Journal: Virology Journal

Article Title: SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication

doi: 10.1186/s12985-024-02460-5

Figure Lengend Snippet: List of primers used in this study for the determination of cellular and viral transcript level by quantitative Real-time polymerase chain reaction

Article Snippet: Plasmids (pcDNA3.1-N protein, pcDNA-HDAC6-FLAG and pcDNA-HDAC6.DC-FLAG) were transfected in A549 cells using Lipofectamine 3000 (Invitrogen, USA).

Techniques: Sequencing

Journal: Virology Journal

Article Title: SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication

doi: 10.1186/s12985-024-02460-5

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Plasmids (pcDNA3.1-N protein, pcDNA-HDAC6-FLAG and pcDNA-HDAC6.DC-FLAG) were transfected in A549 cells using Lipofectamine 3000 (Invitrogen, USA).

Techniques:

Journal: Virology Journal

Article Title: SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication

doi: 10.1186/s12985-024-02460-5

Figure Lengend Snippet: Inhibition of HDAC6 resulted in reduction of SARS CoV-2 replication. A A549 cells were either transfected with shHDAC6 or pcDNA-HDAC6-FLAG followed by SARS-CoV-2 infection (at a MOI = 1). Total RNA was isolated after 24 h of infection followed by qRT-PCR using SARS-CoV-2 orf1a, hdac6 and gapdh -specific primers. Data represent means ± SD of three independent experiments. *** p ≤ .001, unpaired t-test and ** p ≤ .01, unpaired t-test. B Protein was extracted from SARS-CoV-2 infected cells at 24 hpi (MOI = 1) ectopically expressing shHDAC6 or pcDNA-HDAC6-FLAG. S, NSP13 and HDAC6 expression levels were analysed via western blotting. C SARS-CoV-2 infected (24 hpi) A549 cells were treated with increasing doses of tubacin (0–2.5 μM). Cellular extracts were subjected to immunoblot to analyse levels of S and NSP13. GAPDH was used as internal loading control. D Total RNA was isolated from tubacin treated SARS-CoV-2 infected cells (MOI = 1) at 24 hpi followed by qRT-PCR using SARS-CoV-2 orf1a and gapdh specific primers. Data represent means ± SD of three independent experiments. *** p ≤ .001, unpaired t-test and ** p ≤ .01, unpaired t-test. E A549 cells were either transfected with empty vector or pcDNA-HDAC6-FLAG or pcDNA-HDAC6.DC-FLAG followed by SARS-CoV-2 infection (at a MOI = 0.5). Total RNA was isolated after 24 h of infection followed by qRT-PCR using SARS-CoV-2 orf1a , hdac6 and gapdh -specific primers. Data represent means ± SD of three independent experiments. *** p ≤ 0.001, unpaired t-test, * p ≤ 0.1, unpaired t-test and ns = not significant, unpaired t-test. F A549 cells were infected with SARS-CoV-2 (MOI = 1) either in presence or absence of tubacin (1–2.5 μM) for 24, 48 and 72 h followed by plaque assay. Viral titers were measured as plaque forming units [log (pfu/ml)]. Data represent means ± SD of three independent experiments. ** p ≤ .01,multiple t-tests,***p ≤ .001, multiple t-tests and ns = not significant, multiple t-tests. G A549 cells were either transfected with empty vector or shHDAC6 or pcDNA-HDAC6-FLAG or pcDNA-HDAC6.DC-FLAG followed by SARS-CoV-2 infection (at a MOI = 0.5) for 24, 48 and 72 h followed by plaque assay. Viral titers were measured as plaque forming units [log (pfu/ml)]. Data represent means ± SD of three independent experiments. *** p ≤ .001, multiple t-tests, * p ≤ 0.05, multiple t-tests and ns = not significant, multiple t-tests

Article Snippet: Plasmids (pcDNA3.1-N protein, pcDNA-HDAC6-FLAG and pcDNA-HDAC6.DC-FLAG) were transfected in A549 cells using Lipofectamine 3000 (Invitrogen, USA).

