, which is essential for efficient transcription of the integrated provirus following HIV-1 replication and for disease progression [1,2]. Tat binds cooperatively to transactivation-responsive (TAR) RNA, which is found at the 5’ end of all HIV-1 viral RNAs with cellular factors, followed by transcriptional elongation [3]. It is believed that Tat–host cellular factor interactions, transcription, and epigenetic factors form a complex regulatory network for the regulation of HIV-1 gene expression in a diverse range of host cells and in response to a variety of extracellular stimuli. Identification of key factors for Tat-mediated HIV-1 gene expression may be required for understanding HIV replication and for exploring potential targets of antiviral drugs [29,30]
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