Contribution of E3-ubiquitin ligase activity to HIV-1 restriction by TRIM5 rh: structure of the RING domain of TRIM5

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Journal of Virology (Impact Factor: 4.44). 09/2011; 85(17):8725-37. DOI: 10.1128/JVI.00497-11
Source: PubMed


TRIM5α(rh) is a cytosolic protein that potently restricts HIV-1 before reverse transcription. TRIM5α(rh) is composed of four different domains: RING, B-box 2, coiled coil, and B30.2(SPRY). The contribution of each of these domains to restriction has been extensively studied, with the exception of the RING domain. The RING domain of TRIM5α exhibits E3-ubiquitin ligase activity, but the contribution of this activity to the restriction of HIV-1 is not known. To test the hypothesis that the E3-ubiquitin ligase activity of the RING domain modulates TRIM5α(rh) restriction of HIV-1, we correlated the E3-ubiquitin ligase activity of a panel of TRIM5α(rh) RING domain variants with the ability of these mutant proteins to restrict HIV-1. For this purpose, we first solved the nuclear magnetic resonance structure of the RING domain of TRIM5α and defined potential functional regions of the RING domain by homology to other RING domains. With this structural information, we performed a systematic mutagenesis of the RING domain regions and tested the TRIM5α RING domain variants for the ability to undergo self-ubiquitylation. Several residues, particularly the ones on the E2-binding region of the RING domain, were defective in their self-ubiquitylation ability. To correlate HIV-1 restriction to self-ubiquitylation, we used RING domain mutant proteins that were defective in self-ubiquitylation but preserve important properties required for potent restriction by TRIM5α(rh), such as capsid binding and higher-order self-association. From these investigations, we found a set of residues that when mutated results in TRIM5α molecules that lost both the ability to potently restrict HIV-1 and their self-ubiquitylation activity. Remarkably, all of these changes were in residues located in the E2-binding region of the RING domain. Overall, these results demonstrate a role for TRIM5α self-ubiquitylation in the ability of TRIM5α to restrict HIV-1.

