Ubiquitin ligase ITCH regulates life cycle of SARS-CoV-2 virus
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, United States
- Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, United States
- Department of Veterinary Medicine, University of Maryland, United States
- Department of Neuroscience, School of Medicine, Johns Hopkins University, United States
Figures
Figure 1 with 2 supplements
ITCH promotes the ubiquitination of the envelope protein E.
(A, B) Denatured lysates from HEK293 cells expressing Flag-tagged E and HA-tagged ubiquitin with or without elevated ITCH or ITCH-CS were subjected to Flag IP. Immunoblotting of ubiquitinated E using ubiquitin antibody (A) or Ub-K63 antibody (B) showed that WT ITCH, but not the inactive ITCH-CS, promoted the ubiquitination of E through K63 polyubiquitin chains (n = 3). (C) Denatured lysates from vT2-WT and vT2-ITCH-KO cells expressing HA-tagged ubiquitin, Flag-2×Strep tagged E were subjected to Strep affinity precipitation (AP). Immunoblotting analysis showed a reduction of ubiquitinated E in the absence of ITCH. (D) Lysates from HEK293 cells expressing s-tag-fused E with Flag-tagged ITCH were subjected to Flag IP. (E) was present in ITCH’s immunoprecipitates. (E) Lysates from HEK293 cells expressing Flag-tagged E were subjected to Flag IP, followed by immunodetection of coprecipitated endogenous ITCH. ITCH was detected in E precipitates. (F) Colocalization and interaction of ITCH with E was investigated in HEK293 cells by proximity ligation assay (PLA). Flag (rabbit) antibody was paired with ITCH (mouse) antibody. Negative controls comprised non-specific mouse IgG or rabbit IgG coupled with Flag or ITCH antibodies, respectively. Cells were visualized by confocal microscopy to detect red fluorescent dots, indicative of the colocalization of the respective antigens, targeted by the complementary species-specific antibodies. Scale bar, 10 μm. (G) Lysates from vT2 cells infected with SARS-CoV-2 at 1 MOI for 10 hr were subjected to E immunoprecipitation, followed by immunodetection using antibody against ITCH, revealing an ITCH signal in the E precipitates. (H) Denatured lysates from vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 1 MOI for 10 hr were subjected to E immunoprecipitation, and immunoblotting analysis revealed a reduction in ubiquitinated E in the absence of ITCH. Error bars represent means ± SEM. **p ≤ 0.01; n.s., non-significant.
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Figure 1—source data 1
PDF file containing original western blots for Figure 1A–E, G, H, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig1-data1-v1.zip
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Figure 1—source data 2
Original files for western blot analysis displayed in Figure 1A–E, G, H.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig1-data2-v1.zip
Figure 1—figure supplement 1
ITCH promotes ubiquitination of SARS-CoV-2 structural proteins.
(A) Components of SARS-CoV-2 virus-like particle (VLP), infectious VLP, and infectious virion. (B) Lysates of HEK293 cells expressing Flag-2×Strep-tagged S/E/M proteins were subjected to Flag immunoprecipitation (IP) and Strep affinity precipitation (AP), followed by detection of proteins by silver staining. (C) The interaction network between structural proteins and ubiquitination related proteins as identified by the proteomic analysis. (D) Lysates from HEK293 cells expressing Flag-2×Strep tagged S/E/M were subjected to Flag IP. Immunoblotting analysis showed that ITCH was pulled down by the structural proteins. (E, F) Denatured lysates from HEK293 cells expressing HA-tagged ubiquitin (Ub), Flag-tagged S, Flag-2×Strep-tagged E/M with ITCH or ITCH-CS were subjected to Flag IP, followed by immunoblotting against ubiquitin (Ub), Ub-K63, and the structural proteins (S antibody for S and Strep antibody for E/M). The immunoblotting analysis indicated that ITCH, but not the inactive ITCH-CS, promoted the ubiquitination of one or more components of the complex formed by the S/E/M structural proteins, through