8c12  (Bioss)

Journal: Nature Communications

Article Title: Linear ubiquitination regulates the KSHV replication and transcription activator protein to control infection

doi: 10.1038/s41467-024-49887-6

Figure Lengend Snippet: a , b iSLK/rKSHV.219 cells with OTULIN overexpression ( a ) or knockdown ( b ) were treated as indicated. RT-qPCR was performed to quantify the expression levels of KSHV lytic genes RTA , ORF59 , ORF9 , and ORF8.1 . a, n = 2 biological replicates and 2 technical replicates, * P = 0.02288, 0.03410, 0.00229, 0.02574 (Cntrl vs. Tet/VPA) for RTA , ORF59 , ORF9 , ORF8.1 ; #, P = 0.02796, 0.03488, 0.00233, 0.03357 (OTULIN+Tet/VPA vs. Tet/VPA) for RTA, ORF59, ORF9, ORF8.1 . b , n = 2 biological replicates and 2 technical replicates, * P = 0.03772, 0.03042, 0.00704 (siCntrl vs. siCntrl+Tet/VPA) for RTA, ORF59, ORF8.1 ; #, P = (0.03734, 0.02794, 0.03573), (0.03299, 0.02145), (0.00646, 0.01030), (0.00027, 0.00276) (siCntrl vs. siOTULIN) for RTA, ORF59, ORF9, ORF8.1 . c OTULIN inhibits RTA transcriptional activities in reporter assays. HEK293T cells were co-transfected with luciferase reporter plasmid pOri-Lyt-Luc, a Renilla luciferase control plasmid, together with or without RTA, in the presence of increased amount of OTULIN or OTULIN C129A , then harvested and their luciferase activities were quantified. n = 3 biological replicates and 2 technical replicates. d OTULIN inhibits, while HOIP enhances, RTA binding to the Ori-Lyt promoter. HEK293T cells were co-transfected with the reporter plasmid pOri-Lyt-Luc, RTA-Flag, and with Myc-OTULIN, Myc-HOIP, or Myc-OTULIN C129A as indicated. Cells were collected for ChIP assay using anti-Flag magnetic agarose beads. Precipitated Ori-Lyt promoter DNAs were quantified using qPCR. n = 2 biological replicates and 2 technical replicates. e , f OTULIN inhibits, while HOIP enhances, RTA binding to its target gene promoters. iSLK/rKSHV.219 cells were transfected with Myc-OTULIN, Myc-OTULIN C129A , or Myc-HOIP followed by treatment with Tet plus VPA. The cells were collected and subjected to ChIP assay using an anti-RTA antibody. The quantities of PAN ( e ) or ORF57 ( f ) promoter DNA in the precipitates were determined by qPCR, e , f , n = 2 biological replicates and 2 technical replicates. Data are displayed as mean ± s.d., two-sided t test. Source data are provided as a Source Data file.

Article Snippet: The antibodies used were as follows: RTA (8C12, Argene, 1:1000), Myc (Huabio, 0912-2, 1:1000), Flag (Huabio, 0912-1, AB_3068715), Histone H3 (Huabio, HA500298, AB_3071393), β-actin (Huabio, R1207-1, AB_3073201), Linear Ubiquitin (Millipore, MABS199, 1:1000), OTULIN (Abcam, ab151117, 1:1000) and GAPDH (Cell Signaling Technology, 2118, 1:10000).

Techniques: Over Expression, Knockdown, Quantitative RT-PCR, Expressing, Transfection, Luciferase, Plasmid Preparation, Control, Binding Assay

Journal: Nature Communications

Article Title: Linear ubiquitination regulates the KSHV replication and transcription activator protein to control infection

doi: 10.1038/s41467-024-49887-6

Figure Lengend Snippet: a RTA is modified by M1 ubiquitination. RTA-Flag and Ub KO were co-transfected into HEK293T cells and subjected to immunoprecipitation with anti-Flag magnetic beads. RTA M1 ubiquitination was determined by a specific anti-linear Ub antibody (Genentech). Ub KO is a ubiquitin mutant that can only form M1 polyUb chains. n = 3 independent experiments. b HOIP/HOIL-1L promotes RTA M1 ubiquitination. RTA-Flag and Myc-HOIP/Myc-HOIL-1L were co-transfected into HEK293T cells. RTA-Flag was immunoprecipitated with anti-Flag magnetic beads and detected with anti-linear Ub and RTA antibodies, respectively. n = 3 independent experiments. c OTULIN reduces RTA M1 ubiquitination. As in ( b ), but the plasmids were replaced with the indicated plasmids. n = 3 independent experiments. d Endogenous RTA displays M1 ubiquitination. iSLK/rKSHV.219 cells were transfected with Myc-OTULIN or Myc-OTULIN C129A and then treated with Tet plus VPA. RTA was immunoprecipitated with anti-RTA antibody and immunoblotted with anti-linear Ub and anti-RTA antibodies, respectively. n = 3 independent experiments. e Diagrams of RTA domains, the amino acid sequence of the RTA NLS2 and the Lysine (K) to Arginine (R) mutants used in the study. f RTA 2KR is defective in M1 ubiquitination. HEK293T cells were transfected with plasmids as indicated and cell lysates were immunoprecipitated using anti-Flag beads. Immunoprecipitated proteins were immunoblotted with the indicated antibodies. n = 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: The antibodies used were as follows: RTA (8C12, Argene, 1:1000), Myc (Huabio, 0912-2, 1:1000), Flag (Huabio, 0912-1, AB_3068715), Histone H3 (Huabio, HA500298, AB_3071393), β-actin (Huabio, R1207-1, AB_3073201), Linear Ubiquitin (Millipore, MABS199, 1:1000), OTULIN (Abcam, ab151117, 1:1000) and GAPDH (Cell Signaling Technology, 2118, 1:10000).

