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RanBP2 regulates the anti-retroviral activity of TRIM5? by SUMOylation at a predicted phosphorylated SUMOylation motif.

ABSTRACT: TRIM5? is a cytoplasmic restriction factor that blocks post-entry retroviral infection. Evidence suggests that its antiviral activity can be regulated by SUMO, but how this is achieved remains unknown. Here, we show that TRIM5? forms a complex with RanGAP1, Ubc9, and RanBP2 at the nuclear pore, and that RanBP2 E3 SUMO ligase promotes the SUMOylation of endogenous TRIM5? in the cytoplasm. Loss of RanBP2 blocked SUMOylation of TRIM5?, altered its localization in primary cells, and suppressed the antiviral activity of both rhesus and human orthologs. In cells, human TRIM5? is modified on K84 within a predicted phosphorylated SUMOylation motif (pSUM) and not on K10 as found in vitro. Non-modified TRIM5? lacked antiviral activity, indicating that only SUMOylated TRIM5? acts as a restriction factor. This work illustrates the importance of the nuclear pore in intrinsic antiviral immunity, acting as a hub where virus, SUMO machinery, and restriction factors can meet.

SUBMITTER: Maarifi G

PROVIDER: S-EPMC6237768 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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RanBP2 regulates the anti-retroviral activity of TRIM5α by SUMOylation at a predicted phosphorylated SUMOylation motif.

Maarifi Ghizlane G Fernandez Juliette J Portilho Débora M DM Boulay Aude A Dutrieux Jacques J Oddos Stéphane S Butler-Browne Gillian G Nisole Sébastien S Arhel Nathalie J NJ

Communications biology 20181115

TRIM5α is a cytoplasmic restriction factor that blocks post-entry retroviral infection. Evidence suggests that its antiviral activity can be regulated by SUMO, but how this is achieved remains unknown. Here, we show that TRIM5α forms a complex with RanGAP1, Ubc9, and RanBP2 at the nuclear pore, and that RanBP2 E3 SUMO ligase promotes the SUMOylation of endogenous TRIM5α in the cytoplasm. Loss of RanBP2 blocked SUMOylation of TRIM5α, altered its localization in primary cells, and suppressed the a ...[more]

PMID: 30456314

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Project description:Host restriction factor TRIM5 inhibits retroviral transduction in a species-specific manner by binding to and destabilizing the retroviral capsid lattice before reverse transcription is completed. However, the restriction mechanism may not be that simple since TRIM5 E3 ubiquitin ligase activity, the proteasome, autophagy, and TAK1-dependent AP-1 signaling have been suggested to contribute to restriction. Here, we show that, among a panel of seven primate and Carnivora TRIM5 orthologues, each of which has potential for potent retroviral restriction activity, all activated AP-1 signaling. In contrast, TRIM family paralogues most closely related to TRIM5 did not. While each primate species has a single TRIM5 gene, mice have at least seven TRIM5 homologues that cluster into two groups, Trim12a, -b, and -c and Trim30a, -b, -c, and -d. The three Trim12 proteins activated innate immune signaling, while the Trim30 proteins did not, though none of the murine Trim5 homologues restricted any of a panel of cloned retroviruses. To determine if any mouse TRIM5 homologues had potential for restriction activity, each was fused to the human immunodeficiency virus type 1 (HIV-1) CA binding protein cyclophilin A (CypA). The three Trim12-CypA fusions all activated AP-1 and restricted HIV-1 transduction, whereas the Trim30-CypA fusions did neither. AP-1 activation and HIV-1 restriction by the Trim12-CypA fusions were inhibited by disruption of TAK1. Overall then, these experiments demonstrate that there is a strong correlation between TRIM5 retroviral restriction activity and the ability to activate TAK1-dependent innate immune signaling.The importance of retroviruses for the evolution of susceptible host organisms cannot be overestimated. Eight percent of the human genome is retrovirus sequence, fixed in the germ line during past infection. Understanding how metazoa protect their genomes from mutagenic retrovirus infection is therefore of fundamental importance to biology. TRIM5 is a cellular protein that protects host genome integrity by disrupting the retroviral capsid as it transports viral nucleic acid to the host cell nucleus. Previous data suggest that innate immune signaling contributes to TRIM5-mediated restriction. Here, we show that activation of innate immune signaling is conserved among primate and carnivore TRIM5 orthologues and among 3 of the 7 mouse Trim5 homologues and that such activity is required for TRIM5-mediated restriction activity.

