virf1  (GenScript corporation)

Journal: Cell Death and Differentiation

Article Title: Lactylation of NAT10 promotes N 4 ‐acetylcytidine modification on tRNA Ser-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation

doi: 10.1038/s41418-024-01327-0

Figure Lengend Snippet: A The total RNAs from iSLK-KSHV NAT10 +/- cells (NAT10 +/- ) and iSLK-KSHV NAT10 +/+ cells (WT) were subjected to dot blot assay with anti-ac 4 C antibody. Methylene blue staining was used as the internal control. B The heatmap for differentially expressed tRNAs in cells shown as in ( A ) by tRNA acRIP-seq analysis. The pseudo-color represented as the fold enrichment (IP/Input) of WT and NAT10 +/− group, respectively. Any tRNAs not detected in tRNA acRIP-seq were plotted as white in the heatmap. C The total RNAs from cells shown as in ( A ) were subjected to Northern blot with probes to tRNA Ser-CGA-1-1 , tRNA Ser-CGA-4-1 , tRNA Leu-TAG-3-1 , tRNA Ser-AGA , tRNA Leu-TAA , and U6 as the control. D The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, ORF45, ORF57, vIRF1, vIL-6, vBCL-2, ORF65, and K8.1) in cells shown as in ( A ). *** P < 0.001 by Student’s t -test. E Schematic representation of the acetylation site mutation of tRNA Ser-CGA-1-1 (Upper) and tRNA Leu-TAG-3-1 (Lower). The C (marked in red) at position 12 was mutated to U (marked in dark blue). F The total RNAs from iSLK-KSHV cells with overexpression of the wild type tRNA Ser-CGA-1-1 or tRNA Leu-TAG-3-1 (WT), mutant tRNA Ser-CGA-1-1 or tRNA Leu-TAG-3-1 (Mut), and their control empty vector ( EV ) for 48 h were respectively subjected to Northern blot with probes to tRNA Ser-CGA-1-1 , tRNA Leu-TAG-3-1 and U6 as the control. G . The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, vIRF1, vIL-6, vBCL-2, and ORF65) in iSLK-KSHV cells with overexpression of the wild type (tRNA Ser-CGA-1-1 -WT) or its mutant (tRNA Ser-CGA-1-1 -Mut) tRNA Ser-CGA-1-1 for 48 h, and their control (tRNA-EV). *** P < 0.001 by Student’s t -test. H The expression levels of vIRF1 and NAT10 in iSLK-KSHV cells with overexpression of the wild type (WT), mutant (Mut) tRNA Ser-CGA-1-1 , and their control (EV) for 48 h were detected by Western blot. I The SDS gel analysis of the in vitro translation reaction supplemented with the wild type (tRNA Ser-CGA-1-1 -WT) or the mutant (tRNA Ser-CGA-1-1 -Mut) tRNA Ser-CGA-1-1 , in comparison with reaction mixtures that not containing mRNA (No mRNA). J By the assessment of ORF26, real-time DNA-PCR for cells shown as in ( H ) was performed to detect viral copy number after doxycycline stimulation for 72 h. *** P < 0.001 by Student’s t -test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibody was from Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine (Baltimore, Maryland, USA) [ – ].

Techniques: Dot Blot, Staining, Control, Northern Blot, Quantitative RT-PCR, Mutagenesis, Over Expression, Plasmid Preparation, Expressing, Western Blot, SDS-Gel, In Vitro, Comparison

Journal: Cell Death and Differentiation

Article Title: Lactylation of NAT10 promotes N 4 ‐acetylcytidine modification on tRNA Ser-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation

