ABSTRACT
Members of the tripartite motif (TRIM) protein family of RING E3 ubiquitin (Ub) ligases promote innate immune responses by catalyzing synthesis of polyubiquitin chains linked through lysine 63 (K63). Here, we investigate the mechanism by which the TRIM5α retroviral restriction factor activates Ubc13, the K63-linkage-specific E2. Structural, biochemical, and functional characterization of the TRIM5α:Ubc13-Ub interactions reveals that activation of the Ubc13-Ub conjugate requires dimerization of the TRIM5α RING domain. Our data explain how higher-order oligomerization of TRIM5α, which is promoted by the interaction with the retroviral capsid, enhances the E3 Ub ligase activity of TRIM5α and contributes to its antiretroviral function. This E3 mechanism, in which RING dimerization is transient and depends on the interaction of the TRIM protein with the ligand, is likely to be conserved in many members of the TRIM family and may have evolved to facilitate recognition of repetitive epitope patterns associated with infection.
Subject(s)
Carrier Proteins/metabolism , Polyubiquitin/biosynthesis , Protein Multimerization/physiology , Ubiquitin-Conjugating Enzymes/metabolism , Animals , Antiviral Restriction Factors , Carrier Proteins/genetics , Cells, Cultured , Dogs , Polyubiquitin/genetics , Retroviridae/genetics , Retroviridae/metabolism , Tripartite Motif Proteins , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Protein Ligases , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Recent findings suggested that the SUMO-interacting motifs (SIMs) present in the human TRIM5α (TRIM5α(hu)) protein play an important role in the ability of TRIM5α(hu) to restrict N-MLV. Here we explored the role of SIMs in the ability of rhesus TRIM5α (TRIM5α(rh)) to restrict HIV-1, and found that TRIM5α(rh) SIM mutants IL376KK (SIM1mut) and VI405KK (SIM2mut) completely lost their ability to block HIV-1 infection. Interestingly, these mutants also lost the recently described property of TRIM5α(rh) to shuttle into the nucleus. Analysis of these variants revealed that they are unable to interact with the HIV-1 core, which might explain the reason that these variants are not active against HIV-1. Furthermore, NMR titration experiments to assay the binding between the PRYSPRY domain of TRIM5α(rh) and the small ubiquitin-like modifier 1(SUMO-1) revealed no interaction. In addition, we examined the role of SUMOylation in restriction, and find out that inhibition of SUMOylation by the adenoviral protein Gam1 did not alter the retroviral restriction ability of TRIM5α. Overall, our results do not support a role for SIMs or SUMOylation in the antiviral properties of TRIM5α.
Subject(s)
Antiviral Agents/metabolism , Antiviral Agents/pharmacology , HIV-1/drug effects , Proteins/metabolism , Proteins/pharmacology , SUMO-1 Protein/metabolism , Amino Acid Motifs , Animals , Antiviral Agents/chemistry , Cell Line , HIV-1/genetics , HIV-1/metabolism , HIV-1/physiology , Humans , Mutation , Proteins/chemistry , Proteins/genetics , SUMO-1 Protein/chemistry , SUMO-1 Protein/genetics , Sumoylation , Transfection , Ubiquitin-Protein LigasesABSTRACT
Rhesus TRIM5α (TRIM5α(rh)) is a cytosolic protein that potently restricts HIV-1 at an early postentry stage, prior to reverse transcription. The ability of TRIM5α(rh) to block HIV-1 infection has been correlated with a decrease of pelletable HIV-1 capsid during infection. To genetically dissect the ability of TRIM5α to block reverse transcription, we studied a set of TRIM5α(rh) RING domain mutants that potently restrict HIV-1 but allow the occurrence of reverse transcription. These TRIM5α(rh) RING variants blocked HIV-1 infection after reverse transcription but prior to integration, as suggested by the routing of nuclear viral DNA to circularization in the form of 2-long terminal repeat (2-LTR) circles. The folding of RING domain variants was similar to that of the wild type, as evaluated by nuclear magnetic resonance. RING domain changes that allowed the occurrence of reverse transcription were impaired in their ability to decrease the amount of pelletable capsid compared with wild-type TRIM5α. Similar effects of this particular group of mutations were observed with human TRIM5α inhibition of N-tropic murine leukemia virus (N-MLV). Interestingly, TRIM5α(rh) RING domain variants also prevented the degradation of TRIM5α(rh) that occurs following cell entry of HIV-1. These data correlated the block of reverse transcription with the ability of TRIM5α to accelerate uncoating. Collectively, these results suggest that TRIM5α(rh) blocks HIV-1 reverse transcription by inducing premature viral uncoating in target cells.
Subject(s)
Carrier Proteins/metabolism , HIV-1/physiology , Mutation , Proteins/metabolism , Transcription, Genetic , Animals , Antiviral Restriction Factors , Base Sequence , Carrier Proteins/genetics , Cell Line , DNA Probes , Dogs , HIV Long Terminal Repeat , Humans , Nuclear Magnetic Resonance, Biomolecular , Proteins/chemistry , Proteins/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcription , Thymocytes/virology , Tripartite Motif Proteins , Ubiquitin-Protein Ligases , Virus UncoatingABSTRACT
Genetic manipulation of the filamentous fungus Penicillium camemberti has been limited by a lack of suitable genetics tools for this fungus. In particular, there is no available homologous transformation system. In this study, the nitrate reductase (niaD) and orotidine-5'-monophosphate decarboxylase (pyrG) genes from Penicillium camemberti were characterized, and their suitability as metabolic molecular markers for transformation was evaluated. The genes were amplified using PCR-related techniques, and sequenced. The niaD gene is flanked by the nitrite reductase (niiA) gene in a divergent arrangement, being part of the putative nitrate assimilation cluster in P. camemberti. pyrG presents several polymorphisms compared with a previously sequenced pyrG gene from another P. camemberti strain, but almost all are silent mutations. Southern blot assays indicate that one copy of each gene is present in P. camemberti. Northern blot assays showed that the pyrG gene is expressed in minimal and rich media, and the niaD gene is expressed in nitrate, but not in reduced nitrogen sources. The functionality of the two genes as transformation markers was established by transforming A. nidulans pyrG- and niaD-deficient strains. Higher transformation efficiencies were obtained with a pyrG-containing plasmid. This is the first study yielding a molecular and functional characterization of P. camemberti genes that would be useful as molecular markers for transformation, opening the way for the future development of a non-antibiotic genetic transformation system for this fungus.