COMMENTARY COMMENTARY Delicate coordination of TRIM21’s dual activity in virus neutralization and signaling Florian Full and Michaela U. Gack1 and whether there is a molecular “switch” Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA that coordinates both activities. 02115 Ubiquitination is a posttranslational pro- tein modification that is involved in a number of cellular processes in eukaryotes. TRIM21 belongs to the tripartite motif demonstrated that several TRIM proteins, in- Ubiquitin is a small protein of 76 amino (TRIM) family of ubiquitin E3 ligases. Ap- cluding TRIM5α and TRIM19, play dual roles acids that is usually attached to lysine proximately 100 TRIM proteins exist in in antiviral immunity by acting both as “ef- residues of proteins by a cascade of en- humans, and they all share a common fectors” that neutralize viral infection, and as zymes. Ubiquitin molecules are first acti- domain structure composed of a RING “ ” sensors that induce innate immune signal- vated by E1 enzymes, then conjugated by domain that confers the E3 ligase enzymatic ing. It has also been shown that the ability of E2 enzymes, and are finally ligated to the activity, one or two B-boxes, and a coiled-coil TRIM proteins to catalyze different types of substrate by E3 ligases (6). Ubiquitination domain (1). Many TRIM proteins play crucial ubiquitination is important for their effector may result in monoubiquitinated or poly- roles in antiviral immune responses, often by and sensor functions. However, the molecular ubiquitinated proteins. For polyubiquitina- targeting specific viral components. For exam- details of how the dual activity of TRIM pro- tion, either the N-terminal methionine or α ple, TRIM5 inhibits the infection of many teins is temporally and mechanistically coor- one of the seven internal lysine residues of retroviruses, including HIV-1, by binding to dinated are not well understood. In PNAS, ubiquitin (K6, K11, K27, K29, K33, K48, incoming viral capsids, leading to capsid dis- Fletcher et al. uncover a mechanism of step- and K63) can be modified in consecutive sociation and inhibition of reverse transcrip- wise ubiquitination and deubiquitination in conjugation cycles, leading to a chain of tion of the viral genome. TRIM19, also known synchronizing the sensor and effector func- ubiquitins. Polyubiquitin chains can be ei- as promyelocytic leukemia protein, is a key tions of TRIM21, providing molecular-level ther attached covalently to the substrate component of subnuclear structures that re- insights into the antiviral activity of TRIM protein or bound noncovalently as unan- strict a variety of RNA and DNA viruses. proteins (3). chored “free” polyubiquitin chains. Whereas Furthermore, TRIM proteins modulate signal TRIM21 is a cytosolic receptor for anti- the E3 ligase determines the substrate speci- transduction pathways that lead to antiviral body-opsonized virus particles that mediates ficity, it is the E2 enzyme that determines the cytokine production, ultimately contributing proteasomal degradation of the virus (4). In type of polyubiquitin linkage that is made. to the establishment of an antiviral state in addition, upon detection of virus, TRIM21 Given the large number of ubiquitinated pro- both the infected and uninfected cell (1). initiates a signaling cascade that results in teins in the cell, it is not surprising that more For example, TRIM25 activates the innate im- NF-κB activation and subsequent production than 600 E3 ligases exist in humans that, ∼ mune sensor RIG-I, potentiating the produc- of proinflammatory cytokines (5). It remains together with 40 different E2 enzymes (and tion of antiviral cytokines, such as type-I unclear whether TRIM21’ssensorandeffec- two E1 enzymes), are responsible for the hu- “ ” interferons (IFN-α/β) (2). Recent studies tor functions are mechanistically connected man ubiquitinome. To reverse the concerted action of E1, E2, and E3 enzymes, the human genome also encodes ∼100 deubiquitinating enzymes (DUB), which remove mono- or pol- yubiquitin from substrate proteins. Over the past several years, significant pro- gress has been made on the roles of different polyubiquitin linkage types in determining thefateofthemodifiedprotein.K48-linked polyubiquitin chains are well known to be recognized by the proteasome, triggering the degradation of the modified substrate. In contrast, K63-linked ubiquitin chains—both the covalently attached and unanchored forms—are generally believed to not trigger the proteasomal degradation of the substrate

Fig. 1. Regulation of TRIM21’s sensor and effector functions through stepwise ubiquitination and deubiquitination. Author contributions: F.F. and M.U.G. wrote the paper. Binding of TRIM21 to virus-immunoglobulin (Ig) complexes results in TRIM21 auto-monoubiquitination mediated by The authors declare no conflict of interest. the E2 enzyme Ube2W. Monoubiquitination of TRIM21 serves as a substrate for K63-linked polyubiquitination me- diated by Ube2N and Ube2V2. Translocation of TRIM21 to the proteasome leads to the degradation of virus–antibody See companion article on page 10014. complexes, as well as release of the K63-linked polyubiquitin chain from TRIM21 by the proteasome-associated 1To whom correspondence should be addressed. Email: Michaela_Gack@ enzyme Poh1. The released unanchored K63-linked polyubiquitin chains then trigger NF-κB activation. hms.harvard.edu.

