TRIM5alpha-Mediated Ubiquitin Chain Conjugation Is Required for Inhibition of HIV-1 Reverse Transcription and Capsid Destabilization Journal Article

Authors: Campbell, E. M.; Weingart, J.; Sette, P.; Opp, S.; Sastri, J.; O'Connor, S. K.; Talley, S; Diaz-Griffero, F; Hirsch, V.; Bouamr, F.
Article Title: TRIM5alpha-Mediated Ubiquitin Chain Conjugation Is Required for Inhibition of HIV-1 Reverse Transcription and Capsid Destabilization
Abstract: Rhesus macaque TRIM5alpha (rhTRIM5alpha) is a retroviral restriction factor that inhibits HIV-1 infection. Previous studies have revealed that TRIM5alpha restriction occurs via a two-step process. The first step is restriction factor binding, which is sufficient to inhibit infection. The second step, which is sensitive to proteasome inhibition, prevents the accumulation of reverse transcription products in the target cell. However, because of the pleotropic effects of proteasome inhibitors, the molecular mechanisms underlying the individual steps in the restriction process have remained poorly understood. In this study, we have fused the small catalytic domain of herpes simplex virus UL36 deubiquitinase (DUb) to the N-terminal RING domain of rhTRIM5alpha, which results in a ubiquitination-resistant protein. Cell lines stably expressing this fusion protein inhibited HIV-1 infection to the same degree as a control fusion to a catalytically inactive DUb. However, reverse transcription products were substantially increased in the DUb-TRIM5alpha fusion relative to the catalytically inactive control or the wild-type (WT) TRIM5alpha. Similarly, expression of DUb-rhTRIM5alpha resulted in the accumulation of viral cores in target cells following infection, while the catalytically inactive control and WT rhTRIM5alpha induced the abortive disassembly of viral cores, indicating a role for ubiquitin conjugation in rhTRIM5alpha-mediated destabilization of HIV-1 cores. Finally, DUb-rhTRIM5alpha failed to activate NF-kappaB signaling pathways compared to controls, demonstrating that this ubiquitination-dependent activity is separable from the ability to restrict retroviral infection. IMPORTANCE: These studies provide direct evidence that ubiquitin conjugation to rhTRIM5alpha-containing complexes is required for the second step of HIV-1 restriction. They also provide a novel tool by which the biological activities of TRIM family proteins might be dissected to better understand their function and underlying mechanisms of action.
Journal Title: Journal of virology
Volume: 90
Issue: 4
ISSN: 1098-5514; 0022-538X
Publisher: American Society for Microbiology. All Rights Reserved  
Journal Place: United States
Date Published: 2015
Start Page: 1849
End Page: 1857
Language: eng
Notes: LR: 20160129; CI: Copyright (c) 2016; GR: R01 AI087390/AI/NIAID NIH HHS/United States; GR: R01 AI120956/AI/NIAID NIH HHS/United States; GR: R56 AI108432/AI/NIAID NIH HHS/United States; JID: 0113724; 2015/08/03 [received]; 2015/11/12 [accepted]; 2015/12/16 [aheadofprint]; epublish