| 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 TRIM5alpha can bind to lentiviral capsids and disrupt this program. Neither TRIM5alpha restriction nor early retroviral replication is understood in mechanistic detail and both were therefore the targets of investigation for research described in this dissertation.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).Chapter 2 describes a system for producing multimilligram quantities of an active TRIM5alpha variant, termed TRIM5-21R. TRIM5-21R is a chimeric protein composed primarily of the TRIM5alpha 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 TRIM5alpha 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. |
