PhD Dissertation Defense by Priya Sridharan "Investigating the Molecular bases of HIV-1 Nef mediated hijacking of Alix and PTPN23"
Abstract
HIV-1 Nef is a multifunctional accessory protein with a molecular mass of 27 kDa that plays a central role in lentiviral pathogenesis by manipulating the host immune response to enhance viral replication. One of Nef’s key functions is the downregulation of cell surface receptors, particularly CD4 and major histocompatibility complex class I (MHCI). In addition to receptor downregulation, Nef modulates various host cellular processes, including signal transduction and vesicular transport pathways. These interactions not only contribute to immune evasion but also create a cellular environment favorable for viral replication. Nef promotes the internalization of CD4 from the cell surface through the clathrin/AP-2 endocytosis pathway. After internalization, Nef directs CD4 into the multivesicular body (MVB) pathway. The MVB pathway, regulated by the ESCRT complex, sorts CD4 into vesicles that fuse with lysosomes for degradation.
Nef directly interacts with Alix (apoptosis-linked gene 2-interacting protein X), a key component of the ESCRT machinery, to transport CD4 into the multivesicular body (MVB) pathway, which ultimately leads to its degradation. In this dissertation project, the interaction between Alix and Nef was studied using biochemical assays. Chapter 2 of the dissertation focuses on these assays, providing new insights into how Nef interacts with Alix and contributing to a deeper understanding of Nef's role in viral pathogenesis.
PTPN23 is a putative protein tyrosine phosphatase and a paralog of Alix. Like Alix, PTPN23 supports ESCRT function and is a key regulator in endosomal sorting and vesicular trafficking. Preliminary findings from our collaborator, Dr. John Guatelli’s lab, suggests that HIV-1 Nef may exploit PTPN23 in a manner similar to its interaction with Alix to misroute 3 host cell surface receptors toward lysosomal degradation. In Chapter 3 of this dissertation, we explored the potential binding interaction between Nef and PTPN23 using biochemical assays. Our results showed that the Nef-PTPN23 interaction is promoted by acidic pH, consistent with the fact that PTPN23 operates in late endosomal compartments.
Chapter 4 of the dissertation describes experimental procedures for protein expression, purification, and assay methodologies. My additional contributions to the study of HIV-1 Nef interactions, including the cloning of MBP-FLAG-CD4(394–433) and Sumo-CD4 10aa-β2(1–25)-FLAG constructs for FRET and FP assay analysis, are also discussed in Chapter 4.
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