Proteasomal targeting leads to rapid degradation of virions in the cytosol before translation of virally encoded genes. 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 usually that it is fast CMKBR7 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 contamination and provide protection against future contamination. 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 usually that it can take 1 to 2 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 computer virus has joined the cytosol of a MLN-4760 cell, antibodies are helpless to prevent its contamination. Recently we described an intracellular cytosolic protein called tripartite motif-containing 21 MLN-4760 (TRIM21) that is capable of binding to an invariant region of antibody molecules via its PRYSPRY domain name (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 usually 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 presence of this mechanism and its operation in preventing contamination by adenovirus. Our data reveal that humoral immunity provides intracellular protection, whereby antibodies function inside cells by engaging with a potent effector mechanism that targets computer virus for degradation. Results TRIM21 Mediates Intracellular Antibody Neutralization. It is assumed that antibodies do not routinely enter the cytosol during viral contamination. To test this, we preincubated adenovirus (a model computer virus that causes respiratory disease) with different antibodies and added the virions to cultured HeLa cells. Adenovirus was chosen because it is usually a nonenveloped computer virus, and its capsid is usually naturally exposed to serum antibody before cellular contamination. This is in contrast to enveloped viruses, in which the capsid is usually guarded from antibody. After 30 MLN-4760 min of contamination the cells were fixed, and a fluorescent anti-IgG antibody was added to detect antibody-coated virions. As can be seen in Fig. 1and and and purified using amylose resin and size-exclusion chromatography. Fluorescence experiments were performed by titrating IgG using a Cary Eclipse fluorescence spectrophotometer. Fluorescence Anisotropy. The PRYSPRY domain name of TRIM21 was expressed and purified as previously described (4, 5). The protein was labeled with Alexa Fluor 488 5-SDP ester (Invitrogen) and its polarized fluorescence measured at 530 nm using a Cary Eclipse fluorescence spectrophotometer. IgM (Athens Research and Technology) was titrated into 50 nM PRYSPRY and the change in anisotropy averaged over 5 s. SEC MALS. MLN-4760 Size-exclusion chromatography (SEC) MALS was performed using a Wyatt Heleos II 18-angle light-scattering instrument coupled to a Wyatt Optilab rEX online refractive index detector. Samples were prepared as described above and exceeded through the light-scattering and refractive index detectors in a standard SEC MALS format. In Vitro Ubiquitination Assays. In vitro assays were carried out largely as previously described (21). Reaction mixtures were incubated at 37 C for 1 h then visualized by Western blot for TRIM21 (1:500, sc-25351; Santa Cruz Biotechnology), Ad5 hexon (donkey anti-goat IgG HRP 1:5,000 sc-2056; Santa Cruz Biotechnology), or ubiquitin (1:1,000, FK-2; Enzo Life Sciences) as indicated. Supplementary Material Supporting Information: Click here to view. Acknowledgments An initial stock of adenovirus Ad5-GFP was kindly provided by Dr. Olivier Danos (Cancer Biology, University College London Cancer Institute, London, United Kingdom). Footnotes The authors declare no conflict of interest. *This Direct Submission MLN-4760 article.