K63 polyubiquitin chains (n = 3). (G) Denatured lysates from HEK293 cells expressing HA-tagged ubiquitin (Ub), s-tag-fused E/M, and Flag-tagged S with ITCH or ITCH-CS were subjected to Flag IP. Immunoblotting analysis of the immunoprecipitates with ubiquitin antibody showed that the ubiquitination of S protein was not altered by ITCH. (H, I) Denatured lysates from HEK293 cells expressing HA-tagged ubiquitin (Ub), Flag-tagged S, Flag-2×Strep-tagged M, and s-tag-fused E with ITCH or ITCH-CS were subjected to s-tag AP. Immunoblotting analysis of the precipitates with ubiquitin (Ub) or Ub-K63 antibodies showed that, in the presence of M/S, the ubiquitination of E was increased by ITCH, but not the inactive ITCH-CS, through K63-linked chains. (J, K) Denatured lysates from HEK293 cells expressing HA-tagged ubiquitin (Ub), Flag-tagged S, Flag-2×Strep-tagged E, and s-tag-fused M with ITCH or ITCH-CS were subjected to s-tag AP. Immunoblotting analysis of the precipitates with ubiquitin (Ub) or Ub-K63 antibodies showed that, in the presence of E/S, the ubiquitination of M was increased by ITCH, but not the inactive ITCH-CS, through K63-linked chains. Error bars represent means ± SEM. **p ≤ 0.01; ***p ≤ 0.001; n.s., non-significant.
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Figure 1—figure supplement 1—source data 1
PDF file containing original western blots for Figure 1—figure supplement 1D–K, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 1—figure supplement 1D–K.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
ITCH interacts with M and promotes its ubiquitination.
(A, B) Denatured lysates from HEK293 cells expressing HA-tagged ubiquitin (Ub), Flag-tagged M with ITCH or ITCH-CS were subjected to Flag IP. Immunoblotting analysis of the immunoprecipitates with ubiquitin (Ub) or Ub-K63 antibodies showed that the ubiquitination of M was increased by ITCH, but not the inactive ITCH-CS, through K63-linked chains WT(n = 3). (C) Validation of the vT2-ITCH-KO cell lines through immunoblotting of ITCH and sequencing of the sgRNA-targeted DNA region. (D) Denaturing Strep AP using lysates from vT2-WT and vT2-ITCH-KO cells expressing HA tagged ubiquitin (Ub), Flag-2×Strep tagged M was performed. A decrease in the ubiquitination of M was shown in the ITCH-KO cells. (E) Non-denatured lysates from HEK293 cells expressing s-tag-fused M with Flag-tagged ITCH were subjected to Flag IP, showing the specific pull-down of M by ITCH. (F) Non-denatured lysates from HEK293 cells expressing RFP or Flag-tagged M were subjected to Flag IP, showing the specific pull-down of endogenous ITCH by M protein. (G) The proximal colocalization of ITCH with M was analyzed in HEK293 cells by proximity ligation assay (PLA) using a rabbit anti-Flag antibody paired with a mouse anti-ITCH antibody. Negative controls included non-specific mouse IgG or rabbit IgG paired with the Flag or ITCH antibody, respectively (n > 15 cells). Scale bar, 10 μm. (H) Lysates from vT2 cells infected with SARS-CoV-2 at 1 MOI for 10 hr were subjected to M IP, followed by immunodetection using antibody against ITCH. An ITCH signal was clearly observed in the M immunoprecipitates. (I) M IP was carried out using denatured lysates from vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 1 MOI for 10 hr, Immunoblotting analysis revealed a reduction in ubiquitinated M in the absence of ITCH. (J) Models depicting the role of ITCH in promoting mutual interactions among the SARS-CoV-2 structural proteins. Error bars represent means ± SEM. *p ≤ 0.05; ****p ≤ 0.0001; n.s., non-significant.
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Figure 1—figure supplement 2—source data 1
PDF file containing original western blots for Figure 1—figure supplement 2A–F, H, I, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Original files for western blot analysis displayed in Figure 1—figure supplement 2A–F, H, I.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig1-figsupp2-data2-v1.zip
Figure 2
ITCH promotes mutual interactions of the structural proteins.
