The relationship between T cell signals and HIV-1: the positives and the negatives
Open Access
- Author:
- Strasner, Amy
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 14, 2006
- Committee Members:
- Andrew Thomas Henderson, Committee Chair/Co-Chair
Avery August, Committee Member
Richard John Frisque, Committee Member
Dr Biao He, Committee Member
Dr Shao Cong Sun, Committee Member - Keywords:
- HIV-1
T cell signals - Abstract:
- CD4+ T cell activation is a prerequisite for productive HIV-1 infection and replication. Therefore, the virus has evolved mechanisms to exploit host cellular factors to promote its own propagation. Relatively little is known regarding the interplay between proteins involved in T cell activation and HIV-1. The purpose of this work was to investigate the relationship between signals in the TcR/CD3/CD28 pathway and HIV-1 replication. T cell stimulation through TcR/CD3 induces low level HIV-1 transcription. However, maximal HIV-1 expression requires the simultaneous ligation of a costimulatory receptor, such as CD28. Although the significance of transcription factors NF-ƒÛB, NF-AT, Sp1, and AP-1 in T cell activation-induced HIV-1 gene expression is well established, little is known regarding the specific cis-acting elements and the HIV long terminal repeat (LTR) region mediating the CD28 related enhancement of HIV-1 transcription. Using deletional analysis of the HIV-LTR in conjunction with electromobility shift assay (EMSA), we determined that the enhanced HIV-1 transcription in response to CD28 ligation was mediated through the binding of a specific protein to a potential CD28 response element (HIV-28RE) in the region spanning-182 to -205 of the HIV-LTR. This finding illustrates the positive regulation of HIV-1 expression by molecules in the T cell signaling pathway. The tight link between HIV-1 transcription and T cell activation and its dependence on host transcription factors suggests that provirus activity would be silenced as an infected cell transitioned from an activated to a resting state, such as during the process of anergy or memory induction. This is thought to be the underlying mechanism of HIV-1 postintegration latency. The population of resting memory CD4+ T cells harboring latent virus represents the primary barrier against eradication of the virus in HAART patients. The cellular mediators of the establishment of this reservoir are largely unknown due to the difficulty in the isolation of latent cells in vivo and the lack of available representative in vitro models. We have developed a novel in vitro system to identify the molecular events involved in silencing the HIV-1 provirus. This model utilizes a virus engineered to express placental alkaline phosphatase (PLAP) on its surface upon virus transcription in conjunction with the removal of this marker by Phospholipase C treatment. Measurement of PLAP re-expression is used as an indicator of the HIV-1 transcriptional response to various signal manipulations. Using this experimental approach, we demonstrated that suboptimal T cell signaling that activates the calcium pathway induces a reversible, long-term suppression of HIV-1 transcription that may represent a latent state. These findings indicate that signals in the TcR/CD28 pathway can negatively regulate HIV-1 gene expression. The role of Lck, an early and integral member of the T cell activation pathway, was assessed via the infection of Lck deficient T cell lines. HIV-1 replication was consistently attenuated in the absence of Lck, suggesting that Lck was positively regulating HIV-1 expression. However, a series of experiments determined that Lck was not influencing HIV-1 transcription or the early stages of the virus life-cycle, but instead was mediating the later stages of the virus life cycle, namely HIV-1 assembly. HIV-1 particles accumulated in the intracellular compartments of cells lacking Lck, suggesting that Lck was facilitating the progression of HIV-1 from the microvesicles to the plasma membrane for efficient virus release. This novel function of Lck did not involve its kinase activity but was mediated through a physical interaction between Lck and Gag. These findings indicate that T cell signaling proteins can both positively and negatively regulate HIV-1 transcription, and can play important unforeseen roles in other stages of the virus life cycle, such as HIV-1 assembly. Insight regarding the interplay between T cell signals and HIV-1 will lead to a better understanding of how the virus exploits host proteins to promote its replication and will ultimately provide targets for future therapeutic intervention.