REQUIREMENT AND APPROPRIATION OF ACTIVATING SIGNALS BY HIV-1 IN INFECTED CD4+ T CELLS

Open Access
Author:
Yang, Polung
Graduate Program:
Integrative Biosciences
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
September 09, 2005
Committee Members:
  • Andrew Thomas Henderson, Committee Chair
  • Avery August, Committee Member
  • Richard John Frisque, Committee Member
  • Biao He, Committee Member
  • Brian Wigdahl, Committee Member
Keywords:
  • Human Immunodeficiency Virus
  • CD4+ T cells
  • signal transduction
  • CD28
  • Nef
  • Cbl
  • PI3K
  • tyrosine phosphorylation
  • transcription
Abstract:
The infection of CD 4+ T cells by HIV-1 is characterized by the dependence of the virus on stimuli of TCR/CD3 and CD28 for activation and proliferation. In return, the HIV-1 accessory proteins effect changes in T cell physiology, including its signaling activities. The goal of this work was to investigate how cellular signals of CD4+ T cells regulate the activity of HIV-1, and how the virus subverts cellular restrictions imposed by signaling requirement with HIV-1 Nef. It was previously shown that CD28 signaling activates HIV transcription by a combination of negative and positive signals emanating from tyrosines 173 and 200, respectively {Cook, 2003 #260}. To determine the roles of tyrosines 188 and 191 in CD28 signaling, we measured the transcriptional activation of a HIV and IL-2 in cells expressing chimeric receptors without Y188 and Y191 (YFFY), as well as receptors without Y173 and Y200 (FYYF). Our results showed that Y188 and Y191 were required for proviral activation by CD28. In addition, signals emanating from Y188 and Y191 were sufficient to induce activation. Similar requirement for Y188 was also seen for the transcriptional activation of IL-2. Treatment with LY294002 restored transcriptional activation of HIV and IL-2 by YFFY and FYYF, consistent with the role of PI3K in negative regulation of CD28 signals. Comparative analysis of CD28 tyrosine mutations revealed redundant signals by Y188, Y191 and Y200 that cooperate to counterbalance negative regulation by Y173. Taken together, these data confirmed negative regulation of CD28 signaling by Y173-associated PI3K and the requirement of Y188 and Y191 in addition to Y200 to overcome this signaling restriction. The multifunctional HIV-1 protein Nef possesses several motifs that interact with signaling molecules in infected T cells. In order to determine whether Nef influences T cell activation, cells were infected with Nef-positive and Nef-negative clones of HIV. CD28 expression and changes in tyrosine phosphorylation were monitored. We observed no Nef-dependent changes in CD28 expression or function. However, infection with Nef-positive virus led to changes in tyrosine phosphorylation. This Nef-induced phosphorylation was observed in unstimulated cells, and c-Cbl was identified as one of the proteins whose phosphorylation was upregulated by Nef. Furthermore, Lck is required for Nef-mediated c-Cbl tyrosine phosphorylation. These results suggest that Nef modifies T cell signaling in the absence of T cell receptor engagement and co-stimulation. In summary, these results demonstrate that efficient activation and replication of HIV is subject to constraints imposed by the requirements of T cell signaling, including regulation by CD28 signals. In adapting to these constraints, HIV has evolved means to manipulate the property of T cell activating signals, potentially to maximize viral response to activating stimuli. Deeper understanding of the interplay of this two aspects of HIV life cycle will provide us with insights not only in the strategy of viral replication, but also in the mechanism of gene regulation by T cell activation.