Techniques: Modification, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Magnetic Beads, Mutagenesis, Sequencing

Journal: Nature Communications

Article Title: Linear ubiquitination regulates the KSHV replication and transcription activator protein to control infection

doi: 10.1038/s41467-024-49887-6

Figure Lengend Snippet: a M1 ubiquitinated RTA is enriched in nucleus. HeLa cells were co-transfected with RTA-Flag and Myc-OTULIN and subjected to fractionation into cytoplasmic and nuclear portions. RTA in the cytoplasm and nucleus was immunoprecipitated with anti-Flag magnetic beads and immunoblotted with an anti-linear Ub antibody. n = 3 independent experiments. b , c OTULIN facilitates relocation of RTA from the nucleus to the cytoplasm. HeLa cells were transfected with RTA together with OTULIN or OTULIN C129A (b) or iSLK/rKSHV.219 cells were transfected with OTULIN or OTULIN C129A and treated with Tet plus VPA ( c ). Cells were then harvested for fractionation into cytoplasmic and nuclear portions and immunoblotted with the indicated antibodies. n = 3 independent experiments. d , e OTULIN alters the intracellular localization of RTA. HEK293T cells were transfected with RTA-Flag alone (top panels), co-transfected with Myc-OTULIN (middle panel image) or with Myc-OTULIN C129A (lower panel image). After 36 hours, cells were subjected to immunofluorescence analysis. Subcellular localizations of RTA (in green) and of OTULIN (in red) are shown in ( d ) and quantification is shown in ( e ). Scale bar: 10 μm. All cell nuclei in the fields were stained with Hoechst (blue). Image is representative of 110 or more cells per condition in total n = 3 independent experiments. f , g RTA 2KR is located in both the cytoplasm and the nucleus and OTULIN has no effect on its subcellular localization. HeLa cells were transfected with RTA 2KR in the presence or absence of OTULIN or OTULIN C129A and harvested for fractionation into cytoplasmic and nuclear portions and subjected to immunoblotting with the indicated antibodies ( f ). HEK293T cells transfected with the indicated plasmids were subjected to immunofluorescence staining to assess the subcellular localization of RTA 2KR (green). Cell nuclei in the fields were stained with Hoechst (blue) ( g ). Scale bar: 10 μm. Image is representative in total n = 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: The antibodies used were as follows: RTA (8C12, Argene, 1:1000), Myc (Huabio, 0912-2, 1:1000), Flag (Huabio, 0912-1, AB_3068715), Histone H3 (Huabio, HA500298, AB_3071393), β-actin (Huabio, R1207-1, AB_3073201), Linear Ubiquitin (Millipore, MABS199, 1:1000), OTULIN (Abcam, ab151117, 1:1000) and GAPDH (Cell Signaling Technology, 2118, 1:10000).

Techniques: Transfection, Fractionation, Immunoprecipitation, Magnetic Beads, Immunofluorescence, Staining, Western Blot

Journal: Nature Communications

Article Title: Linear ubiquitination regulates the KSHV replication and transcription activator protein to control infection

doi: 10.1038/s41467-024-49887-6

Figure Lengend Snippet: a OTULIN inhibits the transcriptional activities of EBV RTA and MHV68 RTA. HEK293T cells were transfected with the firefly luciferase reporter construct pGL3-Ori-Lyt-Luc, a Renilla luciferase control plasmid, RTA by EBV or MHV68, together with OTULIN or OTULIN C129A . After 36 h cells were harvested and subjected to measurement of luciferase activities, n = 2 biological replicates and 2 technical replicates, mean ± s.d., two-sided t test, * P = 0.00041 (Cntrl vs. EBV), * P = 0.00252 (Cntrl vs. MHV68) #, P = 0.00313 (EBV vs. EBV + OTULIN), &, P = 0.01841 (MHV68 vs. MHV68 + OTULIN). b , c OTULIN diminishes M1 ubiquitination of EBV RTA and MHV68 RTA. Flag tagged EBV RTA or MHV68 RTA, together with Myc-OTULIN or OTULIN C129A was expressed in HEK293T cells and immunoprecipitated with anti-Flag magnetic beads. M1 ubiquitination of EBV RTA and MHV68 RTA were determined by immunoblotting with a specific anti-linear Ub antibody. n = 3 independent experiments. d A model for the M1-polyubiquitination-mediated regulation of RTA’s role in KSHV lytic reactivation. LUBAC specifically ubiquitinates RTA on K516 and K518, which enables RTA translocation into nucleus where it can activate the expression of a lytic gene cascade that initiates KSHV lytic reactivation. Opposing this process, OTULIN can cleave the linear ubiquitination modification from RTA and thereby inhibit its nuclear translocation, thus inhibiting KSHV lytic reactivation. Source data are provided as a Source Data file.

Article Snippet: The antibodies used were as follows: RTA (8C12, Argene, 1:1000), Myc (Huabio, 0912-2, 1:1000), Flag (Huabio, 0912-1, AB_3068715), Histone H3 (Huabio, HA500298, AB_3071393), β-actin (Huabio, R1207-1, AB_3073201), Linear Ubiquitin (Millipore, MABS199, 1:1000), OTULIN (Abcam, ab151117, 1:1000) and GAPDH (Cell Signaling Technology, 2118, 1:10000).

Techniques: Transfection, Luciferase, Construct, Control, Plasmid Preparation, Ubiquitin Proteomics, Immunoprecipitation, Magnetic Beads, Western Blot, Translocation Assay, Expressing, Modification