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 antibodybound 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. V iruses and their hosts have been coevolving for millions of years, and this has given rise to a complex system of immunity traditionally divided into innate and adaptive responses. Innate immunity comprises germ-line encoded receptors and effector mechanisms that recognize pathogen-associated molecular patterns (PAMPs) (1). The advantage of innate immunity is that it is fast and generic; however, viruses are adept at avoiding recognition by inhibiting innate immunity or by changing their molecular patterns. In contrast, adaptive immunity can clear a host of infection and provide protection against future infection. Unlike the PAMP receptors of innate immunity, adaptive immunity uses proteins such as antibodies to target pathogens. Antibodies are unique in the human body in that they evolve during the lifetime of an individual and can continue to target evolving pathogens (2). The weakness of adaptive immunity is that it can take 1 to 2 wk to reach full effectiveness. Moreover, the dogma of antibody immunity for the last 100 y has been that antibodies only provide extracellular protection (3). It is thought that once a virus has entered the cytosol of a cell, antibodies are helpless to prevent its infection.Recently we described an intracellular cytosolic protein called tripartite motif-containing 21 (TRIM21) that is capable of binding to an invariant region of antibody molecules via its PRYSPRY domain (4). We found this activity to be structurally, thermodynamically, and kinetically conserved across mammals (5). However, antibodies are extracellular proteins, as are all known mammalian IgG receptors (with the exception of FcRn, which is intracellular but not cytosolic). It therefore seemed incongruous to us that TRIM21 should be a universally conserved intracellular protein and yet be a high-affinity, highly specific IgG receptor. We hypothesized that there might be an antibody effector mechanism inside cells, mediated by TRIM21. Here we demonstrate the existence of...

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.

SummaryMethods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.

Antibodies can be carried into the cell during pathogen infection where they are detected by the ubiquitously expressed cytosolic antibody receptor TRIM21. Here we show that TRIM21 recognition of intracellular antibodies activates immune signaling. TRIM21 catalyses K63-ubiquitin chain formation, stimulating transcription factor pathways NF-κB, AP-1 and IRF3, IRF5, IRF7. Activation results in proinflammatory cytokine production, modulation of natural killer (NK) stress ligands and the induction of an antiviral state. Intracellular antibody signaling is abrogated by genetic deletion of TRIM21 and is recovered by ectopic TRIM21 expression. Antibody sensing by TRIM21 can be stimulated upon infection by DNA or RNA non-enveloped viruses or intracellular bacteria. The antibody-TRIM21 detection system provides potent, comprehensive innate immune activation, independent of known pattern recognition receptors.

During the early stages of infection, the HIV-1 capsid protects viral components from cytosolic sensors, such as cGAS, and nucleases, such as TREX, while allowing access to nucleotides for efficient reverse transcription1. Here we show that each capsid hexamer has a size-selective pore bounded by a ring of six arginine residues and a ‘molecular iris’ formed by the N-terminal β-hairpin. The arginine ring creates a strongly positively charged channel that recruits the four nucleotides with on-rates that near 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 destabilising 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.

The HIV capsid is semipermeable and covered in electropositive pores that are essential for viral DNA synthesis and infection. Here, we show that these pores bind the abundant cellular polyanion IP6, transforming viral stability from minutes to hours and allowing newly synthesised DNA to accumulate inside the capsid. An arginine ring within the pore coordinates IP6, which strengthens capsid hexamers by almost 10°C. Single molecule measurements demonstrate that this renders native HIV capsids highly stable and protected from spontaneous collapse. Moreover, encapsidated reverse transcription assays reveal that, once stabilised by IP6, the accumulation of new viral DNA inside the capsid increases >100 fold. Remarkably, isotopic labelling of inositol in virus-producing cells reveals that HIV selectively packages over 300 IP6 molecules per infectious virion. We propose that HIV recruits IP6 to regulate capsid stability and uncoating, analogous to picornavirus pocket factors. HIV-1/IP6/capsid/co-factor/reverse transcription.

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.

Alzheimer's disease (AD) and other neurodegenerative disorders are associated with the cytoplasmic aggregation of microtubuleassociated 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.T he cell's ability to identify intracellular viruses and bacteria relies on the detection of pathogen-associated molecular patterns (PAMPs) by specialized host receptors. Although highly effective at detecting microbial pathogens, this strategy is poorly equipped to identify host-derived pathogenic species such as aggregated proteins. As an alternative to PAMP detection, recent work has demonstrated that mammalian cells can use hostderived serum proteins, which are normally excluded from the cell interior, to target invading viruses and bacteria in the cytosol. For instance, nonenveloped viruses and bacteria carry antibodies with them into the cytoplasm during infection. These translocated antibodies are then sensed by the cytoplasmically expressed antibody receptor TRIM21 (tripartite motif protein 21), which binds with subnanomolar affinity to the antibody Fc domain (1-4). After binding to antibody, TRIM21 triggers a potent neutralization response that inhibits viral infection. Neutralization of infection is accompanied by degradation of viral components, which requires the activity of the proteasome and the molecular unfoldase, valosincontaining protein (VCP) or p97 (1, 5). Detection of viruses and bacteria by TRIM21 does not rely on microbial PAMPs, as model substrates such as antibody-coated latex beads can be bound and detected by TRIM21 (1, 3). We therefore hypothesized that the intracellular innate immune system could be repurposed to recognize and degrade host-derived pathogenic proteins.Microtubule-associated protein tau occurs in an assembled and hyperphosphorylated state in the cytoplasm of neurons and glial cells in Alzheimer's disease (AD), progressive supranuclear palsy, chronic traumatic encep...