(A) Lysates from HEK293 cells expressing Flag-tagged E and s-tag-fused M with ITCH or ITCH-CS were subjected to Flag IP, followed by immunoblotting for M. No significant change was observed in the interaction between E and unmodified M under these conditions. (B) S-tag AP was performed using lysates from HEK293 cells expressing s-tag-fused E and Flag-tagged S with ITCH or ITCH-CS. ITCH, but not the inactive ITCH-CS, increased the level of both intact and cleaved S precipitated by E. (C) Scheme of the serial precipitation assays isolating E protein from S containing complexes (top panel). Lysates from HEK293 cells expressing Flag-tagged S, s-tag-fused E, and ITCH or ITCH-CS together with HA-tagged ubiquitin were subjected to Flag IP, followed by s-tag AP under the denaturing condition. Immunoblotting analysis showed that S/E complexes contained ubiquitinated E and that the level of ubiquitinated E was significantly increased by ITCH. (D) Lysates from HEK293 cells expressing Flag-tagged S and s-tag-fused M with ITCH or ITCH-CS were subjected to s-tag AP, followed by immunoblotting for S. ITCH did not alter the interaction between M and S. (E) HEK293 cells expressing Flag-tagged M and s-tag-fused E with ITCH or ITCH-CS were subjected to Flag IP. Immunoblotting analysis showed that, while ITCH decreased the level of unmodified M, more E protein was precipitated by M. (F) Scheme of the serial precipitation assays isolating M protein from E containing complexes (top panel). Lysates from HEK293 cells expressing Flag-tagged E and s-tag-fused M with HA-tagged ubiquitin (Ub) and His6-fused ITCH or ITCH-CS were subjected to Flag IP, followed by s-tag AP under denaturing condition. Ubiquitinated M was observed in the E/M precipitates, and ITCH increased the level of ubiquitinated M in these complexes.
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Figure 2—source data 1
PDF file containing original western blots for Figure 2A–F, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig2-data1-v1.zip
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Figure 2—source data 2
Original files for western blot analysis displayed in Figure 2A–F.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig2-data2-v1.zip
Figure 3 with 1 supplement
ITCH promotes the trafficking of E and M to p62-dependent autophagosomes, aiding secretion of E.
(A) Culture medium from vT2-WT and vT2-ITCH-KO cells expressing HA-tagged ubiquitin (Ub) and Flag-2×Strep tagged E was harvested for Strep AP. A decrease of the extracellular E protein induced by ITCH ablation was visualized by immunoblot (left) and dot blot analysis (right). Coomassie Brilliant Blue (CBB) staining of the culture media is presented as a loading control. (B) Lysates from HEK293 cells expressing Flag-tagged E with ITCH or ITCH-CS were subjected to Flag IP, followed by immunoblotting for autophagosome cargo receptors. (E) specifically precipitated p62 and ITCH promoted their interaction. (C) Co-IP analysis of lysates from HEK293 WT and ITCH-KO cells expressing Flag-tagged E revealed that loss of ITCH reduces the specific interaction between M and p62. HEK293 cells were transfected with Flag-tagged E with ITCH or ITCH-CS. 24 hr later, cells were analyzed by immunofluorescence with Flag, ITCH and p62 or LC3B antibodies. ITCH enhanced the colocalization between E and p62 (D) or LC3B (E). Scale bar, 10 μm. (F) Culture media and denatured lysates from control (CTRL) and p62 knock down (#1, #2) HEK293 cells expressing HA-tagged ubiquitin (Ub), Flag-tagged E were subjected to Flag IP (with incorporation of a washing step with urea before elution for culture media samples), followed by immunoblotting or dot blot analysis. p62 depletion resulted in the accumulation of intracellular E (both unmodified and ubiquitinated), while decreasing the level of extracellular E (n = 3). (G) Culture media from HEK293 cells expressing Flag-tagged E together with control vector or p62 were harvested and subjected to Flag IP. The increase in extracellular E protein induced by ectopic p62 expression was detected by dot blot analysis (n = 3). CBB staining of the culture media is presented as a loading control. (H) HEK293 cells expressing E together with ITCH or ITCH-CS were cultured for 12 hr and then treated with CHX (10 μg/ml) and Mg132 (2 nM) or DMSO for 24 hr. Immunoblot analysis showed that MG132 blocked E degradation but did not rescue the ITCH-mediated reduction in E levels. (I, J) vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 1 MOI for 10 hr were subjected to immunofluorescence analysis with E and p62 or LC3B antibodies. ITCH-ablation decreased the colocalization between E and p62 (G) or LC3B (H). Scale bar, 10 μm. (K) A model of the function of ITCH in promoting autophagosome-mediated SARS-CoV-2 virion egress. ITCH-dependent ubiquitin modification enhances E binding with S and M binding with non-ubiquitinated E, resulting in the increase in virion formation and p62-dependent autophagosome targeting for release. Error bars represent means ± SEM. **p ≤ 0.01; n.s., non-significant.
