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Tuberculosis (TB) disease in HIV co-infected patients contributes to increased mortality by activating innate and adaptive immune signaling cascades that stimulate HIV-1 replication, leading to an increase in viral load. Here, we demonstrate that silencing of the expression of the transcription factor nuclear factor of activated T cells 5 (NFAT5) by RNA interference (RNAi) inhibits Mycobacterium tuberculosis (MTb)-stimulated HIV-1 replication in co-infected macrophages. We show that NFAT5 gene and protein expression are strongly induced by MTb, which is a Toll-like receptor (TLR) ligand, and that an intact NFAT5 binding site in the viral promoter of R5-tropic HIV-1 subtype B and subtype C molecular clones is required for efficent induction of HIV-1 replication by MTb. Furthermore, silencing by RNAi of key components of the TLR pathway in human monocytes, including the downstream signaling molecules MyD88, IRAK1, and TRAF6, significantly inhibits MTb-induced NFAT5 gene expression. Thus, the innate immune response to MTb infection induces NFAT5 gene and protein expression, and NFAT5 plays a crucial role in MTb regulation of HIV-1 replication via a direct interaction with the viral promoter. These findings also demonstrate a general role for NFAT5 in TLR- and MTb-mediated control of gene expression., Author Summary The major cause of AIDS deaths globally has been tuberculosis (TB), which is caused by the bacterium Mycobacterium tuberculosis (MTb). Co-infection with MTb exacerbates human immunodeficiency virus type1 (HIV-1) replication and disease progression via both innate and adaptive host immune responses to MTb infection. In this report, we present evidence that the transcription factor NFAT5 plays a crucial role in MTb-induced HIV-1 replication in human peripheral blood cells and monocytes. We also show that MTb infection itself stimulates NFAT5 gene expression in human monocytes and that its expression involves the TLR signalling pathway and requires the downstream adaptor proteins MyD88, IRAK1, and TRAF6. This identification of a novel role for NFAT5 in TB/HIV-1 co-infection reveals that NFAT5 is a major mediator of TLR-dependent gene expression and thus provides a potential new therapeutic target for treatment of HIV-1 and possibly other diseases.

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., Synopsis To replicate, viruses exploit the cellular machinery of infected cells in their hosts. Macrophages, blood cells involved in the body's defense against pathogens, are resistant to the toxic effects of certain viruses, such as HIV-1, the AIDS virus. The drawback, however, is that macrophages become an avenue through which viruses can spread to other tissues and organs of the body. Here, Ranjbar, Tsytsykova, and colleagues have identified a new role for a human protein, nuclear factor of activated T cells 5 (NFAT5), as a host factor important for replication of HIV-1 in macrophages. The binding site for NFAT5 on a region of the HIV-1 DNA critical for its replication, the long terminal repeat, is the same across multiple subtypes of HIV-1 and the related virus HIV-2, and even the simian immunodeficiency virus; thus, disrupting the interaction between NFAT5 and the long terminal repeat provides a potential means of inhibiting replication of multiple versions of HIV.