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. 2023 Jul 4;14(7):396.
doi: 10.1038/s41419-023-05923-9.

E3 ligase HECTD3 promotes RNA virus replication and virus-induced inflammation via K33-linked polyubiquitination of PKR

Jiaying Huang #  1 , Zhou Yu #  2   3 , Xuelian Li  4 , Mingjin Yang  5 , Qian Fang  5 , Zheng Li  5 , Chunmei Wang  4 , Taoyong Chen  5 , Xuetao Cao  6   7   8   9
Affiliations

E3 ligase HECTD3 promotes RNA virus replication and virus-induced inflammation via K33-linked polyubiquitination of PKR

Jiaying Huang et al. Cell Death Dis. .

Abstract

Uncontrolled viral replication and excessive inflammation are the main causes of death in the host infected with virus. Hence inhibition of intracellular viral replication and production of innate cytokines, which are the key strategies of hosts to fight virus infections, need to be finely tuned to eliminate viruses while avoid harmful inflammation. The E3 ligases in regulating virus replication and subsequent innate cytokines production remain to be fully characterized. Here we report that the deficiency of the E3 ubiquitin-protein ligase HECTD3 results in accelerated RNA virus clearance and reduced inflammatory response both in vitro and in vivo. Mechanistically, HECTD3 interacts with dsRNA-dependent protein kinase R (PKR) and mediates Lys33-linkage of PKR, which is the first non-proteolytic ubiquitin modification for PKR. This process disrupts the dimerization and phosphorylation of PKR and subsequent EIF2α activation, which results in the acceleration of virus replication, but promotes the formation of PKR-IKK complex and subsequent inflammatory response. The finding suggests HECTD3 is the potential therapeutic target for simultaneously restraining RNA virus replication and virus-induced inflammation once pharmacologically inhibited.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. HECTD3 deficiency impairs RNA virus-induced innate cytokine production and restrains virus replication.
a, b Hectd3+/+ or Hectd3–/– BMDM (2 × 105 cells per 24-well) were stimulated with the indicated pathogens for 6 h (MOI = 1 for VSV and SeV and MOI = 5 for HSV-1 and VACV). The amount of IL-6 (a) and IFNβ (b) in supernatants were measured by ELISA. c, d Hectd3+/+ or Hectd3–/– BMDM (2×105 cells per 24-well) were infected with VSV (MOI = 1) (c) or HSV-1 (MOI = 5) (d) for 2 h, 4 h, 6 h. The mRNA level of VSV nucleocapsid (nc) (c) or HSV-1 ICP0 (d) gene was examined by Q-PCR. e Hectd3+/+ or Hectd3–/– BMDM (2 × 105 cells per 24-well) were infected with GFP-VSV (MOI = 1) virus for 12 h, then examined by fluorescence microscope (Scale bar, 100 µm). f Hectd3+/+ or Hectd3–/– BMDM (2×105 cells per 24-well) were infected with VSV (MOI = 1) virus and incubated at 4 °C or 37 °C for 1 h as indicated. The mRNA level of VSV nucleocapsid (nc) gene was examined by Q-PCR. Results are presented as mean ± SD of three biological replicates (a–d, f) (one-way ANOVA followed by Bonferroni multiple comparison). One representative experiment of three is shown. ns, no significance; **P < 0.01; ****P < 0.0001.
Fig. 2
Fig. 2. HECTD3 deficiency impairs innate response and restricts virus propagation in vivo.
a Hectd3+/+ or Hectd3–/– mice were intraperitoneally injected with 2 × 107 PFUs of VSV. The survival of mice (n = 10 per group) was monitored by Kaplan and Meier method and analyzed by Log-rank test. b, c Hectd3+/+ or Hectd3–/– mice were intraperitoneally injected with 5 × 106 PFUs of VSV and their serum levels of IL-6 (b) and IFNβ (c) were measured by ELISA 6 h or 12 h after VSV infections as indicated (n = 4 mice per group). d 24 h after VSV infections, viral titers in the homogenate supernatant of indicated organs from Hectd3+/+ or Hectd3–/– mice were determined by TCID50 assay. e H&E staining of lung sections from mice (in e). Scale bars, 100 µm. f Immunofluorescent staining for CD45 in liver sections from mice (in d). Scale bars, 100 µm. Results are presented as mean ± SD (b–d) (one-way ANOVA followed by Bonferroni multiple comparison). One representative experiment of three is shown. *P < 0.05; **P < 0.01; ****P < 0.0001.
