Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A EBOV-GP 1,2 (GP) was expressed with indicated ER proteins in HEK293T cells. Protein expression was detected by western blotting (WB). GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; HSPA5 was detected by anti-FLAG; CANX was detected by anti-HA. Ctrl, control vector; ACTB, β-actin. B Indicated EBOV glycoproteins were expressed with CALR or CANX in HEK293T cells. Protein expression was detected by WB. EBOV glycoproteins were detected by anti-HiBiT; CALR was detected by anti-Myc; CANX was detected by anti-HA. C Indicated EBOV glycoproteins were expressed with CANX or CALR in HEK293T cells, and co-immunoprecipitated (IP) with anti-EBOV-GP from cell lysate (Input). The GP interactions with CANX and CALR were determined by WB. GPs were detected by anti-EBOV-GP; CALR was detected by anti-Myc; CANX was detected by anti-HA. D EBOV-GP 1,2 was expressed in HEK293T wild-type (WT) and PDIA3 -, CALR -, or CANX -knockout (KO) cells. Protein expression was detected by WB. GP and endogenous PDIA3, CALR, and CANX were detected by their specific antibodies. E EBOV-GP 1,2 was expressed with increased amounts of CALR or CANX in HEK293T WT and CALR - or CANX -KO cells. Protein expression was detected by WB. GP was detected by anti-EBOV-GP; CALR and CANX were detected by anti-Myc or anti-HA. F GP 1,2 proteins from indicated ebolaviruses were expressed with CALR or CANX in HEK293T cells. Protein expression was detected by WB. GPs were detected by anti-EBOV-GP; CALR and CANX were determined by anti-Myc or anti-HA. G GP 1,2 proteins from indicated ebolaviruses were expressed in HEK293T WT and CALR - or CANX -KO cells. Protein expression was detected by WB. GPs were detected by anti-EBOV-GP; CALR and CANX were detected by their specific antibodies. H MARV-GP 1,2 was expressed with CANX or CALR in HEK293T cells. Alternatively, MARV-GP 1,2 was expressed in HEK293T WT and CALR - or CANX -KO cells. Protein expression was detected by WB. MARV-GP was detected by anti-HiBiT; ectopic CALR and CANX were detected by anti-Myc or anti-HA; endogenous CALR and CANX were detected by their specific antibodies. I EBOV-GP 1,2 was expressed with CANX or CALR in A549 cells, HeLa cells, Hep G2 cells, primary mouse macrophages (Mϕ), and THP1-derived macrophages (THP1-Mϕ). Protein expression was detected by WB. GP was detected by anti-EBOV-GP; CALR and CANX were detected by anti-Myc or anti-HA.

Article Snippet: Cells were then incubated with rabbit polyclonal anti-Zaire EBOV-GP (Sino Biological, 40442-T48) at 1:1000 dilution and mouse polyclonal anti-HA (Sigma-Aldrich, H3663) in PBS with 5% BSA for 2 h. After being washed 5 times with PBS, cells were stained with Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Invitrogen, A31573) at 1:700 dilution for 1 h. Cells were washed again with PBS 5 times and then incubated with DAPI for 3 min for nuclear staining.

Techniques: Expressing, Western Blot, Plasmid Preparation, Immunoprecipitation, Knock-Out, Derivative Assay