Techniques: Inhibition, Transfection, Infection, Isolation, Quantitative RT-PCR, Expressing, Western Blot, Control, Plasmid Preparation, Plaque Assay

Journal: Virology Journal

Article Title: SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication

doi: 10.1186/s12985-024-02460-5

Figure Lengend Snippet: Increased interaction between G3BP1 and HDAC6 during SARS-CoV-2 infection. A Cellular lysates from A549 cells either infected with SARS-CoV-2 at a MOI of 2 (18–32 hpi) or kept mock-infected were run on SDS-PAGE and protein level expression of acetylated α-tubulin, HDAC6 and NSP13 protein was analysed via western blotting. B SARS-CoV-2-infected (18–32 hpi) or mock-infected lysates were pulled down using HDAC6-specific antibody followed by western blot analysis of the immunoprecipitates by anti-G3BP1, anti-HSP90 and anti-NSP13 antibodies. Inputs were probed with anti-HSP90, anti-G3BP1, anti-HDAC6, anti-NSP13 and β-actin antibodies confirming protein expression. ( C ) Reciprocal co-immunoprecipitation analysis was carried out in SARS-CoV-2-infected (18–32 hpi) or mock-infected lysates; anti-G3BP1 was used for pull down and immunoblot analysis was performed with HDAC6-specific antibody. Inputs were probed with anti-G3BP1, anti-HDAC6 and β-actin antibodies

Article Snippet: Plasmids (pcDNA3.1-N protein, pcDNA-HDAC6-FLAG and pcDNA-HDAC6.DC-FLAG) were transfected in A549 cells using Lipofectamine 3000 (Invitrogen, USA).

Techniques: Infection, SDS Page, Expressing, Western Blot, Immunoprecipitation

Journal: Virology Journal

Article Title: SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication

doi: 10.1186/s12985-024-02460-5

Figure Lengend Snippet: SARS-CoV-2 N Protein interacts with HDAC6. A Cellular lysates from SARS-CoV-2-infected or mock-infected A549 cells were co-immunoprecipitated with anti-HDAC6 antibody. Western blot analysis was performed to check the expression of N protein and SARS-CoV-2 Spike protein within the HDAC6 immunoprecipitates. Inputs were probed with anti-HDAC6, anti-N and anti-Spike antibodies confirming protein expression. B A549 cells were either transfected with pcDNA3.1-N plasmid or empty vector followed by immunoprecipitation with anti-HDAC6 antibody. Immunoblot analysis was performed with anti-N protein and anti-HDAC6 antibody. C Reciprocal co-immunoprecipitation assay from A549 cells either transfected with pcDNA3.1-N or empty vector using anti-N protein antibody was performed. Immunoprecipitates were analysed for the expression of HDAC6 and N protein. Inputs were probed with anti-HDAC6, anti-N and anti-β-actin antibodies confirming protein expression

Article Snippet: Plasmids (pcDNA3.1-N protein, pcDNA-HDAC6-FLAG and pcDNA-HDAC6.DC-FLAG) were transfected in A549 cells using Lipofectamine 3000 (Invitrogen, USA).

Techniques: Infection, Immunoprecipitation, Western Blot, Expressing, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: Virology Journal

