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 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 h 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 h were subjected to E immunoprecipitation, and immunoblotting analysis revealed a reduction in ubiquitinated E in the absence of ITCH.

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.

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).

(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-E) HEK293 cells were transfected with Flag-tagged E with ITCH or ITCH-CS. 24 h later, cells were analyzed by immunofluorescence with Flag, ITCH and p62 or LC3B or LAMP1 antibodies. ITCH enhanced the colocalization between E and p62 (C) or LC3B (D), while no change in the colocalization between E and LAMP1 (E) was noted. 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, H) vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 1 MOI for 10 h 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.

(I) 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.

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 (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) The levels of WT and mutant S, with a substitution at the PPC site, were determined via immunoblotting analysis of lysates from HEK293 cells expressing Flag-tagged S WT or S MUT with ITCH or ITCH-CS, showing that, unlike S WT, the level of uncleavable S MUT was not affected by ITCH (n = 3).

(D) 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.

(E) 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.

(F) 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 TGN to the cytoplasm. Scale bar, 10 μm.

(G) Subcellular localization of furin in vT2-WT and vT2-ITCH-KO cells with SARS-CoV-2 infection at 1 MOI for 10 h 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.

(H) A model depicting ITCH’s role in regulating the Golgi localization of furin and the related function of S cleavage.

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 in vT2-WT and vT2-ITCH-KO cells with SARS-CoV-2 infection 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.

(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 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.

ITCH ablation suppresses SARS-CoV-2 production.

(A, B) The analysis of 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 hours post-infection (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 and D) and the activation of JNK1, as indicated by the increase of p-JNK1 in both vT2-WT and vT2-ITCH-KO cells (C and E).

(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 nineteen 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 h revealed a significant reduction in virus production with ITCH depletion (n=3).

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).