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A HIV-1 firefly luciferase reporter viruses pseudotyped with EBOV-GP 1,2 were produced from HEK293T WT, PDIA3, CALR or CANX-overexpressing, and PDIA3 -, CALR - or CANX -KO cells. After infecting HEK293T cells with an equal number of these different viruses, viral entry was determined by measuring intracellular luciferase activities. Viral entry is shown as relative values, with the entry of viruses produced from HEK293T WT cells in the presence of a control vector set to 100%. B EBOV replication and transcription-competent virus-like particles (trVLPs) were produced (p0) and passaged two times (p1, p2) in HEK293T cells in the presence or absence of PDIA3, CALR, or CANX. EBOV replication was determined by measuring intracellular Renilla luciferase activity. Viral replication was also measured in the absence of EBOV-L [L(-)], which served as a negative control. Results from three independent experiments are presented. C Huh7 cells were transfected with increasing amounts of vectors expressing PDIA3, CALR, or CANX, and infected with EBOV Mayinga strain at 0.01 multiplicity of infection (MOI). Viral RNAs were extracted from the supernatants at indicated times and quantified by real-time PCR. D EBOV virus-like particles (VLPs) were produced from HEK293T WT, CALR or CANX-overexpressing, and CALR - or CANX -KO cells after expression of EBOV-GP 1,2 with EBOV-VP40. VLPs were purified by ultra-centrifugation and protein expression in cell lysate and virions was analyzed by WB. GP, VP40, and endogenous CALR and CANX were detected by their specific antibodies; ectopic CALR and CANX were detected by anti-Myc or anti-HA. E Viral fusion proteins from indicated viruses were expressed with CALR in HEK293T cells (lanes 1–14). Alternatively, they were also expressed in HEK293T WT or CALR -KO cells (lanes 15-28). Protein expression was detected by WB. EBOV-GP 1,2 , MERS-S, SRAS1-S, and SARS2-S were detected by anti-FLAG; IAV (H5N5) HA, VSV-G, HIV-1 Env, and CALR were detected by their specific antibodies. F Viral fusion proteins from indicated viruses were expressed with CANX in HEK293T cells (lanes 1-14). Alternatively, they were also expressed in HEK293T WT or CANX -KO cells (lanes 15-28). Protein expression was detected by WB as in D , except that CANX was detected by its specific antibody. Error bars in A , B , and C represent the standard error of measurements (SEMs) calculated from three independent experiments.

Article Snippet: Cells were then incubated with rabbit polyclonal anti-Zaire EBOV-GP (Sino Biological, 40442-T48) at 1:1000 dilution and mouse polyclonal anti-HA (Sigma-Aldrich, H3663) in PBS with 5% BSA for 2 h. After being washed 5 times with PBS, cells were stained with Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Invitrogen, A31573) at 1:700 dilution for 1 h. Cells were washed again with PBS 5 times and then incubated with DAPI for 3 min for nuclear staining.

Techniques: Luciferase, Produced, Plasmid Preparation, Activity Assay, Negative Control, Transfection, Expressing, Infection, Real-time Polymerase Chain Reaction, Purification, Centrifugation

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A Expression of PDIA3, CALR, and CANX was determined in HEK293T WT, single PDIA3 -, CALR -, or CANX -KO, and dual PDIA3 / CALR - or PDIA3 / CANX -KO cells. Protein expression was determined by WB. PDIA3, CALR, and CANX were detected by their specific antibodies. B GP was expressed with indicated ER proteins in HEK293T WT and indicated KO cells. Protein expression was detected by WB. GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; HSPA5 was detected by anti-FLAG; CANX was detected by anti-HA. C GP was expressed with PDIA3, CALR, or CANX in HEK293T cells. Cells were treated with indicated amounts of Tizoxanide (TIZ), and protein expression was determined by WB. GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA.

Article Snippet: Cells were then incubated with rabbit polyclonal anti-Zaire EBOV-GP (Sino Biological, 40442-T48) at 1:1000 dilution and mouse polyclonal anti-HA (Sigma-Aldrich, H3663) in PBS with 5% BSA for 2 h. After being washed 5 times with PBS, cells were stained with Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Invitrogen, A31573) at 1:700 dilution for 1 h. Cells were washed again with PBS 5 times and then incubated with DAPI for 3 min for nuclear staining.

