Human macrophages are a major reservoir of persistent HIV replication. However, the mechanisms that regulate viral persistence in these cells ar unknown. Their characterization could lead to the eradication of this key viral reservoir. Nuclear factor kB (NF-kB) is a transcription factor that regulates the transcription of HIV. In a model of persistent HIV infection in monocytes and macrophages but not in T lymphocytes, we have demonstrated that NF-kB is specifically induced during the course of the infection. The impact of this cell specific-virus interaction on the immune function and on HIV replication remains unknown. Furthermore, the mechanisms by which extracellular stimuli or persistent HIV replication regulate NF-kB in monocytes are poorly characterized. Our research is directed at characterizing the mechanisms that regulate NF-kB in HIV- infected monocytes and macrophages. Results from these studies will enable us to determine the relative role of NF-kB in viral persistence. Preliminary data is provided focusing on transcriptional and post- transcriptional mechanisms regulating NF-kB. First, increased NF-kB activity in HIV-infected monocytes is secondary to increased synthesis of the p105/p50 NF-kB subunit. Our cloning of the promoter of the gene encoding p105 indicates its autoregulation by NF-kB. Thus, HIV and NF-kB through its p50 subunit engage in a double autoregulatory loop in human monocytes. Detailed studies of the regulatory regions of this promoter through which HIV and extracellular stimuli increase its transcriptional activity in monocytes will be performed. Second, increased nuclear translocations in HIV-infected monocytes follows a specific serine- threonine phosphorylation of the NF-kB inhibitor, MAD3. A novel signal transduction pathway that is functional monocytes and upregulated in HIV- infected cells, phosphatidyl choline-phospholipase C (PC-PLC), results in NF-kB activation. A kinase within this pathway, PKC-zeta ((zeta) is required for HIV-mediated NF-kB activation and phosphorylates MAD3 in vitro. Moreover, the ras-raf pathway, known to be connected to PC-PLC and PKC-zeta also mediates HIV-dependent NF-kB activation. A systematic analysis of the biochemical steps of these pathways, including kinases that phosphorylate IkB will be performed. In HIV-infected monocytes and macrophages, these putative signal transduction pathways result in a specific serine-threonine phosphorylation of MAD3 in vivo with its subsequent proteolysis. This increases NF-kB nuclear translocation which not only activates transcription of p105/p50 and the HIV-LTR, but also of MAD3. Thus, HIV,NF-kB (p150/p50) and MAD3 now form a continuous triple autoregulatory loop in the human monocyte-macrophage. Inhibition of specific mechanisms identified above should confirm and define their role in the regulation of NF-kB and viral replication in monocytes- macrophages. This will be attempted using complementary and alternative approaches. Through this multidisciplinary approach, we expect to identify the molecular mechanisms that regulate NF-kB in uninfected and HIV-infected monocytes and macrophages, in order to better understand the role of NF- kB in viral persistence, latency and in the immune function of these cells.
