Biochemical studies of retroviral replication and TRIM5α restriction

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Publication Type dissertation
School or College School of Medicine
Department Biochemistry
Author Langelier, Charles R.
Title Biochemical studies of retroviral replication and TRIM5α restriction
Date 2010
Description During early stages of infection, HIV-1 and other lentiviruses progress through a series of cytoplasmic events that include capsid uncoating, reverse transcription and formation of the preintegration complex (PIC). In certain primates, the intrinsic immune factor TRIM5α can bind to lentiviral capsids and disrupt this program. Neither TRIM5α restriction nor early retroviral replication is understood in mechanistic detail and both were therefore the targets of investigation for research described in this dissertation. Chapter 2 describes a system for producing multimilligram quantities of an active TRIM5α variant, termed TRIM5-21R. TRIM5-21R is a chimeric protein composed primarily of the TRIM5α from rhesus (rh) monkeys, except for the N-terminal RING domain, which comes from the human TRIM21 protein. Recombinant TRIM5-21R was functional, as judged by its ability to autoubiquitylate in reconstituted ubiquitin transfer reactions and to bind specifically to HIV and EIAV capsid assemblies. Gel filtration and analytical ultracentrifugation experiments demonstrated that TRIM5-21R formed stable dimers, and chemical crosslinking experiments showed that TRIM5α proteins expressed in mammalian cells are also predominantly dimeric. We anticipate that this system for producing pure recombinant TRIM5-21R will facilitate future studies of the biochemistry, mechanism, and structural biology of this important class of antiretroviral proteins. Research described in the remaining chapters of this thesis characterized the requirement for the ESCRT-II complex in HIV budding and endosomal protein sorting (Chapter 4), structural and biochemical properties of the ubiquitin-binding EAP45 GLUE domain (Chapter 5), the molecular mechanism of (ESCRT-II involvement in) Hepcidin-mediated Ferroportin down-regulation (Chapter 6) and self-assembly of pure recombinant ESCRT-III proteins into helical tubes (Chapter 7).
Type Text
Publisher University of Utah
Subject Molecular biology; Biochemistry; Virology
Dissertation Name Doctor of Philosophy
Language eng
Rights Management (c) Charles R. Langelier
Format Medium application/pdf
ARK ark:/87278/s61z8j04
Setname ir_etd
Date Created 2019-03-18
Date Modified 2021-05-06
ID 1404420
Reference URL
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