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Available from: Francesca Di Nunzio, Oct 01, 2015
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    • "Because the use of HIV-1 viruses bearing capsid changes suggested that the HIV-1 capsid is the determinant for MxB restriction, we examined the ability of MxB to associate with the HIV-1 core. For this purpose, we tested the ability of MxB to bind in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes, as described [24-26]. In vitro assembled HIV-1 CA-NC complexes recapitulate the surface of the HIV-1 core, and are an established model to evaluate the ability of cellular factors to interact with the HIV-1 core [25,27-29]. "
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    ABSTRACT: Background The IFN-¿-inducible restriction factor MxB blocks HIV-1 infection after reverse transcription but prior to integration. Genetic evidence suggested that capsid is the viral determinant for restriction by MxB. This work explores the ability of MxB to bind to the HIV-1 core, and the role of capsid-binding in restriction.ResultsWe showed that MxB binds to the HIV-1 core and that this interaction leads to inhibition of the uncoating process of HIV-1. These results identify MxB as an endogenously expressed protein with the ability to inhibit HIV-1 uncoating. In addition, we found that a benzimidazole-based compound known to have a binding pocket on the surface of the HIV-1 capsid prevents the binding of MxB to capsid. The use of this small-molecule identified the MxB binding region on the surface of the HIV-1 core. Domain mapping experiments revealed the following requirements for restriction: 1) MxB binding to the HIV-1 capsid, which requires the 20 N-terminal amino acids, and 2) oligomerization of MxB, which is mediated by the C-terminal domain provides the avidity for the interaction of MxB with the HIV-1 core.Conclusions Overall our work establishes that MxB binds to the HIV-1 core and inhibits the uncoating process of HIV-1. Moreover, we demonstrated that HIV-1 restriction by MxB requires capsid binding and oligomerization.
    Retrovirology 08/2014; 11(1):68. DOI:10.1186/s12977-014-0068-x · 4.19 Impact Factor
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    • "Cellular proteins were extracted with radioimmunoprecipita - tion assay ( RIPA ) buffer as previously described ( Lienlaf et al . , 2011 ) ."
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    ABSTRACT: SAMHD1 is a human restriction factor that prevents efficient infection of macrophages, dendritic cells and resting CD4+ T cells by HIV-1. Here we explored the antiviral activity and biochemical properties of human SAMHD1 polymorphisms. Our studies focused on human SAMHD1 polymorphisms that were previously identified as evolving under positive selection for rapid amino acid replacement during primate speciation. The different human SAMHD1 polymorphisms were tested for their ability to block HIV-1, HIV-2 and equine infectious anemia virus (EIAV). All studied SAMHD1 variants block HIV-1, HIV-2 and EIAV infection when compared to wild type. We found that these variants did not lose their ability to oligomerize or to bind RNA. Furthermore, all tested variants were susceptible to degradation by Vpx, and localized to the nuclear compartment. We tested the ability of human SAMHD1 polymorphisms to decrease the dNTP cellular levels. In agreement, none of the different SAMHD1 variants lost their ability to reduce cellular levels of dNTPs. Finally, we found that none of the tested human SAMHD1 polymorphisms affected the ability of the protein to block LINE-1 retrotransposition.
    Virology 07/2014; 460(461):34-44. DOI:10.1016/j.virol.2014.04.023 · 3.35 Impact Factor
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    • "The observed phenotype of the TRIM5␣ K10R mutant is markedly different from the behavior of mutants affecting the RING finger zinc binding motif, including C15A, H32A and C35A. Indeed, such RING domain mutations abrogate both auto-ubiquitylation (Lienlaf et al., 2011; Maegawa et al., 2010) and the generation of K63-linked ubiquitin chains (Pertel et al., 2011), and they also abrogate the activation of NF␬B and AP-1 (Pertel et al., 2011; Tareen and Emerman, 2011a; Uchil et al., 2013). In contrast, the consensus sumoylation site is not required for RING-mediated ubiquitylation but it acts as a regulator. "
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    ABSTRACT: TRIM5α is a type I interferon-stimulated anti-retroviral restriction factor expressed in most primates and homologous proteins are expressed in other mammals. Through its C-terminal PRYSPRY (B30.2) domain, TRIM5α binds to incoming and intact post-fusion retroviral cores in the cytoplasm. Following this direct interaction, the retroviral capsid core is destabilized and progression of the virus life cycle is interrupted. Specific recognition of its viral target by TRIM5α also triggers the induction of an antiviral state involving the activation of transcription factors NF-κB- and AP-1. In addition to PRYSPRY, several other TRIM5α domains are important for anti-retroviral function, including a RING zinc-binding motif. This domain has “E3” ubiquitin ligase activity and is involved in both the direct inhibition of incoming retroviruses and innate immune activation. A highly conserved sumoylation consensus site is present between the RING motif and the N-terminal extremity of TRIM5α. No clear role in restriction has been mapped to this sumoylation site, and no sumoylated forms of TRIM5α have been observed. Here we confirm that mutating the putatively sumoylated lysine (K10) of the Rhesus macaque TRIM5α (TRIM5αRh) to an arginine has only a small effect on restriction. However, we show that the mutation significantly decreases the TRIM5α-induced generation of free K63-linked ubiquitin chains, an intermediate in the activation of innate immunity pathways. Accordingly, K10R decreases TRIM5α-mediated activation of both NF-κB and AP-1. Concomitantly, we find that K10R causes a large increase in the levels of ubiquitylated TRIM5α. Finally, treatment with the nuclear export inhibitor leptomycin B shows that K10R enhances the nuclear localization of TRIM5αRh, while at the same time reducing its level of association with nuclear SUMO bodies. In conclusion, the TRIM5α sumoylation site appears to modulate the E3 ubiquitin ligase activities of the adjacent RING domain, promoting K63-linked ubiquitin chains at the expense of auto-ubiquitylation which is probably K48-linked. Consistently, we find this sumoylation site to be important for innate immune activation by TRIM5α. In addition, lysine 10 regulates TRIM5α nuclear shuttling and nuclear localization, which may also be related to its role in innate immunity activation.
    Virus Research 05/2014; 184. DOI:10.1016/j.virusres.2014.02.013 · 2.32 Impact Factor
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