Journal: PLOS Pathogens

Article Title: TRIM72 restricts lyssavirus infection by inducing K48-linked ubiquitination and proteasome degradation of the matrix protein

doi: 10.1371/journal.ppat.1011718

Figure Lengend Snippet: (A) pCAGGS or pCA-Trim72-myc together with pCA-N, pCA-P, pCA-M-HA, and pCA-G-flag were co-transfected into N2a cells respectively for 48 h, and the protein levels of N, P, M-HA and G-flag were analyzed by western blotting. (B) pCAGGS or pCA-Trim72-flag were transfected into N2a cells for 12 h, then infected with RABV (MOI = 1) for 36h and RABV-N and RABV-M protein levels were analyzed by western blotting. (C) pCAGGS or pCA-Trim72-flag together with pCA-M-HA were co-transfected into N2a cells respectively. The specific inhibitors for proteasome and lysosome, Mg132 (10 μM) and NH 4 Cl (5 mM) were applied. Then the protein levels of TRIM72 and M were analyzed by western blotting. (D) pCA-M-HA together with pCAGGS or different concentrations of pCA-Trim72-flag were co-transfected into N2a cells for 48 h. Then the protein levels of TRIM72-flag and M-HA were analyzed by western blotting. (E) pCA-M-HA together with pCAGGS or different concentrations of pCA-Trim72-flag were co-transfected into N2a cells. Then Mg132 (10 μM) was applied and the protein levels of TRIM72-flag and M-HA were analyzed by western blotting. (F) pCA-M-HA together with pCAGGS, pCA-Trim72-flag, or Trim72-ΔRING-flag were co-transfected into N2a cells respectively for 48 h, then M-HA level was analyzed by western blotting. (G) pCAGGS or pCA-Trim72-myc together with pCA-M-flag and UB-HA over-expression vectors were co-transfected into N2a cells respectively. Then treated with Mg132 and Co-IP assays were performed with anti-flag antibody. The protein levels of TRIM72-myc, M-flag, and UB-HA were analyzed by western blotting. (H) pCAGGS or pCA-Trim72-flag were transfected into N2a cells respectively. Then infected with RABV (MOI = 1) for 36 h and treated with Mg132 and Co-IP assays were performed with anti-M antibody. The protein levels of TRIM72-flag, M, and UB were analyzed by western blotting. (I) pCAGGS or pCA-Trim72-myc together with pCA-M-flag and UB-K48-HA over-expression vectors were co-transfected into N2a cells respectively. Then treated with Mg132 and Co-IP assays were performed with anti-flag antibody post transfection for 48 h. Then the protein levels of TRIM72-myc, M-flag, and UB-K48-HA were analyzed by western blotting. (J) pCAGGS or pCA-Trim72-myc together with pCA-M-flag and UB-K63-HA over-expression vectors were co-transfected into N2a cells respectively. Then treated with Mg132 and Co-IP assays were performed with anti-flag antibody. The protein levels of TRIM72-myc, M-flag, and UB-K63-HA were analyzed by western blotting. Western blot data are representative of at least two independent experiments.

Article Snippet: The primary antibodies were against RABV N protein (prepared by our lab, 1:5000), RABV P protein (prepared by our lab, 1:5000), RABV M protein (LSbio, LS-C369074), TRIM72 (Boster, A06982-2 ), Flag tag (MBL, M185-3L, 1:10000), HA tag (MBL, M180-3, 1:10000), Myc tag (MBL, M192-3, 1:10000), or GAPDH (ProteinTech, 60004-1-Ig, 1:5000).

Techniques: Transfection, Western Blot, Infection, Over Expression, Co-Immunoprecipitation Assay

Journal: PLOS Pathogens

Article Title: TRIM72 restricts lyssavirus infection by inducing K48-linked ubiquitination and proteasome degradation of the matrix protein

