No abstract

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. Human immunodeficiency virus (HIV)3 proviral expression is regulated at the transcriptional level. Virus transcription is controlled by the upstream long terminal repeat (LTR), which includes cis-elements that recruit both cellular and viral factors. The HIV-1 LTR is often divided into functional elements: the Tat-activating region (TAR), the promoter, the enhancer, and the negative/modulatory regulatory element. The promoter, enhancer, and modulatory elements recruit host transcription factors, such as Sp-1, NF-B and CCAAT/enhancer-binding protein, necessary to initiate transcription (1, 2). These factors also recruit coactivators, including histone acetyltransferases and SWI/SNF complexes, that influence the chromatin structure of integrated provirus (3-7). For example, the 5Ј-untranslated leader region located downstream of the transcriptional start site is associated with a nucleosome in latent HIV proviruses, which is displaced upon induction of virus transcription. Furthermore, agents that inhibit histone deacetylases, such as trapoxin and trichostatin A, activate HIV provirus transcription, suggesting a critical role for chromatin remodeling in the repression of HIV transcription (7,8).Transcription elongation has also been demonstrated to be a limiting step for HIV expression. HIV encodes a transcriptional activator (Tat) that binds the RNA stem-loop structure formed by TAR, and by recruiting P-TEFb to the LTR, HIV enhances processive transcription. P-TEFb, which is composed of cyclin T1 and CDK9, modifies RNA polymerase II (pol II) activity by hyperphosphorylating the C-terminal domain of pol II. In the absence of Tat, transcription elongation by RNA pol II from the HIV promoter is very inefficient (9). In vitro transcription analyses in the absence of chromatin revealed that the majority of elongation complexes init...

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-␤-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.

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.