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Figure 3—source data 1
PDF file containing original western blots for Figure 3A–C, F–H, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig3-data1-v1.zip
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Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3A–C, F–H.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig3-data2-v1.zip
Figure 3—figure supplement 1
ITCH promotes the trafficking of M from trans-Golgi network (TGN) to p62-dependent autophagosomes and enhances its secretion.
(A) Culture media from vT2-WT and vT2-ITCH-KO cells expressing HA-tagged ubiquitin (Ub) and Flag-2×Strep tagged M were harvested for the Strep AP. Proteins were then visualized by immunoblot or dot blot analysis, showing a decrease of the extracellular M protein in the absence of ITCH. (B) The interaction network between the SARS-CoV-2 structural proteins and secretion host proteins as identified by the proteomic analysis. (C) Co-IP analysis of lysates from HEK293 cells expressing Flag-tagged M with ITCH or ITCH-CS showed that ITCH, but not the inactive ITCH-CS, promoted the specific interaction between M and p62. Immunofluorescence analysis of HEK293 cells expressing Flag-tagged M with ITCH or ITCH-CS showed that ITCH, but not the inactive ITCH-CS, enhanced the colocalization between M and p62 (D) or LC3B (E), while the colocalization between M and LAMP1 was not altered (E). Scale bar, 10 μm. (F, G) HEK293 cells were transfected with Flag-tagged E or M with ITCH or ITCH-CS. 24 hr later, cells were analyzed by immunofluorescence with Flag, ITCH and LAMP1 antibodies. ITCH expression did not alter the colocalization of LAMP1 with E (F) or M (G). Scale bar, 10 μm. (H) Flag IP was conducted to precipitate M protein from culture media and denatured lysates of control (CTRL) and p62 knockdown (#1, #2) from HEK293 cells expressing HA-tagged ubiquitin (Ub) and Flag-tagged M, showing that the levels of both unmodified and ubiquitinated intracellular M were increased by p62 depletion, with a concurrent decrease in the level of extracellular M (n = 3). (I) HEK293 cells expressing M together with ITCH or ITCH-CS were cultured for 12 hr, followed by treatment with CHX (10 μg/ml) and MG132 (2 nM) or DMSO for 24 hr. Immunoblotting revealed that proteasome inhibition prevented M turnover but failed to restore M levels reduced by ITCH expression. (J, K) vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 1 MOI for 10 hr were subjected to immunofluorescence with M and p62 or LC3B antibodies. Colocalization between E and p62 (H) or LC3B (I) was significantly reduced with ITCH ablation. Scale bar, 10 μm. Error bars represent means ± SEM. *p ≤ 0.05; ***p ≤ 0.001; n.s., non-significant.
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Figure 3—figure supplement 1—source data 1
PDF file containing original western blots for Figure 3—figure supplement 1A, C, H, I, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 3—figure supplement 1A, C, H, I.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig3-figsupp1-data2-v1.zip
Figure 4 with 3 supplements
ITCH inhibits S cleavage by disrupting the Golgi localization of furin.
