Simultaneous neutralization and innate immune detection of a replicating virus by TRIM21

Abstract

Tripartite motif-containing 21 (TRIM21) is a cytosolic immunoglobulin receptor that mediates antibody-dependent intracellular neutralization (ADIN). Here we show that TRIM21 potently inhibits the spreading infection of a replicating cytopathic virus and activates innate immunity. We used a quantitative PCR (qPCR)-based assay to measure in vitro replication of mouse adenovirus type 1 (MAV-1), a virus that causes dose-dependent hemorrhagic encephalitis in mice. Using this assay, we show that genetic ablation of TRIM21 or chemical inhibition of either the AAA ATPase p97/valosin-containing protein (VCP) or the proteasome results in a >1,000-fold increase in the relative level of infection in the presence of immune serum. Moreover, the TRIM21-mediated ability of antisera to block replication was a consistent feature of the humoral immune response in immunized mice. In the presence of immune sera and upon infection, TRIM21 also activates a proinflammatory response, resulting in secretion of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). These results demonstrate that TRIM21 provides a potent block to spreading infection and induces an antiviral state.

Fig 1

TRIM21 mediates in vitro neutralization of MAV-1 by antisera raised in mice as measured by TCID₅₀ and qPCR. (A) MAV-1 TCID₅₀/ml in MEF cells isolated from wild-type (WT, white bar) and TRIM21^−/− (K21, black bar) C57BL/6 mice. Error bars show standard errors of the means of the results from 3 experiments. (B) Fold change in observed MAV-1 TCID₅₀ value upon addition of MAV-1 antiserum diluted 1:10,000, all determined by endpoint dilution assay in WT (white bar) and K21 (black bar) MEF cells. Error bars show standard errors of the means of the results from 3 experiments. (C) Viral replication following MAV-1 infection of WT MEFs measured between days 0 and 5 postinfection (ND, not detected). Error bars show standard errors of the means of the results from 3 technical replicates. (D) MAV-1 load 4 days postinfection with 500× TCID₅₀ in WT (white bar) and K21 (black bar) MEF cells. Error bars show standard errors of the means of the results from 3 technical replicates. (E) Viral load in WT (white bar) and K21 (black bar) cells 4 postchallenge with MAV-1, as measured by qPCR for viral DNA. Error bars show standard errors of the means of the results from 3 technical replicates. (F) Relative MAV-1 infection levels in WT (white circles) and K21 (black squares) MEFs in the presence of immune mouse antisera. Viral loads were measured by qPCR for viral DNA 4 days postinfection. Error bars show standard errors of the means of the results from 3 technical replicates.

Fig 2

TRIM21-mediated neutralization is ablated by inhibition of VCP or the proteasome and is increased by stimulation with IFN-α. (A) Relative MAV-1 infection levels in WT MEFs treated with DMSO (white circles) or DBeQ (white squares) and K21 MEFs treated with DMSO (black circles) or DBeQ (black squares) in the presence of immune mouse antisera. (B) Relative MAV-1 infection levels in WT MEFs treated with DMSO (white circles) or MG132 (white triangles) and K21 MEFs treated with DMSO (black circles) or MG132 (black triangles) in the presence of immune mouse antisera. (C) Fold change of TRIM21 transcript levels compared to untreated-cell levels upon overnight infection with 500× TCID₅₀ MAV-1 (white bar) or treatment with IFN-α (black bar) in WT or K21 MEF cells (ND, not detected). (D) Neutralization of MAV-1 by antisera in WT (white bars) and K21 (black bars) MEF cells either under unstimulated (UT) conditions or following pretreatment with IFN-α.

Fig 3

TRIM21 dependence of MAV-1 neutralization is a common feature of antisera raised in different individual mice. (A) Levels of specific anti-MAV-1 IgG detected in sera from immunized mice (1 to 5) and the pooled immune serum (P) normalized to absorbance from naive serum (N) from an uninfected mouse. Sera were diluted 1:5,000 (black bars) and 1:10,000 (white bars). (B to F) Relative MAV-1 infection levels in WT (white circles) and K21 (black squares) MEF cells in the presence of antisera from 5 individual mice. (G) Relative MAV-1 infection levels in WT (white circles) and K21 (black squares) MEF cells in the presence of antisera pooled from 30 mice.

Fig 4

TRIM21 mediates antibody-dependent immune signaling. Data represent secretion of TNF-α (A) and IL-6 (B) upon treatment with MAV-1 and antiserum in WT (white bars) and K21 (black bars) MEF cells.