Advisory Committee Chair
Jamil S Saad
Advisory Committee Members
Todd J Green
Terje Dokland
Chad M Petit
Louis M Mansky
Document Type
Dissertation
Date of Award
2023
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Human T-cell leukemia virus 1 (HTLV-1) was the first oncogenic human retrovirus to be discovered. Several serious diseases have been linked to HTLV-1 infection, such as Adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Between 10-20 million people are infected with HTLV-1 making it a major cause of morbidity and mortality worldwide. A comprehensive understanding of the factors that govern retroviral replication precludes the development of efficient antiretroviral therapies (ARTs). In the example of HIV-1, atomic level structural data was paramount to developing and improving upon many of the ARTs used today, underscoring the need for structure guided drug development. However, similar structural and functional data for HTLV-1 is currently lacking, especially regarding Gag targeting and assembly. In this dissertation, we employed nuclear magnetic resonance spectroscopy (NMR), biophysical and biochemical assays, computational tools, and cell-based assays to assess the role of HTLV-1 MA and MA mutants on membrane binding, virus-like particle (VLP) production, and subcellular localization in cells. I solved the structure of the matrix (MA) domain of HTLV-1 Gag, responsible for targeting of Gag to the plasma membrane. I provided first direct evidence of a PI(4,5)P2 binding site on MA and identified and characterized a separate PS binding site in the unstructured N-terminus. We iv further showed that the PI(4,5)P2 and PS binding sites are essential for VLP production and virus replication. I also developed a novel in vitro approach using liposomes and cryo-electron microscopy (cryo-EM) to study lattice formation of HTLV-1 and HIV-1 Gag-like constructs. I obtained structural data for the higher-order assembly of the immature HTLV-1 Gag lattice, revealing a hexameric arrangement. I observed a new, intermediate structure of the immature HIV-1 Gag lattice and showed that IP6 can act as a molecular switch to change the structure and curvature of the lattice. I further solved an 8 Å structure of the immature HIV-1 Gag lattice, using single particle analysis, revealing density for the MA lattice. Finally, we showed that MA mutants that are defective in Envelope incorporation disrupt proper MA lattice formation. These findings significantly enhance our understanding of retroviral Gag targeting and assembly.
Recommended Citation
Herrmann, Dominik, "Molecular Determinants of Retroviral Gag Targeting to the Plasma Membrane for Assembly" (2023). All ETDs from UAB. 8.
https://digitalcommons.library.uab.edu/etd-collection/8
