Date of Award
Doctor of Philosophy (PhD)
Microbiology and Immunology
TRIM5Î± is an anti-viral restriction factor that inhibits the lifecycle of retroviruses. TRIM5Î± binds to and forms a hexameric lattice around the retroviral capsid, thereby initiating its antiviral activities, which include: (1) inhibition of viral infection; (2) inhibition of viral reverse transcription; (3) disassembly of the capsid; and (4), activation of innate signaling pathways. The formation of this assembly also activates the E3 ubiquitin ligase function of TRIM5Î±. Ubiquitin modification is associated with directing substrates to particular cellular pathways. We and others have shown that TRIM5Î± cytoplasmic bodies colocalize with proteins involved in the autophagy pathway, and we hypothesized that autophagy may play a critical role in the function of TRIM5Î± as a retroviral restriction factor. The goal of this dissertation is to define the molecular interactions required for the association of TRIM5 proteins with autophagy effectors and to delineate the roles of ubiquitination and autophagy in retroviral restriction by TRIM5Î±. We demonstrated that, when the autophagy factors ATG5 or Beclin1 are depleted in human cell lines, the restriction of N-MLV by human TRIM5Î±, and HIV-1 by Rhesus macaque TRIM5Î± and owl monkey TRIM-Cyp is preserved. These data indicate that autophagy machinery is not required for retroviral restriction by TRIM5 proteins. However, given TRIM5Î±'s activity as a ubiquitin ligase, we wanted to further probe the ubiquitin-dependent steps during retroviral restriction. We generated fusion proteins in which the catalytic domain of different deubiquitinase (DUb) enzymes, with different specificities for polyubiquitated linkages, was fused to the N-terminal RING domain of Rhesus macaque TRIM5Î±. We assessed the role of ubiquitination in restriction and the degree to which specific types of ubiquitination are required for the association of TRIM5Î± with autophagic proteins. We determined that K63-linked ubiquitination by TRIM5Î± is critical for its association with autophagosome membranes. In the absence of K63-specific ubiquitin ligase activity, TRIM5Î± forms a stable association with the capsid, allowing reverse transcription to proceed, however infection is still blocked. These data favor a model whereby the formation of a TRIM5Î± assembly around a capsid is sufficient to inhibit infection.
Imam, Sabrina, "Molecular Determinants of Trim5α Restriction and Recruitment of Autophagic Effectors" (2018). Dissertations. 2815.
Copyright © 2018 Sabrina Imam