Techniques: Expressing

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A GP was expressed with CALR or CANX in HEK293T cells and treated with 20 μM lactacystin (Lac), 50 μM kifunensine (KIF), or 10 μM eeyarestatin I (EerI). DMSO was used as a vehicle control. Protein expression was detected by WB. GP was detected by anti-EBOV-GP; CALR and CANX were detected by anti-Myc or anti-HA. B GP was expressed with CALR or CANX in HEK293T cells and treated with 20 μM Lac, 20 μM MG132, 100 nM bafilomycin A1 (BafA1), 20 nM concanamycin A (ConA), or 20 mM NH 4 Cl. DMSO was used as a vehicle control. Protein expression was detected by WB. GP was detected by anti-EBOV-GP; CALR and CANX were detected by anti-Myc or anti-HA. C GP was expressed with CALR or CANX in HEK293T cells and treated with 10 mM 3-methyladenine (3-MA), 20 μM LY294002 (LY), or 100 nM wortmannin (Wort). DMSO was used as a vehicle control (Ctrl). Protein expression was detected by WB. GP was detected by anti-EBOV-GP; CALR and CANX were detected by anti-Myc or anti-HA. D GP was expressed with CALR or CANX in HEK293T cells in the presence of LAMP-2A -siRNA. Protein expression was analyzed by WB. GP was detected by anti-EBOV-GP; CALR and CANX were detected by anti-Myc or anti-HA; LAMP-2A was detected by its specific antibody. E GP was expressed with CALR or CANX in HEK293T WT and ATG3 - or ATG5 -KO cells. Protein expression was analyzed by WB. GP was detected by anti-EBOV-GP; CALR and CANX were detected by anti-Myc or anti-HA; LC3 was detected by its specific antibody. F GP was expressed with CALR or CANX in indicated WT and SQSTM1 -KO cells. Protein expression was analyzed by WB. GP was detected by anti-EBOV-GP; CALR and CANX were detected by anti-Myc or anti-HA; SQSTM1 was detected by its specific antibody. G GP was expressed with CALR or CANX in A549 WT and HDAC6 -KO cells. Protein expression was analyzed by WB. GP was detected by anti-EBOV-GP; CALR and CANX were detected by anti-Myc or anti-HA; HDAC6 was detected by its specific antibody. H GP was expressed with CALR or CANX in HEK293T cells. Cells were treated with 50 μM cycloheximide (CHX) in the presence or absence of 100 nM BafA1. Cells were collected at indicated time points and protein expression was detected by WB. Levels of GP expression were quantified from the blots by ImageJ and are presented as relative values, with the values from time 0 set to 1. Error bars represent SEMs calculated from three independent experiments. I GP with a C-terminal GFP tag (GP-GFP) was expressed with LAMP1 that has a dsRed tag in the presence or absence of CALR or CANX in HeLa cells. Cells with ectopic CALR or CANX were treated with DMSO, 100 nM BafA1, or 20 mM NH 4 Cl. The co-localization of EBOV-GP with LAMP1 was determined by confocal microscopy. The scale bar denotes 5 μm.

Article Snippet: Cells were then incubated with rabbit polyclonal anti-Zaire EBOV-GP (Sino Biological, 40442-T48) at 1:1000 dilution and mouse polyclonal anti-HA (Sigma-Aldrich, H3663) in PBS with 5% BSA for 2 h. After being washed 5 times with PBS, cells were stained with Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Invitrogen, A31573) at 1:700 dilution for 1 h. Cells were washed again with PBS 5 times and then incubated with DAPI for 3 min for nuclear staining.

Techniques: Expressing, Confocal Microscopy

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A RNF26 with a C-terminal mCherry tag was expressed with CALR or TGOLN2 (Trans-Golgi network integral membrane protein 2) that has a GFP-tag in HeLa cells. The RNF26 subcellular localization was determined by confocal microscopy. B EBOV-GP 1,2 (GP) with a C-terminal GFP tag was expressed with RNF26-mCherry in HeLa cells, and their co-localization was determined by confocal microscopy. C GP 1,2 proteins from indicated ebolaviruses were expressed with RNF26 in HEK293T cells. Protein expression was detected by WB. GPs were detected by anti-EBOV-GP; RNF26 was detected by anti-FLAG. D A schematic diagram of RNF26 is presented on the top. Five transmembrane (TM1–TM5) domains and a RING-finger are shown. Five RNF26 deletion mutants that target indicated regions and four point-mutation mutants that target each of C395, C399, or C401 are indicated. E EBOV-GP 1,2 (GP) was expressed with RNF26 WT and its mutants in HEK293T cells. Protein expression was determined by WB. GP was detected by anti-EBOV-GP; RNF26 proteins were detected by anti-FLAG. F EBOV-GP 1,2 (GP) with a HiBiT tag was expressed with RNF26 WT and its mutants in HEK293T cells. RNF26 proteins were immunoprecipitated and their interactions with GP was analyzed by WB. GP was detected by anti-EBOV-GP; RNF26 proteins were detected by anti-FLAG; GFP was detected by its specific antibody. G RNF26-VN and EBOV-GP 1,2 -VC were expressed with CALR that has a C-terminal Blue Fluorescent Protein (BFP) tag in HeLa cells. RNF26 was stained with a red-fluorescent antibody. The subcellular localization of the RNF26-GP complex was determined by confocal microscopy. The scale bar in A , B , and G denotes 5 μm.