(A) Sequence comparison of the S proteins from SARS-CoV-2, SARS-CoV, and MERS in a region containing the S1/S2 boundary. SARS-CoV-2 S contains the proprotein convertase cleavage (PPC) motif. The PPC site mutated from ‘TNSPRRA’ to ‘SLL’ (SARS-CoV) is highlighted in yellow. Immunoblotting analysis of wild-type S (S WT) and PPC site mutant (S MUT) proteins were shown using lysates from HEK293 cells expressing the variants. (B) Levels of intact S and proteolytically formed S1/2 subunits in lysates of HEK293 cells expressing Flag-tagged S with ITCH or ITCH-CS were analyzed by immunoblotting. ITCH significantly reduced the ratio of S1/2 subunits to intact S, indicating that ITCH inhibits S protein cleavage (n = 3). (C) Culture media from vT2 cells expressing Flag-tagged S WT (S WT) and S mutant (S MUT) proteins with ITCH or ITCH-CS were subjected to Flag IP (incorporating washing with urea before elution). Immunoblotting analysis showed that ITCH increased the levels of intact S in both intracellular and extracellular fractions, while decreasing the levels of S1/S2 subunits. The non-cleavable S MUT variant showed no changes in protein levels or secretion in response to modulations of ITCH activities. (D) Subcellular localization of s-tagged furin in HEK293 cells coexpressing ITCH or ITCH-CS was analyzed by furin and ITCH immunofluorescence staining. Elevated expression of ITCH, but not its inactive form, led to a dispersion of furin to the cytoplasm. Scale bar, 10 μm. (E) Transfected HEK293 cells expressing s-tagged furin with ITCH or ITCH-CS were analyzed via immunofluorescence staining using s-tag, TGN46, and ITCH antibodies. ITCH induced a dispersed of furin from trans-Golgi network (TGN) to the cytoplasm. Scale bar, 10 μm. (F) Subcellular localization of furin in vT2-WT and vT2-ITCH-KO cells with SARS-CoV-2 infection at 1 MOI for 10 hr was analyzed by furin and ITCH immunofluorescence staining. SARS-CoV-2 infection resulted in a pronounced dispersion of furin from the TGN to the cytoplasm, whereas ITCH ablation significantly inhibited this phenotype. Scale bar, 10 μm. (G) A model depicting ITCH’s role in regulating the Golgi localization of furin and the related function of S cleavage. Error bars represent means ± SEM. *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001; n.s., non-significant.
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Figure 4—source data 1
PDF file containing original western blots for Figure 4A–C, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4A–C.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig4-data2-v1.zip
Figure 4—figure supplement 1
ITCH does not associate with S and does not affect its subcellular localization.
(A) Non-denatured lysates from HEK293 cells expressing Flag-tagged S protein were subjected to Flag IP, showing that ITCH signal was not detected in the S immunoprecipitates. (B) Subcellular localization analysis of Flag-tagged S in HEK293 cells expressing ITCH or ITCH-CS showed that ITCH did not regulate the subcellular localization of S protein. Scale bar, 10 μm.
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Figure 4—figure supplement 1—source data 1
PDF file containing original western blots for Figure 4—figure supplement 1A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 4—figure supplement 1A.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig4-figsupp1-data2-v1.zip
Figure 4—figure supplement 2
ITCH does not affect subcellular localization of S-related TMPRSS and trypsin proteases.
(A) Validation of the expression of various TMPRSS and trypsin proteases in HEK293 cells. (B–H) HEK293 cells were transfected with the indicated Flag-tagged proteases together with ITCH or ITCH-CS. Immunofluorescence analysis revealed no detectable changes in the subcellular distribution of these proteases upon ITCH overexpression, with the exception of furin and CTSL. Scale bar, 10 μm.
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Figure 4—figure supplement 2—source data 1
PDF file containing original western blots for Figure 4—figure supplement 2A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig4-figsupp2-data1-v1.zip
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Figure 4—figure supplement 2—source data 2
Original files for western blot analysis displayed in Figure 4—figure supplement 2A.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig4-figsupp2-data2-v1.zip
Figure 4—figure supplement 3
ITCH does not affect the levels of TMPRSS and trypsin proteases.
