RelA-associated inhibitor (RAI) is an inhibitor of nuclear factor B (NF-B) newly identified by yeast two-hybrid screen as an interacting protein of the p65 (RelA) subunit. In this study, we attempted to examine the effect of RAI on transcription and replication of human immunodeficiency virus type 1 (HIV-1). We found that RAI inhibited gene expression from the HIV-1 long terminal repeat (LTR) even at the basal level. Upon in vitro DNA-binding reactions, RAI could directly block the DNA-binding of p65 subunit of NF-B but not that of the p50 subunit or AP1. We found that RAI could also inhibit the DNA-binding of Sp1 and thus inhibit the basal HIV-1 promoter activity. We further examined the effects of RAI on Sp1 and found that RAI colocalizes with Sp1 in the nucleus and interacts with Sp1 in vitro and in vivo. Moreover, we found that RAI efficiently blocked the HIV-1 replication when cotransfected with a full-length HIV-1 clone. These findings indicate that RAI acts as an efficient inhibitor of HIV-1 gene expression in which both NF-B and Sp1 play major roles.Nuclear factor B (NF-B) and Sp1 are potent cellular activators of human immunodeficiency virus type 1 (HIV-1) gene expression (1,9,22,33). In cells chronically infected with HIV-1, activation of NF-B together with constitutive active Sp1 could trigger the transcription of viral genes including the trans-activator Tat, which would result in an explosive increase in HIV-1 replication (11,19,28; reviewed in reference 12). Thus, downregulation of NF-B activity has long been sought to inhibit the HIV replication and prevent clinical development of AIDS in the HIV infected individuals (reviewed in references 1, 2, and 20).The members of the NF-B family in mammalian cells include p50/p105 (NF-B1), p52/p100 (NFB2), p65 (RelA), c-Rel, and RelB. These proteins share a highly conserved region in the N terminus, known as the Rel homology domain, which is responsible for DNA binding, dimerization, nuclear translocation, and inhibition by IB proteins (1, 8). NF-B is normally present in the cytoplasm in association with its inhibitor, IB (1, 31). Upon stimulation with various stimuli including interleukin 1, tumor necrosis factor alpha (TNF-␣), phorbol esters, radical oxygens, and UV irradiation, it is dissociated from IB and translocated to the nucleus, where it activates target genes (1,20,22,29). Since IB proteins are susceptible to these extracellular signals, various approaches have been attempted in order to block HIV replication, such as using chemical inhibitors and dominant negative IB mutants (13,26,28,33). However, actions of these inhibitors are broad, and more specific inhibition of HIV gene expression is being sought after.We have recently identified a novel inhibitor for NF-B, RelA-associated inhibitor (RAI), by yeast two-hybrid screen using the central region of p65 as bait (39). RAI contains four tandem ankyrin repeats and an SH3 motif, structural features similar with other proteins interacting with NF-B, such as IB family proteins and 53BP2 (38). We showe...

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