Coordinated Neutralization and Immune Activation by the Cytosolic Antibody Receptor TRIM21

Abstract

TRIM21 is a high-affinity antibody receptor uniquely expressed in the cytosol of mammalian cells. Here we summarize its role in extending antibody protection into the intracellular environment and allowing nonprofessional cells to benefit from adaptive immunity. We highlight recent work that has shed light on how TRIM21 acts as both an immune sensor and effector. We also review how TRIM21 synergizes with other innate immune receptors to promote an integrated antiviral response.

INTRODUCTION

TRIM21 belongs to the tripartite motif family of proteins, of which there are some 100 members in humans (1). TRIM21 is found in almost all tissues, although its expression is regulated by interferons. In common with most TRIM proteins, it is normally located diffusely within the cytosol but has the ability to nucleate into cytoplasmic bodies of unknown function (2). TRIM21 is a multidomain protein consisting of a RING E3 ubiquitin ligase domain, a B box domain of unknown function, a coiled-coil region, and a PRYSPRY domain that binds with high affinity to the Fc portion of IgG, IgM, or IgA (3–5). Ironically, TRIM21 (under its alternative designation Ro52) was first described as an autoantigen (6), and its identification in a yeast-two-hybrid screen using IgG as bait was first assumed to be a consequence of this (7). Further work indicated that TRIM21-IgG binding was specific, but it was erroneously concluded that TRIM21 must be released from the cell or exposed on the cell surface for it to have an antibody-dependent function (8). The possibility of TRIM21 sensing antibodies inside the cell or mediating viral restriction was initially overlooked because antibodies were not thought to enter the cytosol or remain functional even if delivered there (9). Thus, it was only in 2010 that TRIM21 was discovered to be not only a cytosolic antibody sensor and the highest-affinity IgG receptor in humans, but also one that mediates potent viral restriction (5). TRIM21 targets a different part of the antibody molecule than Fcγ receptors and complement but one that overlaps with epitopes recognized by the neonatal Fc receptor (FcRn) and protein A (4). Although first thought to be a trimer, TRIM21 dimerizes through its coiled-coil domain, allowing it to engage both heavy chains of IgG simultaneously. This is in contrast to Fcγs, which bind a single heavy chain, and partly explains TRIM21's superior antibody binding affinity.

Antibodies play a major role in viral immunity and are the principle objective of vaccination. Antibody sera “passively transferred” from protected to naive individuals is sufficient to prevent subsequent infection. TRIM21 forms an important part of this protective antibody immunity. Under conditions where antibody sera was sufficient to fully protect mice from mouse adenovirus (MAV), over half of TRIM21-null animals developed fatal viral encephalomyelitis (10). Antibodies provide immunity in part through their neutralization ability, which is a strong correlate of protection. TRIM21 contributes to neutralization, and a potently neutralizing antibody was shown to become nonneutralizing in TRIM21 knockout cells (11).

TRIM21 immunity is unique from other antibody-mediated responses because it occurs inside the cell, after infection. Antibodies are generally unable to access the cytosol because they cannot cross plasma or endosomal membranes. In contrast, pathogens are adept at crossing membranes and, crucially, are able to do so even when opsonized by antibody molecules. TRIM21 therefore exists to defend against these intracellular antibody-bound pathogens just as Fcγs exist to protect against extracellular pathogens. In contrast to Fcγs however, TRIM21 is a sensor of infectious pathogens rather than merely antibody-bound particles (which could be noninfectious, such as an allergen). This is because TRIM21 intercepts virus during its infectious entry into the cell rather than detecting immune complex that has been taken up by phagocytosis. This key difference in function is reflected in tissue expression: Fcγs are expressed on professional cells, whereas TRIM21 is expressed throughout the body in cells of all histogenetic origin. TRIM21 therefore occupies a unique interface between innate and adaptive immunity, where it can act as an early warning system of pathogen exposure triggered by the process of infection itself.

