Cellular Signals Suppress HIV-1 Transcription By Interfering With Chromatin Remodeling and Polymerase Activation
Open Access
- Author:
- Klatt, Alicia J
- Graduate Program:
- Pathobiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 28, 2007
- Committee Members:
- Andrew Thomas Henderson, Committee Chair/Co-Chair
Avery August, Committee Member
Pamela Correll, Committee Member
Richard John Frisque, Committee Member
David Scott Gilmour, Committee Member - Keywords:
- RNA Polymerase II
Transcription
Macrophages
HIV
NELF
RON - Abstract:
- AIDS is a multifactorial disease that affects various tissues and organs throughout the body, with the primary cellular targets being CD4+ T-cells and monocytes/macrophages. Macrophages are early targets of viral infection, and because they are relatively resistant to the cytopathic effects induced by HIV-1 infection, they serve as potential sites of long-term viral infection in addition to playing a major role in the development of pathologies such as AIDS dementia complex. Monocytic precursor cells differentiate in response to antigen-specific stimuli within a tissue microenvironment into phenotypically distinct macrophage subsets. We have investigated the transcriptional response of HIV-1 in two of these populations, classically and alternatively activated macrophages. Our data indicate that HIV-1 transcription is suppressed in alternatively activated populations that were generated by treating bone marrow derived macrophages with IL-4 when compared to classically activated populations cultured in the presence of IFN-ƒ× and LPS. Additionally HIV-1 transcription was increased in cells lacking stk, a cellular receptor noted for its anti-inflammatory activity. These results indicate that the phenotypic characteristics of a macrophage population can directly influence HIV-1 transcription. To specifically explore the mechanisms by which cellular signals can suppress HIV-1 transcription we use a cell line expressing the receptor tyrosine kinase RON (U937 RON), which has been shown to negatively regulate HIV-1 transcription. Our data indicate that RON suppresses HIV-1 replication by promoting RNA Polymerase II (Pol II) pausing, and preventing chromatin re-organization, and nucleosome remodeling. Furthermore, increased binding of the negative elongation factor subunit NELF-E was correlated with decreased Pol II processivity, suggesting a possible mechanism for the ability of RON to suppress HIV-1 transcription elongation. The targeting of Pol II processivity by RON signaling also suggests a more general mechanism which RON may employ to negatively regulate other genes such as IL-12p40, and iNOS. Additionally, we explore the status of the elongation complex on the HIV-1 LTR in a suppressed virus state using the latently infected cell line U1. We show that RNA Pol II pausing is induced early in provirus transcription before formation of the TAR element at approximately +40 bp and that NELF is instrumental in maintaining this pause. Depleting NELF using siRNA results in enhanced transcription elongation, histone acetylation, and nucleosome remodeling, indicating that transcription elongation and chromatin remodeling are coupled events. Our results suggest a novel role for NELF in repression of early HIV-1 provirus transcription that is different from that previously suggested by transient expression assays. Taken together, these results demonstrate that cellular signals can negatively regulate HIV-1 transcription by targeting the elongation complex, leading to a suppressed or latent virus phenotype. Our data, for the first time, indicate a Tat independent role for NELF in maintaining Pol II pausing at the HIV-1 LTR that would be similar to other cellular genes that are regulated by transcription elongation. These studies further show that cellular signals, such as RON, can actively induce a reversible pause of the RNA Pol II complex to suppress HIV-1 replication and affords us a system in which to study signaling mechanisms and biochemical requirements required to establish viral latency.