(A) The levels of furin in HEK293 cells expressing WT or inactivated ITCH were analyzed by immunoblotting, showing that the level of furin was not altered by ITCH overexpression (n = 3). (B–F) HEK293 cells were transfected with Flag-tagged TMPRSS proteases (TMPRSS2, TMPRSS4, TMPRSS11D) and trypsins (ACR, PLAT) with ITCH or ITCH-CS. No change in the levels of these proteases was observed upon ITCH overexpression. (G) Validation of the HEK293 ITCH-KO cell lines through immunoblotting of ITCH and sequencing of the sgRNA-targeted DNA region. (H) The levels of furin in HEK293 WT and ITCH-KO cells were analyzed by immunoblotting, showing that the level of furin was not altered by ITCH ablation (n = 3). (I–M) HEK293 WT and ITCH-KO cells were transfected with Flag-tagged TMPRSS and trypsin proteases, followed by immunoblotting with Flag or ITCH antibodies, showing that ITCH ablation did not change the levels of these proteases. (N) Immunoblotting analysis of lysates from HEK293 cells expressing CTSL and ITCH or ITCH-CS showed that ITCH, but not ITCH-CS, decreased the level of mature CTSL with a concurrent increase of pro-CTSL (n = 3). (O) Immunoblotting analysis of the S1 subunit using lysates from HEK293 cells expressing N-terminally Flag-tagged S1 and ITCH or ITCH-CS showed that ITCH, but not ITCH-CS, increased the level of S1 (n = 3). Error bars represent means ± SEM. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; n.s., non-significant.
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Figure 4—figure supplement 3—source data 1
PDF file containing original western blots for Figure 4—figure supplement 3A–F, H–O, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig4-figsupp3-data1-v1.zip
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Figure 4—figure supplement 3—source data 2
Original files for western blot analysis displayed in Figure 4—figure supplement 3A–F, H–O.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig4-figsupp3-data2-v1.zip
Figure 5
ITCH promotes the trafficking and maturation of CTSL and protects S protein from degradation.
(A) Subcellular localization of Flag-tagged CTSL in HEK293 cells expressing ITCH or ITCH-CS was analyzed by Flag and ITCH staining. ITCH disrupted the cytoplasmic distribution of CTSL. Scale bar, 10 μm. (B) Immunoblotting analysis of endogenous CTSL in HEK293 cells expressing ITCH or ITCH-CS showed that ITCH increased the level of pro-CTSL while decreasing that of mature CTSL (n = 3). (C) Immunoblotting analysis of endogenous CTSL in WT and ITCH-KO HEK293 cells showed that ITCH ablation promoted CTSL maturation (n = 3). (D) Immunoblotting analysis of CTSL levels in vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at a MOI of 0.0001 and harvested at 48 hpi. An increase in CTSL maturation was observed upon ITCH ablation, as indicated by the increased ratio of mature to pro-CTSL, under the condition of SARS-CoV-2 infection (n = 3). (E) Immunoblotting analysis of the S2 subunit and the derived S2 sub-fragments from HEK293 cells expressing S2 and ITCH or ITCH-CS showed that ITCH increased the level of uncleaved S2 while decreasing the levels of S2 sub-fragments (n = 3). (F) Immunoblotting analysis of small proteolytic fragments of S protein in HEK293 cells expressing Flag-tagged S and ITCH or ITCH-CS showed that ITCH reduced the levels of the S proteolytic cleavage fragments (n = 3). (G) Immunoblotting analysis of intact S and proteolytically formed S1/2 subunits from vT2-WT and vT2-ITCH-KO cells with SARS-CoV-2 infection (0.0001 MOI) at 48 hpi showed that ITCH ablation decreased the levels of both intact S and S1/2 subunits (n = 3). (H) A model depicting the function of ITCH in inhibiting CTSL maturation and subsequently reducing S cleavage. Error bars represent means ± SEM. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; n.s., non-significant.
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Figure 5—source data 1
PDF file containing original western blots for Figure 5B–G, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig5-data1-v1.zip
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Figure 5—source data 2
Original files for western blot analysis displayed in Figure 5B–G.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig5-data2-v1.zip
Figure 6 with 1 supplement
ITCH ablation suppresses SARS-CoV-2 production.
