bDespite 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.A ntibody-mediated immunity forms a crucial part of the antiviral immune response, and its induction is a principal objective of vaccination. Reduced antibody (Ab) production, as occurs in X-linked agammaglobulinemia, hypogammaglobulinemia, and dysgammaglobulinemia, leads to persistent bacterial and viral infection (30,31). In vitro, the binding of Abs to virus causes a reduction in infectious titer, termed neutralization, which is independent of effector mechanisms such as complement fixation or Fc-mediated phagocytosis (5). Neutralizing antibodies (NAbs) are thought to play an important role in antiviral immunity, since the passive transfer of strongly neutralizing Abs is associated with both antiviral protection (10, 12) and the abrogation of disease (7, 34). However, modeling and prediction of neutralization are not straightforward (29). For instance, it is unclear how the binding of one or a few Ab molecules per virus is sufficient for neutralization (4). An average of 1.4 NAb molecules is capable of neutralizing human adenovirus (AdV) type 2 (39), an apparently paradoxical finding given that IgG molecules are considerably smaller than adenovirus particles and occupy only a fraction of the viral surface when bound. The binding of a single NAb was also reported to neutralize poliovirus (13,38). A second neutralization phenomenon that is poorly understood is the persistent fraction (PF), i.e., the level of infection that remains at high NAb concentrations. The cause of the PF was previously attributed to aggregated virus, low-affinity Abs, viral heterogeneity, and polyclonal interference (2,19).Recently, we showed that Abs can mediate neutralization intracellularly by recruiting the cytosolic Ig receptor TRIM21 (23). The engagement of NAb-virus complexes by TRIM21 promotes the degradation of both Ab and virus by the proteasome, a process termed antibody-dependent intracellular neutralization (ADIN) (25). In this study, we describe the mechanistic requirements for ADIN. We quantitatively examine ...

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.A ntibody neutralization is a key component of the antiviral response and provides protective immunity. Our recent work has shown that neutralization can occur inside cells, in an effector-driven process mediated by tripartite motif-containing 21 (TRIM21). TRIM21, a cytosolic antibody receptor of ultrahigh affinity to IgG Fc (1, 2), is recruited to antibody-bound virus and targets the complex to the proteasome for degradation (3). This process potently neutralizes viral infection and has been termed antibody-dependent intracellular neutralization (ADIN) (4). ADIN is dependent upon the E3 ubiquitin ligase activity of TRIM21 and can be abrogated by chemical inhibition of the proteasome.Although both proteasomal activity and ubiquitination are necessary for ADIN, the exact mechanism of virus degradation is poorly understood. Specifically, it is not clear how the proteasome can degrade a virion, a compact proteinaceous particle much larger than the proteasome itself. The 26S proteasome has a mass of ∼2.5 MDa (5), and the pore through which substrates must pass to access the proteolytic chamber is no greater than 2 nm in diameter (6, 7). In contrast, human adenovirus (AdV), a virus potently neutralized by TRIM21, has a diameter of ∼100 nm and a mass of 150 MDa (8).Although there are ATPases in the 19S regulatory subunit of the 26S proteasome that may unfold substrates and allow them to enter through the pore (5, 6), AdV virions are much larger than any of the proteasome's known cellular substrates. Because ADIN has been shown to be independent of autophagy but dependent on proteasomal degradation (3), we hypothesized that an additional energydependent step of AdV capsid disassembly and/or unfolding might precede proteasomal degradation of the virus.In recent studies, the ATPase p97/VCP of the AAA (ATPases associated with diverse cellular activities) family has been implicated in the proteasomal degradation of certain cytosolic substrates (9-13). VCP is capable of dissociating proteins from large cellular structures such as the endoplasmic reticulum (14), the mitotic spindle (12), the nuclear envelope (15), and chromatin (1...

Background:The possibility of eradicating cancer by selective destruction of tumour blood vessels may represent an attractive therapeutic avenue, but most pharmaceutical agents investigated so far did not achieve complete cures and are not completely specific. Antibody conjugates now allow us to evaluate the impact of selective vascular shutdown on tumour viability and to study mechanisms of action.Methods:We synthesised a novel porphyrin-based photosensitiser suitable for conjugation to antibodies and assessed anticancer properties of its conjugate with L19, a clinical-stage human monoclonal antibody specific to the alternatively spliced EDB domain of fibronectin, a marker of tumour angiogenesis.Results:Here we show in two mouse model of cancer (F9 and A431) that L19 is capable of highly selective in vivo localisation around tumour blood vessels and that its conjugate with a photosensitiser allows selective disruption of tumour vasculature upon irradiation, leading to complete and long-lasting cancer eradication. Furthermore, depletion experiments revealed that natural killer cells are essential for the induction of long-lasting complete responses.Conclusions:These results reinforce the concept that vascular shutdown can induce a curative avalanche of tumour cell death. Immuno-photodynamic therapy may be particularly indicated for squamous cell carcinoma of the skin, which we show to be strongly positive for markers of angiogenesis.

Antibodies are a key component of the adaptive immune response but their activity has previously thought to be limited to the extracellular environment. We have demonstrated that pathogens traffic surface-bound antibodies to the intracellular compartment where they are recognised and neutralized by the high affinity cytosolic Fc receptor TRIM21. In this study we demonstrate that following detection of pathogen-antibody complexes, TRIM21 is able to synthesise K63-linked ubiquitin chains resulting in signalling via NF-κB, AP-1 and IRF3/5/7 pathways. Detection by TRIM21 is sufficient to induce the production of cytokines including CXCL10, IL-6 and IFN-β. Furthermore a wide range of intracellular pathogens, including Salmonella, non-enveloped DNA viruses and RNA viruses can be detected by TRIM21. TRIM21 therefore provides context-dependent detection of intracellular antibody, mediating potent neutralization and signalling functions.