Article Snippet: Cells were then incubated with rabbit polyclonal anti-Zaire EBOV-GP (Sino Biological, 40442-T48) at 1:1000 dilution and mouse polyclonal anti-HA (Sigma-Aldrich, H3663) in PBS with 5% BSA for 2 h. After being washed 5 times with PBS, cells were stained with Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Invitrogen, A31573) at 1:700 dilution for 1 h. Cells were washed again with PBS 5 times and then incubated with DAPI for 3 min for nuclear staining.

Techniques: Confocal Microscopy, Expressing, Mutagenesis, Immunoprecipitation, Staining

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A Three RNF26 -KO cell lines (1-E4, 3-B8, 3-F10) were generated from HEK293T cells using CRISPR/Cas9. RNF26 expression in these cells was determined by WB using its specific antibody. B EBOV and RSTV GP 1,2 proteins were expressed in HEK293T WT and RNF26 -KO (1-E4) cells. GP expression was detected by WB using anti-EBOV-GP. C EBOV-GP 1,2 (GP) was expressed with PDIA3, CALR, or CANX in HEK293T WT and RNF26 -KO (1-E4) cells. Protein expression was detected by WB. GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA. D RNF26 was expressed with GFP, PDIA3, CALR, or CANX in HEK293T cells. RNF26 was co-immunoprecipitated (IP) from cell lysate (Input) and its interactions with these proteins was determined by WB. GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA; RNF26 was detected by anti-FLAG; GFP was detected by its specific antibody. E GP was expressed with His-tagged ubiquitin (Ub) and PDIA3, CALR, CANX, and RNF26 or its catalytically inactive mutant (3C/3S) in HEK293T cells. GP proteins were immunoprecipitated and GP polyubiquitination was determined by WB. GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA; Ub was detected by anti-His.

Article Snippet: Cells were then incubated with rabbit polyclonal anti-Zaire EBOV-GP (Sino Biological, 40442-T48) at 1:1000 dilution and mouse polyclonal anti-HA (Sigma-Aldrich, H3663) in PBS with 5% BSA for 2 h. After being washed 5 times with PBS, cells were stained with Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Invitrogen, A31573) at 1:700 dilution for 1 h. Cells were washed again with PBS 5 times and then incubated with DAPI for 3 min for nuclear staining.

Techniques: Generated, CRISPR, Expressing, Immunoprecipitation, Mutagenesis

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A GP was expressed with Ub or its mutants bearing indicated single K-to-R mutation, and PDIA3, CALR, or CANX, in HEK293T cells. GP proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. B GP was expressed with Ub or its mutants expressing indicated single lysine residue, and PDIA3, CALR, or CANX, in HEK293T cells. GP proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. In A and B , GP was detected by anti-EBOV-GP; PDIA3, CALR, and CANX were detected by anti-Myc; Ub was detected by anti-HA.

Article Snippet: Cells were then incubated with rabbit polyclonal anti-Zaire EBOV-GP (Sino Biological, 40442-T48) at 1:1000 dilution and mouse polyclonal anti-HA (Sigma-Aldrich, H3663) in PBS with 5% BSA for 2 h. After being washed 5 times with PBS, cells were stained with Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Invitrogen, A31573) at 1:700 dilution for 1 h. Cells were washed again with PBS 5 times and then incubated with DAPI for 3 min for nuclear staining.