Article Title: SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication

doi: 10.1186/s12985-024-02460-5

Figure Lengend Snippet: SARS-CoV-2 N Protein induces expression of HDAC6. A Cellular lysates were prepared from A549 cells either transfected with increasing concentration of pcDNA3.1-N (1–3 μg) or transfected with empty pcDNA3.1 vector. Expression of HDAC6, N protein and GAPDH was analysed via western blotting. B Co-immunoprecipitation analysis was performed in A549 cells either pcDNA3.1-N protein-transfected or empty vector-transfected A549 cells using anti-G3BP1 antibody and immunoblot analysis was performed using HDAC6 and N protein-specific antibodies. C Total RNA was isolated from A549 cells either transfected with empty vector or pcDNA3.1-N protein followed by qRT-PCR using hdac6 and gapdh -specific primers. ns = not significant, unpaired t-test. D A549 cells were either transfected with empty vector or pcDNA3.1 N protein followed by CHX treatment at 18 h post-transfection. Cells were harvested at indicated time post-CHX treatment (0, 6, 12, 18) and HDAC6, SARS-CoV-2 N protein and GAPDH expressions were analysed via immunoblotting. E A549 cells were either transfected with pcDNA3.1-N protein or empty vector followed by immunoprecipitation with anti-K48-ubiquitin antibody. Immunoblot analysis was performed with anti-HDAC6 antibody. F SARS-CoV-2-infected (24–32 hpi) or mock-infected lysates were pulled down using with anti-K48-ubiquitin antibody. Immunoblot analysis was performed with anti-HDAC6 antibody

Article Snippet: Plasmids (pcDNA3.1-N protein, pcDNA-HDAC6-FLAG and pcDNA-HDAC6.DC-FLAG) were transfected in A549 cells using Lipofectamine 3000 (Invitrogen, USA).

Techniques: Expressing, Transfection, Concentration Assay, Plasmid Preparation, Western Blot, Immunoprecipitation, Isolation, Quantitative RT-PCR, Infection

Journal: Virology Journal

Article Title: SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication

doi: 10.1186/s12985-024-02460-5

Figure Lengend Snippet: HDAC6 mediates interaction between G3BP1 and SARS-CoV-2 N protein. A A549 cells were transfected with pcDNA3.1-N and co-transfected with either pcDNA-HDAC6-FLAG or shHDAC6 were analysed by co-immunoprecipitation by using anti-G3BP1 antibody. Immunoprecipitates were subjected to western blot analysis using HDAC6 and SARS-CoV-2 N protein-specific antibody. B A549 cells transfected with pcDNA3.1-N plasmid either in presence or absence of tubacin (2.5 μM), followed by co-immunoprecipitation of cellular lysates with anti-SARS-CoV-2 N protein antibody. Immunoblot analysis was performed to check the expression of G3BP1 and HDAC6. C A549 cells were transfected with pcDNA3.1-N and co-transfected with either pcDNA-HDAC6-FLAG or pcDNA-HDAC6.DC-FLAG. Cellular lysates were subjected to co-immunoprecipitation using anti-G3BP1 antibody. Western blot analysis was performed using SARS-CoV-2 N protein and HDAC6-specific antibodies. Input lanes were probed with anti-G3BP1, anti-HDAC6 and anti-SARS-CoV-2 N antibodies. D A549 cells transfected with pcDNA3.1-N and either treated with tubacin (2.5 μM) or treated with DMSO. Cell lysates were subjected to immunoprecipitation using anti-Ac-Lys antibody. Western blot was performed using G3BP1 and HDAC6-specific antibodies

Article Snippet: Plasmids (pcDNA3.1-N protein, pcDNA-HDAC6-FLAG and pcDNA-HDAC6.DC-FLAG) were transfected in A549 cells using Lipofectamine 3000 (Invitrogen, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Expressing

