TRIM21 mediates intracellular antibody neutralization. (A) Confocal microscopy images of adenovirus-infected HeLa cells. Adenovirus precoated in antibody and detected after infection with an Alexa Fluor 546 secondary (red) can be seen inside cells. The images are a Z-projection. (Scale bars, 10 μm; 2 μm in Insets.) (B) Cells treated with IFN-α, TRIM21 siRNA, siRNA control, or IFN-α and TRIM21 siRNA were infected with GFP adenovirus at different polyclonal antibody concentrations. Adenovirus infection is reduced by 2 logs in cells expressing the highest levels of TRIM21. (C) Western blot of TRIM21 protein levels in each condition.

TRIM21 mediates neutralization using serum IgG and IgM but not Fab2 fragments. (A) Antibody-dependent TRIM21 neutralization of virus requires the Fc domain. (B) TRIM21 binds IgM with an affinity of 16.8 ± 1.5 μM. (C) Serum IgM antibodies can be used by TRIM21 to neutralize virus. Depletion of TRIM21 reverses this effect, and IFN-α increases it. Error bars in all panels were calculated from triplicate experiments.

Mechanism of TRIM21 neutralization. (A) SEC MALS chromatograms of TRIM21 (black), IgG (light gray), and TRIM21 in complex with IgG (dark gray). The continuous traces represent the refractive index signal (left-hand axis), and the short horizontal lines represent the calculated mass at each sampling interval (1 s) within each peak (right-hand axis). Analysis indicates that TRIM21 is dimeric with a mass of 107 kDa, that IgG has a mass of 154 kDa, and that TRIM21:IgG complex yields a peak corresponding to free IgG and a 1:1 complex with mass ≈280 kDa. (B and C) Steady-state fluorescence titration of IgG with full-length TRIM21 (B) and ΔRING-Box TRIM21 (C) reveals an affinity of 0.6 ± 0.1 nM and 0.9 ± 0.2 nM, respectively. (D) TRIM21 neutralization is reversed by the proteasome inhibitor MG132 but not the autophagy inhibitor 3-MA. Error bars were calculated from triplicate experiments. (E) Direct correlation between MG132 concentration and reversal of TRIM21 neutralization. MG132 only reverses neutralization in the presence of antibody. (F) Proteasomal degradation, TRIM21, and antibody are necessary factors in the same pathway of viral neutralization.

TRIM21 E3 ubiquitin ligase function is essential for viral neutralization. (A) Recombinant full-length TRIM21 neutralizes virus, but TRIM21 lacking the RING and B Box domains does not. (B) TRIM21 is an active E3 ligase, but deletion of RING and B Box domains prevents autoubiquitination. (C) TRIM21 does not ubiquitinate viral capsid (hexon) or bound antibody. (D) Confocal microscopy Z-projection showing HeLa cells infected with antibody-coated adenovirus. TRIM21-colocalized virions are positive for ubiquitin.

TRIM21 mediates rapid degradation of virus. Western blots of hexon, antibody, and TRIM21 protein levels 1–6 h after infection. Adenovirus hexon protein and antibody are rapidly degraded in a TRIM21-dependent manner. Addition of MG132 partially rescues degradation.

Intracellular antibody-coated beads recruit TRIM21 and are ubiquitinated. Streptavidin-conjugated latex beads coated with antistreptavidin antibody and transfected into cells recruit TRIM21 and colocalize with ubiquitin. (Scale bars, 10 μm; 5 μm in Insets.)