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.