Journal: Virology Journal

Article Title: SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication

doi: 10.1186/s12985-024-02460-5

Figure Lengend Snippet: Induction of HDAC6 expression during SARS-CoV-2 infection facilities disruption of Stress Granules. A A549 cells were transfected with pcDNA3.1-N protein and/or co-transfected with shHDAC6 followed by either tubacin (2.5 µM) or DMSO treatment. Cells were treated with NaAsO 2 1 h before harvesting. Co-immunoprecipitation was performed from the cellular lysates in using anti-TIA-1 antibody. Western blot was performed using PABP, N protein, G3BP1, HDAC6 and TIA-1-specific antibodies. B A549 cells were either transfected with pcDNA3.1-N protein or empty vector and either treated with tubacin (2.5 µM) or left untreated. 1 h before fixation cells were treated with NaAsO 2 followed by permeabilization and staining with anti-G3BP1 and anti-N protein primary antibody. Secondary staining was performed with Rhodamine-conjugated anti-mouse (for G3BP1) and DyLight™ 488-conjugated anti-rabbit (for N protein) secondary antibodies. DAPI was used for mounting. Imaging was done with a confocal microscope (63× oil immersion) and scale bar was set at 5 μm

Article Snippet: Plasmids (pcDNA3.1-N protein, pcDNA-HDAC6-FLAG and pcDNA-HDAC6.DC-FLAG) were transfected in A549 cells using Lipofectamine 3000 (Invitrogen, USA).

Techniques: Expressing, Infection, Disruption, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Staining, Imaging, Microscopy

Journal: Virology Journal

Article Title: SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication

doi: 10.1186/s12985-024-02460-5

Figure Lengend Snippet: SARS-CoV-2 infection leads to increased expression of HDAC6. A VERO E6 cells (at a MOI of 1), B Calu-3 cells (at a MOI of 1) and C A549 cell (at a MOI of 2) infected with SARS-CoV-2 or kept mock-infected were harvested at indicated time post-infection (18, 32, and 40 hpi) and cellular lysates were prepared. Lysates were run on SDS-PAGE followed by western blot analysis using anti-HDAC6 antibody. GAPDH and NSP13 were used as internal loading control and SARS-CoV-2 infection marker respectively

Article Snippet: Plasmids (pcDNA3.1-N protein, pcDNA-HDAC6-FLAG and pcDNA-HDAC6.DC-FLAG) were transfected in A549 cells using Lipofectamine 3000 (Invitrogen, USA).

Techniques: Infection, Expressing, SDS Page, Western Blot, Control, Marker

Journal: Virology Journal

Article Title: SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication

doi: 10.1186/s12985-024-02460-5

Figure Lengend Snippet: Schematic representation showing crosstalk between HDAC6 and SARS-CoV-2 infection. SARS-CoV-2 N protein interacts with and induces the expression of HDAC6. Subsequently, HDAC6 promotes the deacetylation of the N protein, which facilitates its association with G3BP1, leading to the disruption of cellular stress granules

Article Snippet: Plasmids (pcDNA3.1-N protein, pcDNA-HDAC6-FLAG and pcDNA-HDAC6.DC-FLAG) were transfected in A549 cells using Lipofectamine 3000 (Invitrogen, USA).

Techniques: Infection, Expressing, Disruption

Journal: The Journal of Clinical Investigation

Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

doi: 10.1172/JCI98765

Figure Lengend Snippet: (A) HEK293T cells were transfected with either empty vector (EV) or HA-FLAG(3×)-NEK2. Proteins binding to NEK2 were pulled down by tandem HA and FLAG antibodies and stained with silver prior to mass spectrometry. (B) ARP1 myeloma cells were lysed and NEK2 was immunoprecipitated using NEK2 antibodies. Western blots were probed with NEK2 and USP7 antibodies. FT, LW, and E represent flow through, last wash, and elution of the immunoprecipitation, respectively. (C) ARP1 myeloma cells were transduced with NEK2-HA plasmids. Transduced cells were lysed and NEK2 was immunoprecipitated using HA antibodies. Western blots were probed using NEK2 and USP7 antibodies. (D) H1299 cells were transfected with mock or USP7-FLAG overexpression vector. Endogenous NEK2 was immunoprecipitated and Western blots were analyzed using NEK2 and USP7 antibodies. (E) ARP1 myeloma cells transduced with EV + USP7-shRNA or NEK2-OE + USP7-shRNA were treated with doxycycline (DOX) or vehicle to suppress USP7 expression. After 72 hours, cells were treated with bortezomib (BTZ; 5 nM) for a further 24 hours and cell viability was measured using trypan blue stain. (F) OPM2 cells transduced with NEK2-shRNA were treated with DOX or vehicle to suppress NEK2 expression. ARP1 cells or OPM2 cells with or without silencing of NEK2 were treated with BTZ (2.5, 5, and 10 nM) for a further 24 hours and cell viability was measured using trypan blue stain. Viability experiments were performed in triplicate and a Student’s t test was performed and showed the significance at 10 nM with or without silencing of NEK2. *P < 0.05.

