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NFAT5 regulates HIV-1 in primary monocytes via a highly conserved long terminal repeat site.

ABSTRACT: To replicate, HIV-1 capitalizes on endogenous cellular activation pathways resulting in recruitment of key host transcription factors to its viral enhancer. RNA interference has been a powerful tool for blocking key checkpoints in HIV-1 entry into cells. Here we apply RNA interference to HIV-1 transcription in primary macrophages, a major reservoir of the virus, and specifically target the transcription factor NFAT5 (nuclear factor of activated T cells 5), which is the most evolutionarily divergent NFAT protein. By molecularly cloning and sequencing isolates from multiple viral subtypes, and performing DNase I footprinting, electrophoretic mobility shift, and promoter mutagenesis transfection assays, we demonstrate that NFAT5 functionally interacts with a specific enhancer binding site conserved in HIV-1, HIV-2, and multiple simian immunodeficiency viruses. Using small interfering RNA to ablate expression of endogenous NFAT5 protein, we show that the replication of three major HIV-1 viral subtypes (B, C, and E) is dependent upon NFAT5 in human primary differentiated macrophages. Our results define a novel host factor-viral enhancer interaction that reveals a new regulatory role for NFAT5 and defines a functional DNA motif conserved across HIV-1 subtypes and representative simian immunodeficiency viruses. Inhibition of the NFAT5-LTR interaction may thus present a novel therapeutic target to suppress HIV-1 replication and progression of AIDS.

SUBMITTER: Ranjbar S

PROVIDER: S-EPMC1698943 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140 kb unique coding region flanked by 30-40 copies of 0.8 kb terminal repeat (TR) sequence. KSHV genomes persist in latently infected cells as episomes via tethering to the host cell chromosomes, and KSHV latency associated nuclear antigen (LANA) plays a crucial role in latent episomal DNA replication and segregation during host cell mitosis by binding to TR. While TR's function in plasmid maintenance is well-established, TR’s transcription regulatory roles as gene enhancer has not been fully explored. Gene enhancer harbors transcription enzymes via arrays of transcription factors bindings and often forms phase separate nuclear body in part through recruitment of BRD4 and MED1 that contain intrinsically disordered domain. Here we show KSHV TR possesses transcription regulatory function with LANA. A series of Cleavage Under Targets & Release Using Nuclease (CUT&RUN) demonstrated that TR fragments are occupied by histone modifying enzymes that are known to interact with LANA in naturally infected cells, and the TR possessed characteristic enhancer histone modifications. The H3K4me3 and H3K27Ac modification were also conserved in unique region of the KSHV genome among three PEL cells, and the KSHV Origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with TR's. In the Ori-Lyt region, the LANA protein complex colocalizes with H3K27Ac-modified nucleosome along with paused RNA polymerase II, and the nucleosome is franked by two K-Rta recruitment sites. The isolated reporter assays demonstrated that neighboring TR fragments enhanced viral lytic gene promoter activity independent of orientation in KSHV-infected and non-infected 293FT cells. K-Rta transactivation function was drastically enhanced with TR, while LANA acquired promoter repression function when the reporter was ligated with TR. The deletion of LANA acidic repeat sequence, a highly-disordered protein domain, further increased gene repression functions. Combined, the TR region is (i) an epigenetically active DNA element that is stitched within 12.5 kb (20-40 copies), (ii) has an array of transcription factor (LANA) binding sites, (iii) recruited by transcription related enzymes including BRD4 (bromodomain containing 4), (iv) decorated by histone H3K27Ac marks, and (v) possesses orientation-independent transcription activation function. KSHV TR is therefore an enhancer domain for KSHV inducible genes. However, in contrast to cellular enhancers that are bound by multiple transcription factors, perhaps KSHV enhancer is predominantly regulated by the LANA nuclear body on the TR. We suggest that KSHV evolved a clever mechanism to tightly control the latency-lytic switch with the TR/LANA complex.

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NFAT5 regulates HIV-1 in primary monocytes via a highly conserved long terminal repeat site.

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