This dissertation, "Mechanisms of HIV-1 Tat Induced Immune Response" by Chun-bong, Li, 李振邦, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Mechanisms of HIV-1 Tat induced immune response Submitted by Li Chun Bong for the degree of Doctor of Philosophy at the University of Hong Kong in August 2005 HIV Transactivator protein (Tat) has been known to have multiple regulatory roles including replication of HIV and modulation of cellular kinases. PKR, a double-stranded RNA-activated protein kinase, has been known for mediating the effects of interferon (IFN) and tumor necrosis factor-α (TNF-α), and virus-induced apoptosis. I hypothesize that PKR plays an important role in Tat-induced subversion of host defenses against HIV. The aim of this study is to investigate whether PKR plays a critical role in mediating the Tat-induced dysregulation of cytokine expression against HIV. Following treatment of primary blood monocytes (PBM) with recombinant Tat, cytokines including IL-10 and TNF-α were induced, with concomitant PKR activation. To investigate the role of PKR, I have shown that inhibition of PKR activity abrogates the Tat-induced cytokine induction. I next identified that the mitogen activated protein kinases (MAPK) including ERK-1/2 and p38 MAPK are downstream of PKR in regulating these Tat-induced pathways. Thus, PKR may play a critical role in mediating the subversive effects of HIV Tat resulting in IL-10 induction, which may contribute to immunosuppression. In view of the induction of IL-10 by Tat, a detailed analysis of the structure and function of the promoter of the human IL-10 gene was performed. I delineated that the Tat responsive element was located within 622 base pair (bp) to 610 bp upstream from the transcription start site (+1) using transient transfection of IL-10 promoter driven luciferase constructs. In this region, the binding sites for Ets-1 and Sp-1 as the regulatory elements were identified by electrophoretic mobility shift assays. Furthermore, mutation of the Ets-1 and Sp-1 binding sites in the -625/-595 nucleotides (nt) region abrogated the DNA binding activity as well as the IL-10 promoter activities as measured by induced luciferase expression. Overexpression of Ets-1 led to increases in the promoter activities of IL-10. The next aim was to study the signaling pathways of Tat-induced IL-10 transcription. Results showed that PKR and p38 MAPK are responsible for the Tat- induced pathway. These results demonstrated that coordinated activities of PKR and p38 MAPK, together with the activation of Ets1 and Sp1, may play a crucial role in HIV-1 Tat induced human IL-10 transcription. To further delineate the mechanisms of HIV-induced immune defects, the effects of Tat on the immune recognition and Toll-like receptor (TLR) expression were investigated. The role of TLR in the innate immune system was to recognize microbial antigens, such as lipopolysaccharide (LPS) and mycobacteria, resulting in induction of cytokine expression. Following Tat treatment, there was induction of TLR2 with concomitant downregulation of TLR4 and TLR9 in PBM. The Tat-induced TLR expression was regulated by PKR but independent of the activation of MAP kinases. Furthermore, Tat enhances the TLR2 expression in response to the IL-10 activation. In summary, these results demonstrated that the Tat induction of TLR expression is mediated by PKR, leading to downstream upregulation of cytokines including IL-10. (475 words) DOI: 10.5353/th_b3153716 Subjects: HIV (Virus