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.

During pathogen infection, antibodies can be carried into the cell, where they are detected by the cytosolic antibody receptor TRIM21. McEwan and colleagues show that the recognition of intracellular antibodies by TRIM21 activates immunological signaling. 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.

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. The early steps of HIV-1 infection are poorly understood, in part because of the difficulty in obtaining high-resolution information on encapsidated virus and its interaction with host cofactors. This, in turn, has made it difficult to design effective anti-capsid (CA) drugs. In our present study, we have used stabilized hexamers of HIV-1 CA to obtain complexed crystal structures with two cellular cofactors that are important for HIV-1 infection. These structures and accompanying virology reveal an essential interface in the capsid of HIV-1 that is lost upon viral uncoating. This interface is used to recruit both the nuclear targeting cofactor CPSF6 and NUP153, a nuclear pore component that facilitates nuclear entry. The high-resolution information provided by these structures reveals that the interface is degenerate and CA mutations can be made that selectively perturb sensitivity to each cofactor. This interface is also competed by two antiviral drugs, PF74 and BI-2, whose different mechanisms of action are not fully understood. We show that PF74, but not BI-2, binds across monomers within multimerized capsid affecting an inter-hexamer interface that is crucial for maintaining intact virions and that the addition of saturating concentrations of PF74 causes an irreversible block to viral reverse transcription.

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.

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.

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