Article Snippet: The USP7-Flag vector was obtained from Addgene (plasmid 16655).

Techniques: Transfection, Plasmid Preparation, Binding Assay, Staining, Mass Spectrometry, Immunoprecipitation, Western Blot, Transduction, Over Expression, shRNA, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

doi: 10.1172/JCI98765

Figure Lengend Snippet: (A and B) Knockdown of USP7 decreases NEK2 protein. ARP1 (A) and OCI-MY5 (B) myeloma cells were transfected with EV, NEK2, or NEK2 + USP7-shRNA. After 72-hour induction with doxycycline, cells were lysed. NEK2 and USP7 protein levels were analyzed by Western blot. (C) OCI-MY5, Delta-47, JJN3, OPM2, and ARP1 myeloma cell lines were treated with 16 μM P5091 for 24 hours. Cells were lysed and NEK2 levels analyzed by Western blot. (D) H1299 cells were transfected with mock or USP7-FLAG–overexpressing vectors, lysed, and NEK2 and USP7 levels were determined by Western blot. (E) ARP1 myeloma cells were treated with the proteasome inhibitor MG132 (10 μM) alone for 30 minutes or in combination with P5091 (16 and 25 μM) for an additional 5 hours. Cells were lysed and NEK2 levels were analyzed by Western blot. (F) OPM2 cells were treated with or without P5091 (25 μM for 2 hours) and protein was extracted with lysis buffer supplemented with NEM. Endogenous NEK2 was immunoprecipitated and analyzed by Western blot using NEK2 and ubiquitin antibodies. FT, LW, and E represent flow through, last wash, and elution of the immunoprecipitation, respectively. (G) H1299 cells were transfected with EV and HA-ubiquitin (HA-UB) or FLAG-USP7 and HA-UB. Cells were lysed and endogenous NEK2 was immunoprecipitated (IP) by NEK2 antibodies and ubiquitination levels were analyzed by Western blot. The higher-molecular-weight band is nonspecific IgG. (H) H1299 cells were transfected with NEK2-OE, HA-UB, and FLAG-USP7 or NEK2-OE and HA-UB. Cells were lysed and total NEK2 protein, including both endogenous and exogenous, was immunoprecipitated (IP) by anti-NEK2 antibodies and ubiquitination levels were analyzed using HA antibodies by Western blot.

Article Snippet: The USP7-Flag vector was obtained from Addgene (plasmid 16655).

Techniques: Transfection, shRNA, Western Blot, Lysis, Immunoprecipitation, Molecular Weight

Journal: The Journal of Clinical Investigation

Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

doi: 10.1172/JCI98765

Figure Lengend Snippet: (A) Primary myeloma samples from 16 patients (Pts) were lysed and analyzed by Western blot using NEK2, p-p65-S536, total p65 (p65), USP7, and GAPDH antibodies. (B) CD138-positive myeloma cells isolated from 4 primary myeloma patients were mounted on cytospin slides and analyzed by immunofluorescence using NEK2 and p-p65-S536 antibodies. DAPI staining was used to visualize nuclei. Yellow arrowheads indicate myeloma cells coexpressing NEK2 and p-p65-S536. Blue arrowheads show myeloma cells expressing p-p65-S536 with undetectable NEK2 levels. (C) EV and NEK2-OE ARP1 cells were treated with vehicle, BAY11-7082 (0.5 or 1.0 μM), and bortezomib (5 nM) alone or in combination. After 48 hours, cell viability was assessed by trypan blue staining and Dunnett’s method was used to calculate the multiplicity-adjusted P values for each treatment and control group pair. **P = 0.0023; ****P = 0.0001. NS, no significance. Experiment was performed in triplicate. (D) A model for NEK2 deubiquitination and stabilization by interacting with USP7. USP7 prevents E3 ligase APC/C (30) to ubiquitinate NEK2 resulting in its stabilization.

