William A. McEwan

Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21)

Antibodies provide effective antiviral immunity despite the fact that viruses escape into cells when they infect. Here we show that antibodies remain attached to viruses after cell infection and mediate an intracellular immune response that disables virions in the cytosol. We have discovered that cells possess a cytosolic IgG receptor, tripartite motif-containing 21 (TRIM21), which binds to antibodies with a higher affinity than any other IgG receptor in the human body. TRIM21 rapidly recruits to incoming antibody-bound virus and targets it to the proteasome via its E3 ubiquitin ligase activity. Proteasomal targeting leads to rapid degradation of virions in the cytosol before translation of virally encoded genes. Infection experiments demonstrate that at physiological antibody concentrations TRIM21 neutralizes viral infection. These results reveal an intracellular arm of adaptive immunity in which the protection mediated by antibodies does not end at the cell membrane but continues inside the cell to provide a last line of defense against infection.

Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21

During pathogen infection, antibodies can be carried into the infected cell, where they are detected by the ubiquitously expressed cytosolic antibody receptor TRIM21. Here we found that recognition of intracellular antibodies by TRIM21 activated immune signaling. TRIM21 catalyzed the formation of Lys63 (K63)-linked ubiquitin chains and stimulated the transcription factor pathways of NF-κB, AP-1, IRF3, IRF5 and IRF7. Activation resulted in the production of proinflammatory cytokines, modulation of natural killer stress ligands and induction of an antiviral state. Intracellular antibody signaling was abrogated by genetic deletion of TRIM21 and was restored by ectopic expression of TRIM21. The sensing of antibodies by TRIM21 was stimulated after infection by DNA or RNA nonenveloped viruses or intracellular bacteria. Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system independently of known pattern-recognition receptors.

Host Cofactors and Pharmacologic Ligands Share an Essential Interface in HIV-1 Capsid That Is Lost upon Disassembly.

The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.

AAA ATPase p97/VCP is essential for TRIM21-mediated virus neutralization

Tripartite motif-containing 21 (TRIM21) is a cytosolic IgG receptor that mediates intracellular virus neutralization by antibody. TRIM21 targets virions for destruction in the proteasome, but it is unclear how a substrate as large as a viral capsid is degraded. Here, we identify the ATPase p97/valosin-containing protein (VCP), an enzyme with segregase and unfoldase activity, as a key player in this process. Depletion or catalytic inhibition of VCP prevents capsid degradation and reduces neutralization. VCP is required concurrently with the proteasome, as addition of inhibitor after proteasomal degradation has no effect. Moreover, our results suggest that it is the challenging nature of virus as a substrate that necessitates involvement of VCP, since intracellularly expressed IgG Fc is degraded in a VCP-independent manner. These results implicate VCP as an important host factor in antiviral immunity.