Project description:Restriction factors are intrinsic cellular defense proteins that have evolved to block microbial infections. Retroviruses such as HIV-1 are restricted by TRIM5 proteins, which recognize the viral capsid shell that surrounds, organizes, and protects the viral genome. TRIM5α uses a SPRY domain to bind capsids with low intrinsic affinity (KD of >1 mM) and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition. TRIMCyp, on the other hand, binds HIV-1 capsids through a cyclophilin A domain, which has a well-defined binding site and higher affinity (KD of ∼10 μM) for isolated capsid subunits. Therefore, it has been argued that TRIMCyp proteins have dispensed with the need for higher-order assembly to function as antiviral factors. Here, we show that, consistent with its high degree of sequence similarity with TRIM5α, the TRIMCyp B-box 2 domain shares the same ability to self-associate and facilitate assembly of a TRIMCyp hexagonal lattice that can wrap about the HIV-1 capsid. We also show that under stringent experimental conditions, TRIMCyp-mediated restriction of HIV-1 is indeed dependent on higher-order assembly. Both forms of TRIM5 therefore use the same mechanism of avidity-driven capsid pattern recognition.IMPORTANCE Rhesus macaques and owl monkeys are highly resistant to HIV-1 infection due to the activity of TRIM5 restriction factors. The rhesus macaque TRIM5α protein blocks HIV-1 through a mechanism that requires self-assembly of a hexagonal TRIM5α lattice around the invading viral core. Lattice assembly amplifies very weak interactions between the TRIM5α SPRY domain and the HIV-1 capsid. Assembly also promotes dimerization of the TRIM5α RING E3 ligase domain, resulting in synthesis of polyubiquitin chains that mediate downstream steps of restriction. In contrast to rhesus TRIM5α, the owl monkey TRIM5 homolog, TRIMCyp, binds isolated HIV-1 CA subunits much more tightly through its cyclophilin A domain and therefore was thought to act independently of higher-order assembly. Here, we show that TRIMCyp shares the assembly properties of TRIM5α and that both forms of TRIM5 use the same mechanism of hexagonal lattice formation to promote viral recognition and restriction.

Project description:TRIM5 proteins can restrict retroviral infection soon after delivery of the viral core into the cytoplasm. However, the molecular mechanisms by which TRIM5? inhibits infection have been elusive, in part due to the difficulty of developing and executing biochemical assays that examine this stage of the retroviral life cycle. Prevailing models suggest that TRIM5? causes premature disassembly of retroviral capsids and/or degradation of capsids by proteasomes, but whether one of these events leads to the other is unclear. Furthermore, how TRIM5? affects the essential components of the viral core, other than capsid, is unknown. To address these questions, we devised a biochemical assay in which the fate of multiple components of retroviral cores during infection can be determined. We utilized cells that can be efficiently infected by VSV-G-pseudotyped retroviruses, and fractionated the cytosolic proteins on linear gradients following synchronized infection. The fates of capsid and integrase proteins, as well as viral genomic RNA and reverse transcription products were then monitored. We found that components of MLV and HIV-1 cores formed a large complex under non-restrictive conditions. In contrast, when MLV infection was restricted by human TRIM5?, the integrase protein and reverse transcription products were lost from infected cells, while capsid and viral RNA were both solubilized. Similarly, when HIV-1 infection was restricted by rhesus TRIM5? or owl monkey TRIMCyp, the integrase protein and reverse transcription products were lost. However, viral RNA was also lost, and high levels of preexisting soluble CA prevented the determination of whether CA was solubilized. Notably, proteasome inhibition blocked all of the aforementioned biochemical consequences of TRIM5?-mediated restriction but had no effect on its antiviral potency. Together, our results show how TRIM5? affects various retroviral core components and indicate that proteasomes are required for TRIM5?-induced core disruption but not for TRIM5?-induced restriction.

Dataset Information

RanBP2 regulates the anti-retroviral activity of TRIM5? by SUMOylation at a predicted phosphorylated SUMOylation motif.

Publications

RanBP2 regulates the anti-retroviral activity of TRIM5α by SUMOylation at a predicted phosphorylated SUMOylation motif.

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