doi: 10.1371/journal.ppat.1011718

Figure Lengend Snippet: (A) pCAGGS or pCA-Trim72-flag together with pCA-M-HA were co-transfected into N2a cells respectively. Then Mg132 (10 μM) was treated, and Co-IP assays were performed with anti-flag antibody post-transfection for 48 h. The protein levels of TRIM72-flag and M-HA were analyzed by western blotting. (B) pCAGGS or pCA-M-flag together with pCA-Trim72-HA were co-transfected into N2a cells respectively for 48 h. Then Co-IP assays were performed with anti-flag antibody. The protein levels of TRIM72-flag and M-HA were analyzed by western blotting. (C) N2a cells were infected with RABV (MOI = 1) for 36 h. Then Co-IP assays were performed with anti-TRIM72 antibody. The protein levels of TRIM72 and M were analyzed by western blotting. (D) TRIM72 truncations were designed and constructed based on its functional domain. (E) The over-expression vectors of TRIM72-flag truncations together with pCA-M-HA were co-transfected into N2a cells respectively for 48 h. Then Co-IP assays were performed with anti-flag antibody. The protein levels of TRIM72 truncations and M-HA were analyzed by western blotting. (F) RABV-M truncations were designed and constructed based on its secondary structure. (G) The over-expression vectors of M-HA truncations together with pCA-Trim72-SPRY-flag were co-transfected into N2a cells for 48 h. Then Co-IP assays were performed with anti-HA antibody. The protein levels of M truncations and SPRY-flag were analyzed by western blotting. (H) A structural model of RABV-M was built using SWISS-MODEL online software ( https://swissmodel.expasy.org/interactive ) based on the crystal structure of Lagos bat virus M protein (PDB code: 2W2S). (I) An interaction model of the human TRIM72-SPRY domain and RABV-M was built with GalaxyWEB online software ( https://galaxy.seoklab.org/ ) based on hTRIM72 structure (PDB code: 6NPY), the potential interaction sites were labeled. (J) Over-expression vectors of RABV-M-HA mutations together with TRIM72-SPRY-flag over-expression vector were co-transfected into N2a cells respectively for 48 h. Then Co-IP assays were performed with anti-HA antibody. The protein levels were analyzed by western blotting. (K) The protein sequences of SPRY domain between hTRIM72 and mouse TRIM72 were compared and analyzed with ESPript 3.0 online software ( https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi ). (L) Over-expression vectors of TRIM72-SPRY-flag mutations together with M-HA over-expression vectors were co-transfected into N2a cells respectively for 48 h. Then Co-IP assays were performed with anti-flag antibody and protein levels were analyzed by western blotting. (M) Over-expression vectors of TRIM72-flag mutations together with M-HA over-expression vector were co-transfected into N2a cells respectively for 48 h. The protein levels were analyzed by western blotting. (N) Over-expression vectors of TRIM72-flag mutations or empty vectors were transfected into N2a cells respectively for 12 h, then infected with rRABV (MOI = 0.01) for 48 h and the viral titers in the supernatants were analyzed. Statistical analysis of grouped comparisons was carried out by student’s t-test (*P < 0.05; **P<0.01; ***P<0.001; ****P<0.0001). The bar graph represents means ± SD, n = 3. Western blot data are representative of at least two independent experiments.

Article Snippet: The primary antibodies were against RABV N protein (prepared by our lab, 1:5000), RABV P protein (prepared by our lab, 1:5000), RABV M protein (LSbio, LS-C369074), TRIM72 (Boster, A06982-2 ), Flag tag (MBL, M185-3L, 1:10000), HA tag (MBL, M180-3, 1:10000), Myc tag (MBL, M192-3, 1:10000), or GAPDH (ProteinTech, 60004-1-Ig, 1:5000).

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Infection, Construct, Functional Assay, Over Expression, Software, Virus, Labeling, Plasmid Preparation

Journal: PLOS Pathogens

Article Title: TRIM72 restricts lyssavirus infection by inducing K48-linked ubiquitination and proteasome degradation of the matrix protein

doi: 10.1371/journal.ppat.1011718

Figure Lengend Snippet: (A) Illustration of the lysine (K) sites in RABV-M. (B) The lysine residues in RABV-M were mutated to alanine and over-expression vectors were constructed and together with pCAGGS or pCA-Trim72-flag were co-transfected into N2a cells respectively for 48 h. The protein levels of M-HA mutations and TRIM72-flag were analyzed by western blotting. (C) WT M-HA or M-K195A-HA was over-expressed in N2a cells respectively for 48 h, and the protein level was analyzed by western blotting. (D) pCA-M-flag or pCA-M-K195A-flag together with UB-HA and pCAGGS or pCA-Trim72-myc were co-transfected into N2a cells and treated with Mg132 (10 μM), then Co-IP assays were performed with anti-flag antibody. The protein levels were analyzed by western blotting. (E) pCAGGS or pCA-Trim72-myc together with pCA-M-K195A-HA and pCA-G-flag were co-transfected into HEK-293T cells respectively for 48 h, the protein levels of M-K195A-HA and G-flag in the supernatants and cells were analyzed by western blotting. (F) Schematic diagram of the recombinant rRABV-M-K195A. (G) N2a cells were infected with rRABV or rRABV-M-K195A (MOI = 0.01) for the indicated time and their growth kinetics were compared. (H) pCAGGS or pCA-Trim72-flag was transfected into N2a cells respectively for 12 h, then infected with WT rRABV (MOI = 0.01) or rRABV-M-K195A (MOI = 0.01) for 48 h. The supernatants were collected and viral titers were analyzed. Statistical analysis of grouped comparisons was carried out by student’s t-test (*P < 0.05; **P<0.01; ***P<0.001; ****P<0.0001). The bar graph represents means ± SD, n = 3. Western blot data are representative of at least two independent experiments.

Article Snippet: The primary antibodies were against RABV N protein (prepared by our lab, 1:5000), RABV P protein (prepared by our lab, 1:5000), RABV M protein (LSbio, LS-C369074), TRIM72 (Boster, A06982-2 ), Flag tag (MBL, M185-3L, 1:10000), HA tag (MBL, M180-3, 1:10000), Myc tag (MBL, M192-3, 1:10000), or GAPDH (ProteinTech, 60004-1-Ig, 1:5000).

Techniques: Over Expression, Construct, Transfection, Western Blot, Co-Immunoprecipitation Assay, Recombinant, Infection