Intracellular sensing of complement C3 activates cell autonomous immunity

Introduction Intracellular pathogens, which include viruses and some bacteria, typically disseminate through extracellular fluids before entering their target cells and beginning replication. While in the extracellular environment, pathogens can be intercepted by humoral immunity or by professional immune cells. However, immune surveillance is not always sufficient to prevent infection, and all cells need innate mechanisms to detect and disable pathogens. Intracellular complement C3 activates innate immunity. Complement component C3 covalently attaches to pathogens in the extracellular space. Upon pathogen entry into the cytosol, the cell senses attached C3. Sensing of C3 triggers a dual sensor and effector response, involving mitochondrial antiviral signaling (MAVS)–dependent immune signaling and proteasome-mediated viral degradation. Rationale We hypothesized that one method of pathogen detection may be to take advantage of the pathogen’s transition between extracellular and intracellular environments. Complement is a system of immune serum proteins with the ability to attach covalently to pathogens. We investigated whether this irreversible tagging of pathogens results in complement component C3 being carried into the cytosol during infection. Given that C3 should not otherwise be present inside the cell, we tested whether this could act as an invasion signal. Results Antibodies and complement components C3 and C4, but not other serum proteins, were found to associate with adenovirus. During adenoviral infection, deposited C3 was carried into cells, resulting in potent nuclear factor–κB (NF-κB) activation. Activation of NF-κB by C3 required viral entry into the cytosol, and no activity was observed when C3-coated adenovirus was trapped in endosomes. In addition to NF-κB, C3 also activated the activating protein 1 (AP-1) and interferon regulatory factor 3 (IRF3)/IRF5/IRF7 transcription pathways. Induction of these signaling pathways resulted in robust cytokine secretion, including interferon-β. Cytosolic C3 sensing was dependent on a number of signaling hubs known to be involved in innate immunity. In addition to activating immune signaling, C3 targeted cytosolic adenovirus for rapid degradation via the AAA–adenosine triphosphatase (ATPase) valosin-containing protein (VCP) and the proteasome. This degradation pathway potently restricted viral infection. C3-dependent intracellular sensing was widely conserved in mammals. Moreover, C3 activated NF-κB upon infection of diverse cell lines and primary cells including human lung cells, a physiologically relevant adenovirus target. Intracellular C3 also activated NF-κB in response to infection by diverse nonenveloped viruses—including papillomavirus, astrovirus, calicivirus, rhinovirus, poliovirus, coxsackievirus, enterovirus, and the facultative cytosolic bacteria Salmonella—but not enveloped respiratory syncytial virus. Picornaviruses were much less susceptible to complement sensing as a result of antagonism by their 3C protease, which cleaved C3 and prevented NF-κB activation and proteasome-mediated restriction. However, treatment with the 3C antagonist rupintrovir prevented 3C cleavage and restored full complement sensing of rhinovirus and poliovirus. Conclusion Complement mediates a potent intracellular immune response to nonenveloped viruses and cytosolic bacteria. The deposition and covalent attachment of C3 onto pathogens results in its translocation into cells during infection, in which it simultaneously induces an antiviral state and directs the degradation of viral particles. Intracellular complement immunity is highly effective against a range of pathogens, occurs in a variety of cell types, is independent of professional immune cells, and is highly conserved in mammals. Bringing in the agent of your own destruction Cells need mechanisms to detect and disable pathogens that infect them. Tam et al. now show that complement C3, a protein that binds to pathogens in the blood, can enter target cells together with the pathogen. Once inside the cell, the presence of C3 triggers both immune signaling and degradation of the internalized pathogen. The discovery of this pathway reveals that cells possess an early warning system of invasion that works against a diverse array of pathogens and does not require recognition of any specific pathogen molecules. Science, this issue 10.1126/science.1256070 A serum protein that tags along when pathogens enter cells triggers an immune reaction to them. Pathogens traverse multiple barriers during infection, including cell membranes. We found that during this transition, pathogens carried covalently attached complement C3 into the cell, triggering immediate signaling and effector responses. Sensing of C3 in the cytosol activated mitochondrial antiviral signaling (MAVS)–dependent signaling cascades and induced proinflammatory cytokine secretion. C3 also flagged viruses for rapid proteasomal degradation, preventing their replication. This system could detect both viral and bacterial pathogens but was antagonized by enteroviruses, such as rhinovirus and poliovirus, which cleave C3 using their 3C protease. The antiviral rupintrivir inhibited 3C protease and prevented C3 cleavage, rendering enteroviruses susceptible to intracellular complement sensing. Thus, complement C3 allows cells to detect and disable pathogens that have invaded the cytosol.

Truncation of TRIM5 in the Feliformia Explains the Absence of Retroviral Restriction in Cells of the Domestic Cat

ABSTRACT TRIM5α mediates a potent retroviral restriction phenotype in diverse mammalian species. Here, we identify a TRIM5 transcript in cat cells with a truncated B30.2 capsid binding domain and ablated restrictive function which, remarkably, is conserved across the Feliformia. Cat TRIM5 displayed no restriction activity, but ectopic expression conferred a dominant negative effect against human TRIM5α. Our findings explain the absence of retroviral restriction in cat cells and suggest that disruption of the TRIM5 locus has arisen independently at least twice in the Carnivora, with implications concerning the evolution of the host and pathogen in this taxon.