Fig. 3
Fig. 3. HECTD3 interacts with PKR and impairs the activation of PKR-EIF2α pathway.
a Wild-type BMDM were infected with VSV (MOI = 1), SeV (MOI = 1), PR8 (MOI = 1), HSV-1 (MOI = 5) for 2 h or stimulated with liposome-packaged poly (I:C) (1 µg/ml), naked poly (I:C) (5 µg/ml), or LPS (100 ng/ml) for 0.5 h or 2 h as indicated. Whole-cell extracts (WCE) were immunoprecipitated with anti-HECTD3 antibody plus protein A/G beads then immunoblotted to detected indicated proteins. b, c Hectd3+/+ or Hectd3–/– BMDM were infected with VSV (MOI = 1) (b) or HSV-1 (MOI = 5) (c) for 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h as indicated and the activation of PKR-EIF2α signaling pathway were examined by Western blotting. d Hectd3+/+ or Hectd3–/– RAW264.7 cells with reconstitution of Myc-HECTD3 or Myc-HECTD3(C823A) were infected with VSV (MOI = 1) for 0 h or 6 h as indicated and the activation of the signaling mediators were examined by Western blotting.
Fig. 4
Fig. 4. The DOC domain of HECTD3 binds the RNA-binding domain of PKR.
a Schematic illustration of domains in the full-length (FL) or fragments (F) of Myc-HECTD3 or Flag-PKR. DOC, DOC domain; HECT, HECT domain; R, RNA binding domain; KD, kinase domain. Numerical numbers indicate for the site of amino acids. b, c HEK293T cells were transiently transfected with plasmids expressing Flag-tagged wild-type PKR or PKR mutants and Myc-tagged wile type HECTD3 or HECTD3 mutants as indicated for 48 h. Whole-cell extracts (WCE) were immunoprecipitated with anti-Myc Sepharose Beads (b) or anti-Flag Sepharose Beads (c) then immunoblotted for indicated proteins. d RAW264.7 cells grown on cover slides were transiently transfected with a plasmid expressing N-terminal red fluorescent protein (Nred)-tagged HECTD3 for 48 h, and then infected with VSV (MOI = 1) viruses for 2 h. After counterstained with PKR (Alexa Flour 488) and DAPI, cells were examined by confocal microscope (Scale bar, 5 µm). One representative experiment of three is shown.
Fig. 5
Fig. 5. HECTD3 mediates K33-linked polyubiquitination of PKR during RNA virus infection.
a–c, f Hectd3+/+ or Hectd3–/– BMDM were infected with VSV (MOI = 1) for 2 h, whole-cell extracts (WCE) were heated in buffer containing 1% SDS then immunoprecipitated (IP) with anti-PKR antibody plus protein A/G beads. Polyubiquitination (wild-type) (a), K48-linked polyubiquitination (b), K63-linked polyubiquitination (c) and K33-linked polyubiquitination (f) of PKR were examined by Western blotting. d HEK293T cells were transiently transfected with plasmids expressing Myc-tagged HECTD3, Flag-tagged PKR and HA-tagged wild-type or mutant Ub as indicated for 48 h. Then polyubiquitination of PKR was examined by Western blotting after immunoprecipitations with anti-Flag Sepharose Beads. e HEK293T cells were transiently transfected with plasmids expressing HA-tagged K33O ub, Myc-tagged wild-type or mutant HECTD3 and Flag-tagged PKR as indicated for 48 h. Then polyubiquitination of PKR was examined by Western blotting against HA after immunoprecipitations with anti-Flag Sepharose Beads. One representative experiment of three is shown.
Fig. 6
Fig. 6. Polyubiquitination of PKR catalyzed by HECTD3 inhibits PKR dimerization.
