The receptor tyrosine kinase RON represses HIV-1 transcription by targeting RNA polymerase II processivity.

Efficient HIV-1 transcription requires the induction of cellular transcription factors, such as NF-kappaB, and the viral factor Tat, which through the recruitment of P-TEFb enhances processive transcription. However, whether cellular signals repress HIV-1 transcription to establish proviral latency has not been well studied. Previously, it has been shown that the receptor tyrosine kinase RON inhibits HIV transcription. To gain insights into the biochemical mechanisms by which RON inhibits transcription we examined the binding of transcription factors to the HIV provirus long terminal repeat using chromatin immunoprecipitation. RON expression decreased basal levels of NF-kappaB and RNA polymerase II (Pol II) binding to the HIV provirus long terminal repeat but did not prevent the induction of these complexes following treatment with cytokines. However, RON did decrease efficient transcription elongation because reduced RNA Pol II was associated with HIV-1 genomic sequences downstream of the transcriptional start site. There was a correlation between RON expression and increased binding of factors that negatively regulate transcription elongation, NELF, Spt5, and Pcf11. Furthermore, the ability of RON to inhibit HIV-1 transcription was sensitive to a histone deacetylase inhibitor and was associated with nucleosome remodeling. These results indicate that RON represses HIV transcription at multiple transcriptional check points including initiation, elongation and chromatin organization and are the first studies to show that cellular signaling pathways target Pol II pausing to repress gene expression.

Transcription termination factor Pcf11 limits the processivity of Pol II on an HIV provirus to repress gene expression.

Many elongation factors in eukaryotes promote gene expression by increasing the processivity of RNA polymerase II (Pol II). However, the stability of RNA Pol II elongation complexes suggests that such complexes are not inherently prone to prematurely terminating transcription, particularly at physiological nucleotide concentrations. We show that the termination factor, Pcf11, causes premature termination on an HIV provirus. The transcription that occurs when Pcf11 is depleted from cells or an extract is no longer sensitive to 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a compound that causes premature termination. Hence, Pcf11 can act as a negative elongation factor to repress RNA Pol II gene expression in eukaryotic cells.

Negative elongation factor NELF represses human immunodeficiency virus transcription by pausing the RNA polymerase II complex.

Human immunodeficiency virus (HIV) transcription requires virally encoded Tat and the P-TEFb protein complex, which together associate with the Tat-activating region, a structured region in the nascent transcript. P-TEFb phosphorylates Proteins in the transcription elongation complex, including RNA polymerase II (pol II), to stimulate elongation and to overcome premature termination. However, the status of the elongation complex on the HIV long terminal repeat (LTR) in a repressed state is not known. Chromatin immunoprecipitation demonstrated that NELF, a negative transcription elongation factor, was associated with the LTR. Depleting NELF increased processive HIV transcription and replication. Mapping pol II on the LTR showed that pol II was paused and that NELF depletion released pol II. Decreasing NELF also correlated with displacement of a positioned nucleosome and increased acetylation of histone H4, suggesting coupling of transcription elongation and chromatin remodeling. Previous work has indicated that the Tat-activating region plays a critical role in regulating transcription from the LTR. Our results reveal an earlier stage, mediated by NELF, when repression occurs at the HIV LTR.

Function of small hydrophobic proteins of paramyxovirus.

Mumps virus (MuV), a rubulavirus of the paramyxovirus family, causes acute infections in humans. MuV has seven genes including a small hydrophobic (SH) gene, which encodes a type I membrane protein of 57 amino acid residues. The function of the SH protein is not clear, although its expression is not necessary for growth of MuV in tissue culture cells. It is speculated that MuV SH plays a role in viral pathogenesis. Simian virus 5 (SV5), a closely related rubulavirus, encodes a 44-amino-acid-residue SH protein. Recombinant SV5 lacking the SH gene (rSV5DeltaSH) is viable and has no growth defect in tissue culture cells. However, rSV5DeltaSH induces apoptosis in tissue culture cells and is attenuated in vivo. Neutralizing antibodies against tumor necrosis factor alpha (TNF-alpha) and TNF-alpha receptor 1 block rSV5DeltaSH-induced apoptosis, suggesting that SV5 SH plays an essential role in blocking the TNF-alpha-mediated apoptosis pathway. Because MuV is closely related to SV5, we hypothesize that the SH protein of MuV has a function similar to that of SV5, even though there is no sequence homology between them. To test this hypothesis and to study the function of MuV SH, we have replaced the open reading frame (ORF) of SV5 SH with the ORF of MuV SH in a SV5 genome background. The recombinant SV5 (rSV5DeltaSH+MuV-SH) was analyzed in comparison with SV5. It was found that rSV5DeltaSH+MuV-SH was viable and behaved like wild-type SV5, suggesting that MuV SH has a function similar to that of SV5 SH. Furthermore, both ectopically expressed SV5 SH and MuV SH blocked activation of NF-kappaB by TNF-alpha in a reporter gene assay, suggesting that both SH proteins can inhibit TNF-alpha signaling.

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.

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-kappaB 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.