REGULATION OF HIV TRANSCRIPTION IN T CELLS
Open Access
- Author:
- Natarajan, Malini
- Graduate Program:
- Immunology and Infectious Diseases
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 20, 2010
- Committee Members:
- Avery August, Dissertation Advisor/Co-Advisor
Andrew Thomas Henderson, Committee Member
Avery August, Committee Chair/Co-Chair
Anthony Paul Schmitt, Committee Member
Pamela Hankey Giblin, Committee Member
David Scott Gilmour, Committee Member - Keywords:
- Pcf11
Transcription
NELF
HIV
CD28
T cell activation - Abstract:
- ABSTRACT CD4+T cells are the primary target of HIV infection, and depletion in CD4+T cell count corresponds with disease progression leading to aquired immunodeficiency syndrome (AIDS). The current treatment for AIDS employs the administration of highly active antiretroviral therapy (HAART), which has not been successful in completely eliminating HIV infection since the virus persists in a subset of cells harboring latent provirus. A number of events contribute to HIV transcriptional latency including lack of transcription factors, repressive chromatin structure, epigenetic modifications of the DNA, the inability of Tat to recruit functional PTEFb or poor processivity of RNA polymerase II. Latent provirus can be reactivated when T cells are activated by signals downstream of the T cell receptor and the costimulatory molecule CD28. I have studied two facets of HIV transcription; how signals downstream CD28 induce HIV transcription and the role of NELF and Pcf11 in repressing HIV transcription by regulation transcription elongation. Activation through the T cell receptor (TCR) and the costimulatory receptor CD28 supports efficient HIV transcription as well as reactivation of latent provirus. In order to characterize critical signals associated with CD28 that regulate HIV-1 transcription, I generated a library of chimeric CD28 receptors that harbored different combinations of key tyrosine (Y) residues in the cytoplasmic tail, Y173, Y188, Y191 and Y200. I found that Y191 and Y200 induce HIV-1 transcription via the activation of NF- κB and its recruitment to the HIV-long terminal repeat. Y188 modifies positive and iv negative signals associated with CD28. Importantly, signaling through Y188, Y191 and Y200 is required to overcome the inhibition posed by Y173. CD28 also regulates PTEFb activity, which is necessary for HIV-1 transcription processivity, by limiting the release of PTEFb from the HEXIM1-7SK inhibitory complex in response to T cell receptor signaling. My studies reveal that CD28 regulates HIV-1 provirus transcription through a complex interplay of positive and negative signals that may be manipulated to control HIV-1 transcription and replication. We have previously established an important role of negative elongation factor (NELF) in regulating HIV transcription in latent cell lines by inducing promoter proximal pausing. I have extended these studies by evaluating the role of NELF in infected T cell populations, including primary CD4+ T cells. Depleting NELF in HIV infected CD4+ T cells increased the release of virus. This increase in virus production corresponded to enhanced transcription elongation, emphasizing the role of NELF in maintaining a paused RNA polymerase II complex and limiting HIV transcription. Furthermore, I have shown that NELF interacts with Pcf11, a transcription termination factor. Pcf11 has been shown to cause premature termination of the paused elongation complex and I demonstrated that depletion of Pcf11 in CD4+T cells induces HIV elongation and transcription. In fact, depletion of both NELF and Pcf11 increases basal HIV transcription, suggesting that they act in concert in the same biochemical pathway. I propose that NELF recruits Pcf11 to the paused polymerase complex, coupling promoter proximal pausing with premature termination. These studies confirm that NELF mediated promoter proximal pausing and premature termination by Pcf11 are critical early check points in HIV transcription. v Overall, my studies provide new insights into the factors that regulate RNA polymerase II processivity and their overall impact on HIV transcription and latency, as well as identifying signals downstream of T cell activation which reactivate latent provirus. How these results may lead to the development of novel strategies to purge latent provirus from cellular reservoirs that harbor HIV are discussed.