title: TRIM5α-Mediated Ubiquitin Chain Conjugation Is Required for Inhibition of HIV-1 Reverse Transcription and Capsid Destabilization.
abstract: Rhesus macaque TRIM5α (rhTRIM5α) is a retroviral restriction factor that inhibits HIV-1 infection. Previous studies have revealed that TRIM5α 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 rhTRIM5α, 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-TRIM5α fusion relative to the catalytically inactive control or the wild-type (WT) TRIM5α. Similarly, expression of DUb-rhTRIM5α resulted in the accumulation of viral cores in target cells following infection, while the catalytically inactive control and WT rhTRIM5α induced the abortive disassembly of viral cores, indicating a role for ubiquitin conjugation in rhTRIM5α-mediated destabilization of HIV-1 cores. Finally, DUb-rhTRIM5α failed to activate NF-κB signaling pathways compared to controls, demonstrating that this ubiquitination-dependent activity is separable from the ability to restrict retroviral infection.
  
  
  OBJECTIVE
  These studies provide direct evidence that ubiquitin conjugation to rhTRIM5α-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.
authors.affiliation: Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA ecampbell@luc.edu bouamrf@mail.nih.gov.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Campbell
authors.affiliation: Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA.
authors.forename: Jared
authors.initials: J
authors.lastname: Weingart
authors.affiliation: Laboratory of Molecular Microbiology, National Institutes of Allergy and Infectious Disease, Bethesda, Maryland, USA.
authors.forename: Paola
authors.initials: P
authors.lastname: Sette
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Silvana
authors.initials: S
authors.lastname: Opp
authors.affiliation: Laboratory of Molecular Microbiology, National Institutes of Allergy and Infectious Disease, Bethesda, Maryland, USA.
authors.forename: Jaya
authors.initials: J
authors.lastname: Sastri
authors.affiliation: Laboratory of Molecular Microbiology, National Institutes of Allergy and Infectious Disease, Bethesda, Maryland, USA.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: O'Connor
authors.affiliation: Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Talley
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Felipe
authors.initials: F
authors.lastname: Diaz-Griffero
authors.affiliation: Laboratory of Molecular Microbiology, National Institutes of Allergy and Infectious Disease, Bethesda, Maryland, USA.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Hirsch
authors.affiliation: Laboratory of Molecular Microbiology, National Institutes of Allergy and Infectious Disease, Bethesda, Maryland, USA ecampbell@luc.edu bouamrf@mail.nih.gov.
authors.forename: Fadila
authors.initials: F
authors.lastname: Bouamr
mesh_terms: D000818:Animals; D002213:Capsid / Q000378:metabolism; D002460:Cell Line; D015497:HIV-1 / Q000276:immunology* / Q000502:physiology*; D006801:Humans; D008253:Macaca mulatta; D011506:Proteins / Q000378:metabolism*; D048348:Reverse Transcription; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D019065:Virus Assembly
chemical_list: D011506:Proteins; D025801:Ubiquitin; C482587:TRIM5(alpha) protein, rhesus monkey; D044767:Ubiquitin-Protein Ligases