Permanent occupancy of the human immunodeficiency virus type 1 enhancer by NF-kappa B is needed for persistent viral replication in monocytes

Abstract

This work aimed to ascertain the role of kappaB-responsive elements of the human immunodeficiency virus type 1 (HIV-1) enhancer not only in early initiation but also in long-term maintenance of proviral transcription in cells of the monocytic lineage. For this purpose, we used three main approaches. The first was to abruptly terminate tumor necrosis factor-induced NF-kappaB binding to the enhancer sequences in U1 monocytic cells, using a short pulse of exogenous tumor necrosis factor. This resulted in concomitant decrease in nuclear NF-kappaB DNA-binding activity and endogenous long terminal repeat transcriptional activity. The second was to suppress the permanent NF-kappaB translocation induced by HIV-1 replication itself in chronically infected U937 cells, using a specific proteasome inhibitor (Z-LLL-H). As early as 2 h after addition of the inhibitor to the culture medium, there was an inhibition of both constitutive activation of NF-kappaB and HIV-1 genome expression. The third approach was to monitor the replication competence in U937 cells of an infectious HIV-1 provirus carrying point mutations in the kappaB-responsive elements of both long terminal repeats. Compared with its wild-type counterpart, this mutated provirus showed a profoundly decreased, Z-LLL-H-insensitive transcriptional and replicative activity in U937 monocytes. Together, our results indicate that occupancy of the viral enhancer by NF-kappaB (p50/p65) heterodimers is required for ongoing transcription of integrated HIV provirus in monocytes, even in cells chronically infected and permanently producing functional HIV Tat protein. Thus, the ability of HIV-1 replication to activate NF-kappaB is crucial to the intense self-perpetuated viral transcription observed in cells of the monocytic lineage.