Once they have infected a cell and entered the cytosol, antibody-bound pathogens are promptly detected by TRIM21. Two events are known to follow. In one, TRIM21 drives rapid degradation of the incoming pathogen/antibody complex, by recruiting the AAA ATPase VCP/p97 and the 26S proteasome (5, 12), resulting in viral neutralization. In the other, TRIM21 activates innate immune signaling pathways (including NF-κB, AP-1, and IRF3/5/7), by generating a ubiquitin signal that stimulates signal-inducing kinases like TAK1 (13). In contrast to classical sensor-then-effector immune responses, these two events occur concurrently, resulting in robust expression of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interferons, and CXCL10, independently from the restriction of viral replication (14).

TRIM21-mediated neutralization is distinct from classical receptor-independent mechanisms, and this has important implications for neutralizing antibody stoichiometry, kinetics, and efficacy (15). For instance, as TRIM21 does not require a critical threshold of antibody molecules, it functions according to an incremental rather than liminal model of neutralization (11). Moreover, TRIM21 results in elimination of the virus rather than simply inhibiting infection. TRIM21 is also distinct from classical sensing mechanisms as it is pathogen-associated molecular pattern (PAMP) independent. However, the ability of TRIM21 to detect incoming virions rather than replication products (such as occurs through RIG-I sensing of progeny genomes [16]) means that sensing is rapid. During viral infection of mice, interferon expression was activated within 6 h of MAV infection, but only in animals expressing TRIM21 and in the presence of a protective antibody serum (17). Consistent with its role in providing an early warning of pathogen exposure, TRIM21 has been shown to operate at low viral multiplicities, where infection is otherwise undetected (11).

The range of viruses susceptible to TRIM21 neutralization remains to be established but appears restricted to nonenveloped viruses, including adenoviruses and picornaviruses (5, 18). It may be the case that enveloped viruses escape TRIM21 because their capsids are not directly bound by serum antibodies and so are undetectable by cytosolic TRIM21. Direct viral antagonism of TRIM21 has not been demonstrated, although pathogen entry may provide one effective countermeasure. Many picornaviruses have evolved an entry mechanism in which their RNA genomes are extruded through a pore in endocytosed compartments, rather than causing endosomal collapse; this could have the consequence of evading TRIM21 detection. Beyond viruses, Salmonella was shown to carry attached antibodies into cells during infection (13). TRIM21 localized to salmonellae that escaped their vacuoles, leading to immune stimulation.

Recent work on TRIM21 has focused on two central questions: (i) how is TRIM21 able to act as both an immune sensor and effector, and (ii) how does TRIM21 integrate with the gamut of pattern recognition receptors in the cell? The key to the first question has been the discovery that TRIM21 synthesizes an immune second messenger—free K63-linked ubiquitin chains—only upon proteasome recruitment and thus pathogen degradation (Fig. 1) (19). Moreover, degradation is only possible after the precursor form of this messenger has been made, meaning that sensor and effector functions are inextricably linked. Free “K63 chains” activate several innate immune pathways (20), although their synthesis and regulation are poorly understood. Recent work has identified that TRIM21 is first activated following monoubiquitination by the E2 enzyme Ube2W (19). Monoubiquitinated TRIM21 becomes a substrate for the E2 heterodimer Ube2N/V2, which catalyzes the synthesis of TRIM21-anchored K63 chains. Current data suggest that K63-ubiquitinated TRIM21 undergoes a process of degradation-coupled deubiquitination by the proteasome-associated deubiquitinase Poh1/Rpn11. This enzyme specializes in the wholesale liberation of ubiquitin chains from substrates, which allows their translocation into the 20S proteolytic core. Importantly, while neither virus nor bound antibody molecules are themselves ubiquitinated, they are nevertheless part of the complex that is degraded by the proteasome (5). In this respect, TRIM21 is analogous to TRIM5 in that both E3 ligases direct viruses for degradation indirectly by first capturing them and then undergoing autoubiquitination (21). Tying the generation of free K63 chains with pathogen destruction neatly couples TRIM21-mediated immune stimulation to the presence of an antibody-coated pathogen. This system also avoids a scenario whereby the pathogen is degraded before K63 chain synthesis, which would dampen maximal immune activation.