(A, B) The analysis of infectious virus titers (TCID50) and virus copy numbers in culture media of vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 0.0001 MOI, at 48 hpi, showed a robust inhibition of virus production by ITCH ablation (n = 3). (C–E) Immunoblotting analysis of phosphorylated ITCH (p-ITCH), ITCH and p-JNK1 in vT2-WT and vT2-ITCH-KO cells with SARS-CoV-2 infection at 48 hpi showed that the viral infection induced the activation of ITCH, as indicated by the significance increase in its phosphorylation (C, D) and the activation of JNK1, as indicated by the increase of p-JNK1 in both vT2-WT and vT2-ITCH-KO cells (C, E, n = 3). (F) Immunoblotting analysis of phosphorylated ITCH in vT2 cells, infected with SARS-CoV-2 at 0.01 MOI at various time points, showed time-dependent activation of ITCH, as indicated by its phosphorylation (n = 3). (G–K) Analysis of published single-nucleus RNA sequencing data from the lungs of 19 individuals who died from COVID-19 and seven control individuals showed that ITCH is ubiquitously expressed in major lung cell clusters (G) and that SARS-CoV-2 infection significantly elevates ITCH mRNA levels in B cells, fibroblasts, epithelial cells, and endothelial cells compared to controls. (L) The analysis of virus copy numbers in culture media of control and ITCH-depletion Calu-3-ACE2 cells infected with SARS-CoV-2 at 1 MOI for 48 hr revealed a significant reduction in virus production with ITCH depletion (n = 3). Error bars represent means ± SEM. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; n.s., non-significant.
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Figure 6—source data 1
PDF file containing original western blots for Figure 6C, F, L, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig6-data1-v1.zip
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Figure 6—source data 2
Original files for western blot analysis displayed in Figure 6C, F, L.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig6-data2-v1.zip
Figure 6—figure supplement 1
Pharmacological inhibition of ITCH suppresses SARS-CoV-2 production.
(A) Culture media from vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 for 48 hr were collected and subjected to trichloroacetic acid (TCA) precipitation. S proteins (1× input from WT and 10× input from ITCH-KO samples) were analyzed by immunoblotting, revealing a marked increase in S protein cleavage in the absence of ITCH. (B) Immunoblotting analysis of vT2 cells expressing ubiquitin and Flag-tagged ITCH and treated with various concentrations of Clom (18 hr) showed that Clom inhibited the activity of ITCH, as indicated by its self-ubiquitination, at the concentration of 15 μM or above. (C) Analysis of vT2 cells infected with SARS-CoV-2 at 0.0001 MOI showed that the SARS-CoV-2-induced cytopathic effect (CPE) and virus production as indicated by virus copy number (48 hpi) were significantly inhibited by the Clom treatment (n = 3). (D, E) Analysis of vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 0.0001 MOI showed that SARS-CoV-2-induced viral copy numbers in the culture medium (48 hpi) were reduced by several hundred-fold in vT2-ITCH-KO cells treated with Clom (n = 3). (F–J) ITCH expression analysis using published single-nucleus RNA sequencing data from the COVID-19 patients and control individuals revealed no significant difference in ITCH mRNA levels in T cells, neurons, mast cells, myeloid cells, or APC-like cells in the lungs with or without SARS-CoV-2 infection. Error bars represent means ± SEM. **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; n.s., non-significant.
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Figure 6—figure supplement 1—source data 1
PDF file containing original western blots for Figure 6—figure supplement 1A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/105105/elife-105105-fig6-figsupp1-data2-v1.zip
Figure 7
A model for the multiple roles of ITCH in the SARS-CoV-2 life cycle.
The activation of JNK1 occurs during the late stage of the SARS-CoV-2 life cycle, which in turn activates the E3 ligase activity of ITCH (pink arrows). ITCH promotes ubiquitination of the E and M proteins, resulting in increased virion formation and p62-dependent autophagosome targeting (blue arrows), aiding SARS-CoV-2 egress. ITCH disrupts furin and CTSL protease activities, resulting in increased incorporation of intact S protein into the virion and enhanced virion infectivity and stability (black arrows).
Author response image 1
Vero-TMPRSS2 (A) or Vero-ITCH-KO (B) cells were treated with DMSO or chloroquine (Clo) for 48 h, and cell viability was assessed by calcein AM staining (n = 3).
Author response image 2
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Ubiquitin ligase ITCH regulates life cycle of SARS-CoV-2 virus
eLife 14:RP105105.
https://doi.org/10.7554/eLife.105105.3