Article Snippet: The USP7-Flag vector was obtained from Addgene (plasmid 16655).

Techniques: Western Blot, Isolation, Immunofluorescence, Staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

doi: 10.1172/JCI98765

Figure Lengend Snippet: (A) USP7 was knocked down in ARP1 cells transduced with NEK2-OE after 72 hours induction with doxycycline (DOX). Nuclear and cytosolic fractionations were carried out. p65 levels were analyzed between EV and NEK2-OE with or without USP7 shRNA by Western blot. β-Actin and histone H3 (H3) were used as cytosolic and nuclear markers, respectively. (B–D) EV and NEK2-OE ARP1, OCI-MY5, and H1299 cells were lysed. NEK2, p65-S536 phosphorylation, IKK phosphorylation, and IκBα were analyzed by Western blot. (E) H1299 cells transiently transfected with EV or NEK2-OE (WT) or NEK2-K37R mutant (NEK2-Dead) were lysed, and NEK2 and p65-S536 phosphorylation was analyzed by Western blot. (F) ARP1 and OCI-MY5 cells transfected with EV or NEK2-OE were treated with vehicle or MK-2206 2HCl, an AKT inhibitor, for 30 minutes and then cells were lysed. p65-S536 phosphorylation was analyzed by Western blot. (G) NEK2-shRNA ARP1 cells were induced with DOX for 48 hours and then treated with tautomycin, a PP1α inhibitor, for another 24 hours. NEK2, p-p65-S536, p-PP1α, and p-AKT were analyzed by Western blot.

Article Snippet: The USP7-Flag vector was obtained from Addgene (plasmid 16655).

Techniques: Transduction, shRNA, Western Blot, Transfection, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

doi: 10.1172/JCI98765

Figure Lengend Snippet: (A) EV and NEK2-OE ARP1 and OCI-MY5 cells were treated with vehicle or BSM-345541, and HSPE mRNA levels were analyzed by qRT-PCR. (B) HPSE mRNA levels were analyzed by qRT-PCR in EV, NEK2-OE, or NEK2-OE + USP7-shRNA ARP1 and OCI-MY5 myeloma cells. **P < 0.01 by Student’s t test. (C) ARP1 and (D) OPM2 cells were treated with P5091 (16 μM overnight), INH1 (25 μM for 24 hours), or NEK2-shRNA DOX (48 hours). Cells were lysed and proteins were analyzed by Western blot with NEK2, p-p65-S536, HPSE, and GAPDH antibodies.

Article Snippet: The USP7-Flag vector was obtained from Addgene (plasmid 16655).

Techniques: Quantitative RT-PCR, shRNA, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

doi: 10.1172/JCI98765

Figure Lengend Snippet: USP7 binds to and stabilizes NEK2 by deubiquitination, allowing it to accumulate in myeloma cells. Accumulated NEK2 binds to and phosphorylates PP1α, resulting in loss its AKT-suppressing activity. Active AKT triggers the canonical NF-κB pathway by phosphorylating IKK, with subsequent phosphorylation and degradation of IκBα. p65 released from the complex with IκBα translocates into the nucleus, where it activates its target genes leading to drug resistance in myeloma. Ub, ubiquitin.

Article Snippet: The USP7-Flag vector was obtained from Addgene (plasmid 16655).

Techniques: Activity Assay