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. 2020 Oct 16;295(42):14343-14351.
doi: 10.1074/jbc.RA119.011006. Epub 2020 Aug 12.

HDAC6 regulates antibody-dependent intracellular neutralization of viruses via deacetylation of TRIM21

Songbo Xie  1 , Linlin Zhang  2 , Dan Dong  3 , Ruixin Ge  3 , Qianqian He  2 , Cunxian Fan  3 , Wei Xie  3 , Jun Zhou  3   2 , Dengwen Li  2 , Min Liu  1
Affiliations

HDAC6 regulates antibody-dependent intracellular neutralization of viruses via deacetylation of TRIM21

Songbo Xie et al. J Biol Chem. .

Abstract

Tripartite motif-containing protein 21 (TRIM21) is a cytosolic antibody receptor that targets the internalized virus-antibody complex to the proteasome for degradation. However, the precise mechanism regulating TRIM21 activity is unknown. Here we show that TRIM21 is a substrate of histone deacetylase 6 (HDAC6) and that its function is regulated by acetylation. HDAC6 interacts with TRIM21 through its PRYSPRY motif and deacetylates TRIM21 at lysine 385 and lysine 387, thus promoting its homodimerization. Inhibiting HDAC6 activity increases TRIM21 acetylation, and hyperacetylation blocks TRIM21 dimerization and ubiquitination, preventing its binding to the virus-antibody complex and its degradation via the ubiquitin-proteasome pathway. HDAC6 depletion or inhibition increases virus accumulation in cells, indicative of an impaired capacity for antibody-dependent intracellular neutralization of viruses, whereas TRIM21 acetylation-deficient K385/387R mutant rescues HDAC6 depletion-caused ADIN impairment. These findings provide evidence for HDAC6 as a novel regulator of TRIM21-mediated intracellular innate immunity.

Keywords: HDAC6; TRIM21; acetylation; antibody-dependent intracellular neutralization; histone deacetylase 6; infection; virus.

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Conflict of interest statement