www.pnas.org/cgi/doi/10.1073/pnas.1512642112 PNAS | August 11, 2015 | vol. 112 | no. 32 | 9797–9798 Downloaded by guest on September 28, 2021 protein, but rather have important roles ing unanchored K63-linked ubiquitin chains; which subsequently induces proinflammatory in activating signal transduction pathways however, unexpectedly, these unanchored signal-transduction (Fig. 1). (6). Mechanistically, K63-linked polyubiquitin K63-linked chains did not trigger NF-κB The work of Fletcher et al. (3) indic- can activate signaling pathways by either activation in the cell. This finding suggested ates that monoubiquitination of TRIM21 is a stabilizing the substrate protein or inducing that regulation of TRIM21-mediated proin- prerequisite for subsequent K63-polyubiqui- its multimeric active form, or by facilitating flammatory signal activation is governed by tination, and that two different E2 enzymes— the recruitment of other signaling proteins. an additional mechanism. Ube2W and Ube2N/Ube2V2—are needed In particular, TRIM proteins have been im- How does TRIM21 induce innate immune for the stepwise activation of TRIM21. This plicated in the regulation of immune- signaling? Fletcher et al. (3) hypothesized that unveils a complex regulatory mechanism for defense pathways by modifying key signal- a DUB enzyme associated with the proteasome TRIM21 activation, which could be critical ing proteins with K63-linked polyubiquitin. for controlling aberrant signaling to prevent Upon viral infection, TRIM25 mediates the The work of Fletcher unwanted inflammatory responses in the K63-linked ubiquitination of the intracellular et al. proposes an absence of infection. Although their study viral RNA sensor RIG-I, thereby inducing has considerably advanced our understand- RIG-I tetramerization, which is necessary for intriguing model of ing of TRIM21 activity, the findings of RIG-I translocation to mitochondria where TRIM21 activation Fletcher et al. also raise several important signaling is perpetuated (2, 7). In the case through stepwise questions. It remains unclear how the protea- of TRIM21, in vitro experiments indicated someisrecruitedtoTRIM21,andinpar- that TRIM21, together with the E2 enzymes autoubiquitination ticular how K48-linked ubiquitin chains, Ube2N/Ube2V1, can induce the forma- and Poh1-mediated which are also conjugated to TRIM21 (4, tion of unanchored K63-polyubiquitin chains deubiquitination. 10), contribute to TRIM21-mediated degra- (5), which are known to play a role in TAK1- dation of antibody–pathogen complexes. dependent NF-κB activation (8). The mecha- cleaves off anchored K63-polyubiquitin Furthermore, Fletcher et al. (3) do not ad- nistic details, however, of how TRIM21 in- from TRIM21, releasing it to generate un- dress which downstream targets are activated duces innate signaling in a virus-infected cell anchored K63-polyubiquitin, which then in- by K63-polyubiquitin chains released by have not been well characterized. In particu- duces NF-κB activation. The authors focused Poh1 and their contributions to proinflam- lar, it remains unclear how the dual activity of on Poh1, a proteasome-associated DUB that matory cytokine induction. TRIM21 in virus neutralization and proin- is known to remove ubiquitin chains en bloc, In conclusion, the work of Fletcher et al. flammatory signal initiation is coordinated. thereby generating unanchored ubiquitin (3) proposes an intriguing model of TRIM21 Fletcher et al. (3) first tested whether chains (9). Depletion of endogenous Poh1 activation through stepwise autoubiquiti- NF-κB activation by TRIM21 is a prerequi- increased the levels of K63-polyubiquitin co- nation and Poh1-mediated deubiquitina- site for its ability to neutralize virus particles. valently attached to TRIM21, indicating that tion. Interestingly, a similar activation Using various inhibitors of the proteasome Poh1 indeed removes