FIG 1.

TRIM21 sequential ubiquitination and deubiquitination drives dual sensor and effector functions. Infectious entry by a nonenveloped virus is depicted, in which virus engages with a specific cell surface receptor, is endocytosed, and escapes into the cytosol. Importantly, any antibody molecules that are bound to the virion are also carried into the cell. Once an antibody-coated virion escapes into the cytosol, it is bound by TRIM21. TRIM21 catalyzes its own monoubiquitination, via the E2 enzyme Ube2W, which primes anchored K63-ubiquitin (Ub) chain extension via Ube2N/V2. Autoubiquitination of TRIM21 targets the entire immune complex for degradation by the proteasome. TRIM21/immune complexes are resolved in a proteasome-dependent process in which the deubiquitinase Poh1 liberates ubiquitin chains en bloc, coupling the release of free K63-polyubiquitin for immune signaling with viral degradation.

Another string in TRIM21's antiviral bow was recently revealed in the observation that TRIM21 promotes the activities of additional pattern recognition receptors (PRRs). Interplay between PRRs is poorly understood, yet it seems probable that during any given viral infection—involving multiple species of nucleic acid replication intermediates, cytosolic DNA, repetitive oligomeric viral capsids, DNA repair pathways, alien enzymatic activities, and mislocalized host proteins or damaged membranes—multiple host sensors will be triggered. In theory, nucleic acid detectors cGAS (detecting DNA) and RIG-I (detecting RNA) could recognize both incoming as well as progeny viral genomes. However, incoming viral genomes are generally hard to detect, due in part to the encapsidation strategy utilized by almost all viruses, in which a protein shell protects their genomes. Where viral genomes must be exposed to the cytosol, as occurs during well-timed capsid disassembly, nonstructural viral proteins may continue to cloak genomes from detection. However, it has now been shown that TRIM21 prematurely exposes incoming viral genomes through its ability to recruit the proteasome and force catastrophic capsid disassembly (Fig. 2) (17). This process was shown to allow sensing of incoming adenovirus and rhinovirus by cGAS and RIG-I in a manner dependent upon downstream adaptors STING and MAVS. An intriguing aspect of this data is that by integrating with other PRRs, an initially generalized cytokine response is differentiated by virus-derived patterns. Thus, rather than representing redundancy in the system, it may be that the simultaneous activation of multiple PRRs is used to tailor a pathogen-specific response. Synergy with specific PRRs may be particularly important for TRIM21, which by recognizing only the antibody molecule on the immune complex is atypical of mammalian viral sensors.

FIG 2.

TRIM21 potentiates RIG-I and cGAS sensing by exposing the genomes of incoming virions. TRIM21 directly activates immune signaling in a K63-linked ubiquitin chain-dependent manner but independently of viral PAMPs. TRIM21 recruitment of VCP and the proteasome results in catastrophic viral uncoating, exposing the genomes of incoming virions to nucleic acid sensors. This facilitates a second PAMP-dependent immune signaling event. The combination of these two signaling events may amplify immune transcription and allow a pathogen-specific response to be stimulated by an initial generic cytosolic detection of antibody.

The presence of antibody-coated pathogens in the cytosol presents a unique opportunity for the cell to detect infection at the earliest opportunity. By recognizing such immune complexes, TRIM21 establishes an important innate immune response that can protect the host from lethal viral infection. However, such a potent proinflammatory response must be regulated, and it will be important to determine how TRIM21 is controlled. Understanding the mechanisms behind TRIM21 function may also benefit our comprehension of the roles and action of similar TRIM proteins implicated in innate immunity.