Techniques: Mutagenesis, Immunoprecipitation, Expressing

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A GP was expressed with His-tagged Ub in the presence of indicated E3 Ub ligases or their catalytically inactive mutants in HEK293T cells. GP and E3 proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. GP was detected by anti-EBOV-GP; RNF26, RNF185, and TRIM25 were detected by anti-FLAG; MARCH8 was detected by anti-HA; Ub was detected by anti-His. B GP was expressed with indicated proteins in HEK293T cells. Protein expression was determined by WB. GP, RNF26, RNF185, TRIM25, and MARCH8 were detected as in A ; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA. C GP was expressed with RNF185 or RNF26 and treated with indicated inhibitors as we did previously. Protein expression was determined by WB. GP was detected by anti-EBOV-GP; RNF26 and RNF185 proteins were detected by anti-FLAG. D A schematic diagram of RNF185 is presented. Two transmembrane (TM1, TM2) domains and a RING-finger are shown. Indicated RNF26 mutants were constructed. E GP was expressed with RNF185 and its mutants in HEK293T cells. Protein expression was determined by WB. GP was detected by anti-EBOV-GP; RNF185 proteins were detected by anti-FLAG. F GP was expressed with GFP, RNF185, and indicated RNF185 mutants in HEK293T cells. GFP and RNF185 proteins were immunoprecipitated and their interaction with GP was analyzed by WB. GP was detected by anti-EBOV-GP; GFP and RNF185 proteins were detected by anti-FLAG. G GP was expressed with Ub with a His-tag and RNF185 or its indicated mutants in HEK293T cells. GP and RNF185 proteins were immunoprecipitated and GP polyubiquitination was determined by WB. GP was detected by anti-EBOV-GP; RNF185 proteins were detected by anti-FLAG; Ub was detected by anti-His.

Article Snippet: Cells were then incubated with rabbit polyclonal anti-Zaire EBOV-GP (Sino Biological, 40442-T48) at 1:1000 dilution and mouse polyclonal anti-HA (Sigma-Aldrich, H3663) in PBS with 5% BSA for 2 h. After being washed 5 times with PBS, cells were stained with Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Invitrogen, A31573) at 1:700 dilution for 1 h. Cells were washed again with PBS 5 times and then incubated with DAPI for 3 min for nuclear staining.

Techniques: Immunoprecipitation, Expressing, Construct

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A GP and its mutants K673A or ∆CT were expressed with indicated E3 Ub ligases in HEK293T cells. GP and E3 proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. GP was detected by anti-EBOV-GP; RNF26, RNF185, and TRIM25 were detected by anti-FLAG; MARCH8 was detected by anti-HA; Ub was detected by anti-His. B GP and its two mutants were expressed with His-tagged Ub in the presence of PDIA3, CALR, or CANX in HEK293T cells. GP proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA; Ub was detected by anti-His. C GP and its two mutants were expressed with indicated E3 ubiquitin ligases or ER proteins in HEK293T cells. Protein expression was determined by WB. GP was detected by anti-EBOV-GP; MARCH8 and CANX were detected by anti-HA; RNF26, RNF185, and TRIM25 were detected by anti-FLAG; PDIA3 and CALR were detected by anti-Myc. D RNF185 was expressed with a control (Ctrl) or RNF185 -specific siRNAs in HEK293 cells. RNF185 expression was determined by WB using anti-FLAG. E GP was expressed with PDIA3, CALR, or CANX in the presence of Ctrl or RNF185 -specific siRNAs in HEK293T cells. GP expression was determined by WB using anti-EBOV-GP. F GP was expressed with CALR, CANX, or PDIA3, and indicated siRNAs in HEK293T cells. GP expression was determined by WB using anti-EBOV-GP.

Article Snippet: Cells were then incubated with rabbit polyclonal anti-Zaire EBOV-GP (Sino Biological, 40442-T48) at 1:1000 dilution and mouse polyclonal anti-HA (Sigma-Aldrich, H3663) in PBS with 5% BSA for 2 h. After being washed 5 times with PBS, cells were stained with Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Invitrogen, A31573) at 1:700 dilution for 1 h. Cells were washed again with PBS 5 times and then incubated with DAPI for 3 min for nuclear staining.

Techniques: Immunoprecipitation, Expressing