this modification from mechanism has been recently reported for and of NF-κB activation, the authors show TRIM21. Silencing of Poh1 also resulted in TRIM5α (11). It has been shown that that the two functions of TRIM21 are indeed a dramatic reduction in NF-κB activation, TRIM5α monoubiquitination mediated by tightly linked. Furthermore, upon depletion suggesting that the Poh1-mediated release Ube2W and subsequent K63-polyubiquiti- of Ube2N, the virus could no longer be neu- of K63-linked polyubiquitin induces activa- nation by Ube2N/Ube2V2 is required for tralized by TRIM21, indicating that the syn- tion of NF-κB signaling. Fletcher et al. (3) TRIM5α’s ability to inhibit HIV reverse thesis of K63-linked polyubiquitin precedes further show that production of proin- transcription. A comprehensive view of viral neutralization. In a subsequent series of flammatory cytokines triggered by virus–Ig how ubiquitination/deubiquitination con- experiments the authors depleted Ube2W, complexes is dependent on Poh1. The au- trols the antiviral and immunomodulatory an E2 enzyme that has been reported to thors thus propose a model in which co- functions of TRIM proteins will greatly en- facilitate covalent K63-linked polyubiquiti- valently bound K63-linked polyubiquitin hance our understanding of antiviral re- nation mediated by Ube2N/Ube2V2. Deple- chains on TRIM21 are cleaved off by Poh1, striction mechanisms and may lead to tion of Ube2W abolished both the TRIM21- generating “free” K63-linked polyubiquitin, novel antiviral therapies. mediated degradation of virus–Ig complexes as well as NF-κB activation. Collectively, these results suggest that both Ube2W and Ube2N/ 1 Rajsbaum R, García-Sastre A, Versteeg GA (2014) TRIMmunity: The 6 Davis ME, Gack MU (2015) Ubiquitination in the antiviral immune Ube2V2 play roles in regulating TRIM21’s roles of the TRIM E3-ubiquitin ligase family in innate antiviral response. Virology 479-480:52–65. immunity. J Mol Biol 426(6):1265–1284. 7 Peisley A, Wu B, Xu H, Chen ZJ, Hur S (2014) Structural basis for dual antiviral activity. 2 Gack MU, et al. (2007) TRIM25 RING-finger E3 ubiquitin ligase is ubiquitin-mediated antiviral signal activation by RIG-I. Nature In a series of elegant in vitro experiments, essential for RIG-I-mediated antiviral activity. Nature 446(7138): 509(7498):110–114. Fletcher et al. (3) demonstrate that Ube2W 916–920. 8 Xia ZP, et al. (2009) Direct activation of protein kinases by 3 Fletcher AJ, Mallery DL, Watkinson RE, Dickson CF, James LC unanchored polyubiquitin chains. Nature 461(7260):114–119. first facilitates TRIM21 auto-monoubiquiti- (2015) Sequential ubiquitination and deubiquitination enzymes 9 Yao T, Cohen RE (2002) A cryptic protease couples nation, and that TRIM21 monoubiquitination synchronize the dual sensor and effector functions of TRIM21. Proc deubiquitination and degradation by the proteasome. Nature Natl Acad Sci USA 112:10014–10019. 419(6905):403–407. then serves as a substrate for the covalent 4 Mallery DL, et al. (2010) Antibodies mediate intracellular immunity 10 Bidgood SR, Tam JC, McEwan WA, Mallery DL, James LC (2014) attachment of K63-polyubiquitin chains to through tripartite motif-containing 21 (TRIM21). Proc Natl Acad Sci Translocalized IgA mediates neutralization and stimulates innate immunity TRIM21 catalyzed by Ube2N/Ube2V2. With- USA 107(46):19985–19990. inside infected cells. Proc Natl Acad Sci USA 111(37):13463–13468. 5 McEwan WA, et al. (2013) Intracellular antibody-bound pathogens 11 Fletcher AJ, et al. (2015) TRIM5alpha requires Ube2W to out monoubiquitination of TRIM21 by stimulate immune signaling via the Fc receptor TRIM21. Nat Immunol anchor Lys63-linked ubiquitin chains and restrict reverse Ube2W, TRIM21 was still capable of catalyz- 14(4):327–336. transcription. EMBO J, 10.15252/embj.201490361.

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