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.
HDAC6 interacts with TRIM21. a, representative image of HeLa cells labeled with antibodies against TRIM21 (red) and HDAC6 (green) and stained with DAPI (blue). b, representative image of HeLa cells cotransfected with HA-TRIM21 and GFP-HDAC6 followed by staining with antibodies against HA (red) and GFP (green) and DAPI (blue). Scale bars in panels a and b, 10 μm. c, HEK293T cell lysates were immunoprecipitated with control rabbit IgG or anti-HDAC6 antibody and probed with the indicated antibodies. d, HEK293T cells were transfected with HA-TRIM21 or HA constructs, and cell lysates were subjected to immunoprecipitation with an anti-HA antibody and then probed with the indicated antibodies. e, HEK293T cells were cotransfected with HA-TRIM21 and GFP or GFP-HDAC6. Anti-GFP immunoprecipitates were probed with the indicated antibodies. f, HEK293T cells were cotransfected with GFP-HDAC6 and HA or HA-TRIM21 constructs, and anti-HA immunoprecipitates were probed with the indicated antibodies. g, His-tagged TRIM21 and Myc/DDK tagged HDAC6 were incubated with IgG or anti-His antibody together with protein A beads, and the pulldown precipitates were probed with an anti-His antibody.
Figure 2.
Figure 2.
HDAC6 deacetylates TRIM21. a, HEK293T cells were cotransfected with HA-TRIM21 and the indicated GFP-tagged HDAC6 constructs. Cell lysates were subjected to immunoprecipitation with an anti-HA antibody and probed with the indicated antibodies. b, HEK293T cells were cotransfected with GFP-HDAC6 and the indicated FLAG-tagged TRIM21 constructs; cell lysates were subjected to immunoprecipitation with an anti-GFP antibody and probed with the indicated antibodies. c, HEK293T cells were treated with tubacin or vehicle for 8 h; anti-AcK immunoprecipitates were probed with an anti-TRIM21 antibody. The relative TRIM21 acetylation level was determined by normalizing to the corresponding input. d and e, HEK293T cells were transfected with HA-TRIM21 (d) or FLAG-TRIM21 (e) and treated with tubacin or vehicle for 8 h. Anti-AcK and -HA immunoprecipitates were probed with the indicated antibodies (d), or anti-FLAG immunoprecipitates were subjected to competitive elution with FLAG peptide, and the elution was probed with the indicated antibodies (e). The relative TRIM21 acetylation level was determined by normalizing to the corresponding input (d) or the corresponding elution probed with anti-FLAG antibody (e). f, HEK293T cells were cotransfected with FLAG-TRIM21 and GFP or GFP-HDAC6; anti-AcK immunoprecipitates were probed with an anti-FLAG antibody. The relative TRIM21 acetylation level was determined by normalizing to the corresponding input probed with anti-FLAG antibody. g, HEK293T cells were cotransfected with HA-TRIM21 and GFP or GFP-HDAC6; anti-HA immunoprecipitates were probed with the indicated antibodies. The relative TRIM21 acetylation level was determined by normalizing to the corresponding immunoprecipitate probed with anti-HA antibody. h, HEK293T cells were transfected with GFP-HDAC6 and various FLAG-TRIM21 mutants, anti-FLAG immunoprecipitates were probed with the indicated antibodies. The relative TRIM21 acetylation level was determined by normalizing to the corresponding immunoprecipitate probed with anti-FLAG antibody.*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.
Figure 3.
Figure 3.
Hyperacetylation of TRIM21 blocks its dimerization and ubiquitination. a and b, HEK293T cells were cotransfected with FLAG-TRIM21 and HA-TRIM21 and treated with tubacin or vehicle for 8 h; anti-HA immunoprecipitates (a) and anti-FLAG immunoprecipitates (b) were probed with an anti-FLAG or -HA antibody. The relative TRIM21 level was determined by normalizing to the corresponding immunoprecipitate probed with an anti-HA (a) or -FLAG (b) antibody. c, HEK293T cells were cotransfected with FLAG-TRIM21 and HA-TRIM21 and either GFP-HDAC6-WT, the inactive mutant (MT), or the GFP-only construct. Anti-HA immunoprecipitates were probed with the indicated antibodies. The relative TRIM21 level was determined by normalizing to the corresponding immunoprecipitate probed with anti-HA antibody. d, HeLa cells were transfected with HA-TRIM21, treated with tubacin or vehicle, and labeled with an anti-HA antibody (red) followed by staining with DAPI (blue). e, experiments were performed as in (d), and the percentage of cells with rodlike TRIM21 was quantified (n = 200 for each dataset). f, HEK293T cells were cotransfected with HA-TRIM21 and His-Myc-ubiquitin and treated with the proteasome inhibitor MG132 and tubacin or vehicle for 8 h. Anti-HA immunoprecipitates were probed with the indicated antibodies. g, HEK293T cells were cotransfected with HA-TRIM21 and His-Myc-ubiquitin along with GFP-HDAC6-WT, GFP-HDAC6-MT, or the GFP-only construct and treated with MG132. Anti-HA immunoprecipitates were probed with the indicated antibodies. h, HEK293T cells were cotransfected with His-Myc-ubiquitin and various FLAG-TRIM21 mutants and treated with the proteasome inhibitor MG132 for 8 h. Anti-FLAG immunoprecipitates were probed with the indicated antibodies. i, HEK293T cells were cotransfected with the indicated FLAG-TRIM21 and the indicated HA-TRIM21 plasmids and treated with tubacin for 8 h; anti-FLAG immunoprecipitates were probed with an anti-FLAG or -HA antibody. j, HeLa cells were cotransfected with HA-TRIM21 and His-Myc-ubiquitin (Ub) and labeled with anti-HA (green) and anti-Myc (red) antibodies. The relative TRIM21 level was determined by normalizing to the corresponding immunoprecipitate probed with an anti-FLAG antibody. Scale bars in panels c and g, 10 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.
Figure 4.
Figure 4.
Depletion of HDAC6 or inhibition of its activity impairs viral clearance. a, protocol for cellular infection with antibody-coated AdVs. Cells were seeded at the indicated density on day 1. On day 2, AdVs carrying the GFP gene were preincubated with an antibody against hexon. Cells were incubated with AdV-antibody complexes for 48 h. On day 4, the cells were harvested and infection rate was analyzed by flow cytometry. b and c, WT and HDAC6 KO MEFs were incubated with AdV-antibody complexes. d, Western blot analysis of the knockdown efficiency of siRNAs used in (e) and (f). Ctrl, control; HD6, HDAC6. The relative HDAC6 level was determined by normalizing to the corresponding α-tubulin. e–h, HeLa cells transfected with control or HDAC6-specific siRNA without (e and f) or with (g and h) tubacin or vehicle pretreatment were incubated with AdV-antibody complexes. i, Western blot analysis of the expression efficiency of the indicated FLAG-TRIM21 plasmids used in (j and k). j and k, HeLa cells transfected with HDAC6-specific siRNA and the indicated plasmids were incubated with AdV-antibody complexes. Representative plots of GFP-positive cells detected by flow cytometry (b, e, g, and j) and quantification of GFP-positive cells (c, f, h, and k) are shown (n = 6 for each dataset). Data represent the mean ± S.D. of three independent experiments. ns, not significant; *, p < 0.05, **, p < 0.01; ***, p < 0.001.
Figure 5.
Figure 5.
Model of the role of HDAC6 in TRIM21-mediated ADIN. TRIM21 senses the entry of antibody-bound AdVs and recruits HDAC6, which deacetylates and promotes the dimerization of TRIM21 via the coiled-coil domain. TRIM21 homodimers bind AdV-antibody complexes via the Fc domain of the antibody and target the complexes to the proteasome for degradation through automonoubiquitination and subsequent polyubiquitination. In the absence of HDAC6, TRIM21 is hyperacetylated, which inhibits the formation of TRIM21 homodimers; antibody-bound AdVs fail to be removed through TRIM21-mediated ADIN, resulting in viral replication and expression of viral proteins.
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