a The dimerization of PKR in Hectd3+/+ or Hectd3–/– BMDM infected with VSV (MOI = 1) as indicated was examined by native page and Western blot. b Quantification analysis of western blotting results in (a). β-actin was used as a control. c The dimerization of PKR in Hectd3+/+ or Hectd3–/– RAW264.7 cell with reconstitution of Myc-HECTD3 or Myc-HECTD3 (C823A) and infected with VSV (MOI = 1) for 0 h or 4 h as indicated was examined by native page and Western blot. Results are presented as mean ± SD of three biological replicates (b) (one-way ANOVA followed by Bonferroni multiple comparison). One representative experiment of three is shown. **P < 0.01; ****P < 0.0001.
Fig. 7
Fig. 7. PKR is required for HECTD3-mediated anti-viral responses against RNA virus.
a Pkr–/– Raw264.7 cells were transfected with plasmids expressing Flag-tagged PKR (wt) or PKR (K68R). The efficiency of reconstitution was evaluated by Western blotting. b Pkr+/+ or Pkr–/– Raw264.7 cells reconstituted with PKR (wt) or PKR (K68R) were transfected with control (Ctrl) or Hectd3 siRNA (#2) for 48 h and infected with VSV (MOI = 1) as indicated. The mRNA level of VSV nucleocapsid (nc) was examined by Q-PCR. c, d Pkr+/+ or Pkr–/– Raw264.7 cells reconstituted with PKR (wt) or PKR (K68R) were transfected with control (Crtl) or Hectd3 siRNA(#2) as indicated and infected with VSV (MOI = 1) for 8 h. Amounts of IL-6 (c) and IFNβ (d) in supernatants were measured by ELISA. e Quantification of the percentage of ANNEXIN V+ Pkr+/+ or Pkr–/– Raw264.7 cells which were transfected with control (Ctrl) or Hectd3 siRNA (#2) as indicated and infected with VSV (MOI = 1) for 24 h as indicated time. Cells were washed by PBS, stained with ANNEXIN V-FITC for 15 min at RT, and subjected to cell apoptosis analysis by flow cytometry. f Pkr+/+ or Pkr–/– Raw264.7 cells were transfected with control (Ctrl) or Hectd3 siRNA (#2) as indicated and infected with VSV(MOI = 1) for 18 h as indicated and the level of cleaved caspase3 and caspase8 were examined by Western blotting. Results are presented as mean ± SD of four biological replicates (b–e) (One-way ANOVA followed by Bonferroni multiple comparison). One representative experiment of three is shown. n.s., no significance; ****P < 0.0001.
Fig. 8
Fig. 8. K33-linked PKR forms a complex with IKK and exerts proinflammatory function.
a–c Hectd3+/+ or Hectd3–/– BMDM (2 × 105 cells per 24-well) were pretreated with DMSO or ISRIB (3.5 nM) for 1 h and infected with VSV (MOI = 1) for 4 h, 6 h as indicated. The mRNA level of VSV nucleocapsid (VSV-nc) gene was examined by Q-PCR (a) and the amounts of IL-6 (b) and IFNβ (c) in supernatants were measured by ELISA. d, e Hectd3+/+ or Hectd3–/– BMDM (2 × 105 cells per 24-well) were stimulated with cytosolic nucleic acids sensor agonists for 6 h (500 ng/ml 5’ppp-dsRNA for RIG-I, 500 ng/ml poly (I:C) for MDA5). The amounts of IL-6 (d) and IFNβ (e) in supernatants were measured by ELISA. f Wild-type BMDM were infected with VSV (MOI = 1), SeV (MOI = 1), PR8 (MOI = 1), stimulated with liposome-packaged poly (I:C) (1 µg/ml), naked poly (I:C) (5 µg/ml), HSV-1 (MOI = 5) or LPS (100 ng/ml) for 0.5 h or 2 h. Then whole-cell extracts (WCE) were immunoprecipitated with anti-PKR antibody plus protein A/G beads. Components in the PKR complex were examined by Western blotting. g Hectd3+/+ or Hectd3–/– Raw264.7 cells reconstituted by Mock, Myc-HECTD3 or Myc-HECTD3 (C823A) were infected with VSV (MOI = 1) for 2 h as indicated. Then whole-cell extracts (WCE) were immunoprecipitated with anti-PKR antibody plus protein A/G beads. Components in the PKR complex were examined by Western blotting. h Pkr+/+ or Pkr–/– Raw264.7 cells were transfected with a plasmid expressing Myc-HECTD3 and infected with VSV (MOI = 1) for 4 h or 6 h as indicated. The activation of the signaling mediators were examined by Western blotting. Results are presented as mean ± SD of three biological replicates (a–e) (one-way ANOVA followed by Bonferroni multiple comparison). One representative experiment of three is shown. n.s., no significance; *P < 0.05; ****P < 0.0001.
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