GRHAL1, a novel lncRNA, regulates HIV-1 gene expression by modulating Tat- and Sp1-mediated HIV-1 LTR activation.

We recently reported that GADD34, an integrated stress response (ISR)-associated protein expressed at low levels in many cell lines, functions as a novel HIV-1 restriction factor. To elucidate the mechanism underlying GADD34-mediated inhibition of HIV replication, we investigated the role of long non-coding RNAs (lncRNAs). Here, we identify and characterize a novel lncRNA, GRHAL1 (GADD34-regulated HIV accessory lncRNA-1), which is highly expressed in GADD34-knockout (GADD34-KO) cells relative to WT cells. GRHAL1 is expressed at low basal levels in Jurkat and MT-2 CD4+ T cell lines and in primary human CD4+ T cells and is upregulated following HIV-1 infection. T cell activation signals that promote HIV-1 replication also induced GRHAL1 expression; however, this induction was independent of IFN and ISR signalling. Transfection of in vitro - synthesized GRHAL1 significantly enhanced HIV-1 LTR-driven gene expression from both integrated and unintegrated promoters. GRHAL1 stimulated both Tat-independent and Tat-dependent LTR activation, with a more pronounced effect observed at suboptimal Tat levels, indicating functional synergy between GRHAL1 and Tat under limiting Tat conditions. Mutational analysis of the LTR demonstrated that GRHAL1-mediated activation requires an intact Tat-responsive TAR element and upstream Sp1-binding sites, linking GRHAL1 activity to Tat and Sp1 in transcriptional regulation. This synergy was further confirmed using a minimal HIV-1 promoter containing Sp1 sites and a TAR sequence. Moreover, GRHAL1 alone was sufficient to activate a heterologous promoter containing Sp1-binding elements. The Sp1-selective inhibitor mithramycin suppressed both Tat-independent LTR activation and the cooperative activation mediated by GRHAL1 and Tat. Electrophoretic mobility shift assays, RNA immunoprecipitation and RNA-pulldown experiments demonstrated that GRHAL1 directly interacts with Sp1 and that Tat enhances this interaction, suggesting that GRHAL1 modulates Sp1 activity through direct binding. In summary, we identify GRHAL1 as a novel HIV-1-induced lncRNA that regulates viral gene expression by interacting with Sp1 and synergizing with Tat to enhance HIV-1 transcription.