RON Receptor Tyrosine Kinase, a Negative Regulator of Inflammation, Inhibits HIV-1 Transcription in Monocytes/Macrophages and Is Decreased in Brain Tissue from Patients with AIDS

Abstract

Activation of macrophages and microglia cells after HIV-1 infection and their production of inflammatory mediators contribute to HIV-associated CNS diseases. The mechanisms that initiate and maintain inflammation after HIV-1 infection in the brain have not been well studied. Furthermore, it is not understood why in HIV-associated CNS disease, macrophages and microglia are biased toward inflammation rather than production of mediators that control inflammation. We have focused on the receptor tyrosine kinase RON, a critical negative regulator of macrophage function and inflammation, to determine whether this receptor regulates HIV-1 expression. Overexpressing RON in monocytes/macrophages demonstrates that RON inhibits HIV-1 proviral transcription in part by decreasing the binding activity of NF-κB to the HIV-1 long terminal repeat. Because macrophages and microglia cells are a critical reservoir for HIV-1 in the CNS, we examined brain tissues for RON expression and detected RON in astrocytes, cortical neurons, and monocytoid cells. RON was detected in all control patients who were HIV seronegative (n = 7), whereas six of nine brain samples obtained from AIDS patients exhibited reduced RON protein. These data suggest that RON initiates signaling pathways that negatively regulate HIV-1 transcription in monocytes/macrophages and that HIV-1 suppresses RON function by decreasing protein levels in the brain to assure efficient replication. Furthermore, HIV-1 infection would compromise the ability of RON to protect against inflammation and consequent CNS damage.

The Receptor Tyrosine Kinase RON Represses HIV-1 Transcription by Targeting RNA Polymerase II Processivity

“…1). Consistent with previous studies (22), wild-type RON inhibited HIV transcription; however, mutating either Y1353, Y1360, or the kinase domain ablated the ability of RON to repress HIV-1 transcription (Fig. 1) demonstrating that signals emanating from RON actively repress provirus transcription.…”

“…RON is primarily expressed on human tissue resident macrophages which are not readily accessible or amendable to biochemical studies (22,23,30,31). Therefore, to gain further insight into mechanisms by which RON repress HIV-1 provirus transcription we used site-directed mutagenesis to generate several mutations in the RON cytoplasmic domain (25,26).…”

“…We have previously demonstrated that the receptor tyrosine kinase RON represses HIV-1 transcription (22). RON, which is expressed on tissue resident macrophages, regulates macrophage differentiation, apoptosis, and cell movement and in general suppresses activities associated with inflammation (23).…”

“…Cell lines used in these studies were the monocytic cell line U937 and U937-RON, which overexpress RON (22). RON mutations were introduced using a QuikChange Mutagenesis kit (Stratagene) and then were subcloned into murine stem cell virus-derived retroviral transfer vector MSCV2.1 as previously described (25,26).…”

“…The pNL43-Luc ϩ Env Ϫ virus, a replication-defective virus (27), was generated by transfecting 293T cells with 15 g of pNL43-Luc ϩ Env Ϫ DNA, 3 g of VSV-G-Env (vesicular stomatitis virus glycoprotein envelope) DNA, and 3 g of Rev DNA with calcium phosphate transfection (22). One milliliter of virus stock was added to 1.0 ϫ 10 6 cells for 18 and 48 h postinfection virus transcription was measured using a luciferase assay (Promega).…”