A Novel Role For ITK, A Tec Family Tyrosine Kinase in HIV-1 Replication

Open Access
Schiralli, Gillian M.
Graduate Program:
Immunology and Infectious Diseases
Doctor of Philosophy
Document Type:
Date of Defense:
March 02, 2009
Committee Members:
  • Andrew Thomas Henderson, Dissertation Advisor/Co-Advisor
  • Andrew Thomas Henderson, Committee Chair/Co-Chair
  • Avery August, Committee Chair/Co-Chair
  • Pamela Hankey Giblin, Committee Member
  • Neil David Christensen, Committee Member
  • Michael N Teng, Committee Member
  • HIV replication
  • ITK
  • Kinase inhibitor
HIV is highly dependent on T cell activation for its replication. The virus has evolved to hijack host machinery and signals to facilitate its entry, expression, assembly and release. Which T cell specific signals regulate what specific steps of the viral life cycle are still being discovered. Understanding how these cellular factors are critical for HIV replication will provide insights into viral pathogenesis. Though HIV has evolved to manipulate the intracellular environment of its host, the virus needs to first gain entry into the cell. To do this, HIV must overcome a cellular barrier, the cytoskeleton. Not only is viral entry dependent on dynamic cytoskeleton changes, so is the egress of viral particles. Despite the obvious need for HIV to traverse and interact with the cytoskeleton, how these events are regulated in the context of HIV infection is still not appreciated. Thus, the purpose of this thesis was to examine T cell specific signals which were both involved in T cell activation, and the regulation of cytoskeleton dynamics, to identify potential key check points in HIV replication, in particular entry and egress. The Tec family tyrosine kinase, IL-2 inducing tyrosine kinase (ITK), is an important amplifier of T cells signals which emanate from TCR/antigen-presenting cell interactions. In addition to its role in T cell activation, ITK regulates actin cytoskeleton changes. ITK activates Vav-1, a guanine exchange factor, which in turn regulates GTPases Rac, Rho, and Cdc42, resulting in F-actin polarization. We investigated the role of ITK in HIV replication using several different strategies such as siRNA depletion of ITK, chemical inhibition of ITK, and the expression of several ITK mutants. Our data suggest that inhibition of ITK blocks HIV infection by affecting multiple steps of HIV replication. The loss of ITK function resulted in marked reductions in intracellular p24 levels upon HIV infection. Loss of ITK function after the establishment of HIV infection decreased viral spread within the culture. Chemical inhibition of ITK partially blocked HIV viral entry, which correlated with decreased actin polarization to gp120. ITK is required for efficient HIV transcription. Over-expression of ITK results in increased HIV transcription along with increased release of virus-like particles. ITK requires its Pleckstrin homology and Src homology 2 domains for regulating VLP formation whereas its kinase domain is dispensable. Further investigation of the role of ITK in viral particle assembly and release provided evidence of colocalization between ITK and Gag at the plasma membrane in a distinct punctate staining pattern. ITK co-staining with Gag was altered when ITK activation was inhibited. However, we have not detected a direct physical interaction between these molecules. These findings indicate that the Tec family tyrosine kinase, ITK, enhances HIV replication. ITK expression is necessary for efficient HIV entry, transcription, and viral release. In addition these data indicate that kinase-dependent and independent activities of ITK influence HIV replication. Understanding the interplay between T cell signals and HIV, will provide more insight into mechanisms that regulate HIV and identify new therapeutic strategies to aid in the fight against HIV.