Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

TRIM5α is an anti-viral restriction factor that inhibits the lifecycle of retroviruses. TRIM5α 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 TRIM5α. Ubiquitin modification is associated with directing substrates to particular cellular pathways. We and others have shown that TRIM5α cytoplasmic bodies colocalize with proteins involved in the autophagy pathway, and we hypothesized that autophagy may play a critical role in the function of TRIM5α 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 TRIM5α. We demonstrated that, when the autophagy factors ATG5 or Beclin1 are depleted in human cell lines, the restriction of N-MLV by human TRIM5α, and HIV-1 by Rhesus macaque TRIM5α and owl monkey TRIM-Cyp is preserved. These data indicate that autophagy machinery is not required for retroviral restriction by TRIM5 proteins. However, given TRIM5α'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 TRIM5α. We assessed the role of ubiquitination in restriction and the degree to which specific types of ubiquitination are required for the association of TRIM5α with autophagic proteins. We determined that K63-linked ubiquitination by TRIM5α is critical for its association with autophagosome membranes. In the absence of K63-specific ubiquitin ligase activity, TRIM5α 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 TRIM5α assembly around a capsid is sufficient to inhibit infection.

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