Regulation of Virus Neutralization and the Persistent Fraction by TRIM21

ABSTRACT Despite a central role in immunity, antibody neutralization of virus infection is poorly understood. Here we show how the neutralization and persistence of adenovirus type 5, a prevalent nonenveloped human virus, are dependent upon the intracellular antibody receptor TRIM21. Cells with insufficient amounts of TRIM21 are readily infected, even at saturating concentrations of neutralizing antibody. Conversely, high TRIM21 expression levels decrease the persistent fraction of the infecting virus and allows neutralization by as few as 1.6 antibody molecules per virus. The direct interaction between TRIM21 and neutralizing antibody is essential, as single-point mutations within the TRIM21-binding site in the Fc region of a potently neutralizing antibody impair neutralization. However, infection at high multiplicity can saturate TRIM21 and overcome neutralization. These results provide insight into the mechanism and importance of a newly discovered, effector-driven process of antibody neutralization of nonenveloped viruses.

Intracellular antibody-mediated immunity and the role of TRIM21

Protection against bacterial and viral pathogens by antibodies has always been thought to end at the cell surface. Once inside the cell, a pathogen was understood to be safe from humoral immunity. However, it has now been found that antibodies can routinely enter cells attached to viral particles and mediate an intracellular immune response. Antibody‐coated virions are detected inside the cell by means of an intracellular antibody receptor, TRIM21, which directs their degradation by recruitment of the ubiquitin‐proteasome system. In this article we assess how this discovery alters our view of the way in which antibodies neutralise viral infection. We also consider the antiviral function of TRIM21 in the context of its other reported roles in immune signalling and autoimmunity. Finally, we discuss the conceptual implications of intracellular antibody immunity and how it alters our view of the discrete separation of extracellular and intracellular environments.

HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

During the early stages of infection, the HIV-1 capsid protects viral components from cytosolic sensors and nucleases such as cGAS and TREX, respectively, while allowing access to nucleotides for efficient reverse transcription. Here we show that each capsid hexamer has a size-selective pore bound by a ring of six arginine residues and a ‘molecular iris’ formed by the amino-terminal β-hairpin. The arginine ring creates a strongly positively charged channel that recruits the four nucleotides with on-rates that approach diffusion limits. Progressive removal of pore arginines results in a dose-dependent and concomitant decrease in nucleotide affinity, reverse transcription and infectivity. This positively charged channel is universally conserved in lentiviral capsids despite the fact that it is strongly destabilizing without nucleotides to counteract charge repulsion. We also describe a channel inhibitor, hexacarboxybenzene, which competes for nucleotide binding and efficiently blocks encapsidated reverse transcription, demonstrating the tractability of the pore as a novel drug target.

Cytosolic Fc receptor TRIM21 inhibits seeded tau aggregation

Significance The mammalian cell cytoplasm contains numerous proteins with direct antimicrobial activity. Although these have been extensively studied in the context of viral and bacterial infection, it is unknown whether pathogenic self-propagating proteins, proposed to underlie common neurodegenerative diseases, can be targeted in a similar manner. We studied the ability of tripartite motif protein 21 (TRIM21), a newly identified intracellular antibody receptor, to intercept assemblies of misfolded tau, a cytoplasmic protein that aggregates in patients with Alzheimer’s disease. We developed tau “seeding” assays in human cells and found that TRIM21 could intercept and potently neutralize antibody-labeled tau assemblies. These findings demonstrate that the intracellular immune system can act against self-propagating misfolded proteins, with implications for ongoing attempts to develop antibody-based therapies for neurodegenerative disorders. Alzheimer’s disease (AD) and other neurodegenerative disorders are associated with the cytoplasmic aggregation of microtubule-associated protein tau. Recent evidence supports transcellular transfer of tau misfolding (seeding) as the mechanism of spread within an affected brain, a process reminiscent of viral infection. However, whereas microbial pathogens can be recognized as nonself by immune receptors, misfolded protein assemblies evade detection, as they are host-derived. Here, we show that when misfolded tau assemblies enter the cell, they can be detected and neutralized via a danger response mediated by tau-associated antibodies and the cytosolic Fc receptor tripartite motif protein 21 (TRIM21). We developed fluorescent, morphology-based seeding assays that allow the formation of pathological tau aggregates to be measured in situ within 24 h in the presence of picomolar concentrations of tau seeds. We found that anti-tau antibodies accompany tau seeds into the cell, where they recruit TRIM21 shortly after entry. After binding, TRIM21 neutralizes tau seeds through the activity of the proteasome and the AAA ATPase p97/VCP in a similar manner to infectious viruses. These results establish that intracellular antiviral immunity can be redirected against host-origin endopathogens involved in neurodegeneration.