doi: 10.1038/s41418-024-01327-0

Figure Lengend Snippet: A Schematic representation of DUF1726 domain (marked in blue), RNA helicase domain (marked in yellow), N -acetyltransferase domain (marked in green) and RNA-binding domain (marked in purple) of NAT10. The identified lactylation lysine site ( K ) within the RNA helicase domain was marked in red, which was mutated to arginine ( R ) and marked in dark blue. B The iSLK-KSHV cells transduced with the wild type (NAT10-Flag), the mutant NAT10 (K290R-Flag) or its control (pCDH) for 48 h were subjected to the anti-Flag immunoprecipitation, and then examined by Western blot with the anti-Pan Kla antibody. C The sequences around NAT10 Lys290 from different species were aligned. Conserved Lys290 was marked in red. D The total RNAs from cells shown as in ( B ) were subjected to dot blot assay with anti-ac 4 C antibody. Methylene blue staining was used as the internal control. E The iSLK-KSHV cells with THUMPD1 overexpression (THUMPD1-Myc) were transduced with the wild type (NAT10-Flag), the mutant NAT10 (K290R-Flag) or its control (pCDH) for 48 h. Cells were subjected to the anti-Flag immunoprecipitation and analyzed by Western blot using anti-Myc antibody. F The total RNAs from iSLK-KSHV cells transduced with the wild type (NAT10) and the mutant (K290R) NAT10 for 48 h were subjected to Northern blot with probes to tRNA Ser-CGA-1-1 , and U6 as the control. G The expression levels of vIRF1 and K-bZIP in cells treated as in ( B ) were examined by Western blot with the corresponding antibodies. H The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, ORF45, ORF57, vIRF1, vIL-6, vBCL-2, ORF65, and K8.1) in cells shown as in ( B ). * P < 0.05, ** P < 0.01 and *** P < 0.001 by Student’s t -test. I The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, vIRF1, vIL-6, vBCL-2, and ORF65) in iSLK-KSHV cells with the wild type (NAT10-WT) or the mutant (NAT10-K290R) NAT10 overexpression, which were complemented with the wild type (tRNA Ser-CGA-1-1 -WT) or the mutant (tRNA Ser-CGA-1-1 -Mut) tRNA Ser-CGA-1-1 for 48 h. * P < 0.05, **, P < 0.01 and *** P < 0.001 by Student’s t -test. J By the assessment of ORF26, real-time DNA-PCR for cells treated as in ( B ) was performed to examine viral copy number after doxycycline stimulation for 72 h. *** P < 0.001 by Student’s t -test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibody was from Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine (Baltimore, Maryland, USA) [ – ].

Techniques: RNA Binding Assay, Transduction, Mutagenesis, Control, Immunoprecipitation, Western Blot, Dot Blot, Staining, Over Expression, Northern Blot, Expressing, Quantitative RT-PCR

Journal: Cell Death and Differentiation

Article Title: Lactylation of NAT10 promotes N 4 ‐acetylcytidine modification on tRNA Ser-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation

doi: 10.1038/s41418-024-01327-0

Figure Lengend Snippet: A The expression levels of vIRF1 and K-bZIP in iSLK-KSHV cells transduced with lentiviral sgATAT1 (sgATAT1 #2, sgATAT1 #3) or control lentivirus (Lenti-V2) for 72 h were examined by Western blot with the corresponding antibodies. B The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (K5, K8, vIRF1, vIL-6, vBCL-2, and ORF65) in cells treated as in ( A ). * P < 0.05, ** P < 0.01 and *** P < 0.001 by Student’s t -test. C By the assessment of ORF26, real-time DNA-PCR for cells treated as in ( A ) was performed to examine viral copy number after doxycycline stimulation for 72 h. ***, P < 0.001 by Student’s t -test. D The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, ORF45, ORF57, vIRF1, vIL-6, vBCL-2, ORF65, and K8.1) in iSLK-KSHV cells transduced with lentiviral ATAT1 (ATAT1) or its control (pCDH) for 48 h. ***, P < 0.001 by Student’s t -test. E By the assessment of ORF26, real-time DNA-PCR for cells treated as in ( D ) was performed to detect viral copy number after doxycycline stimulation for 72 h. ** P < 0.01 by Student’s t -test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibody was from Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine (Baltimore, Maryland, USA) [ – ].

Techniques: Expressing, Transduction, Control, Western Blot, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Sperm associated antigen 9 promotes oncogenic KSHV-encoded interferon regulatory factor-induced cellular transformation and angiogenesis by activating the JNK/VEGFA pathway

doi: 10.1371/journal.ppat.1008730

Figure Lengend Snippet: (A). Western-blotting analysis of SPAG9 in HUVECs transduced with lentiviral-vIRF1 or its control lentiviral-pHAGE (MOI of 2). (B). Western-blotting analysis of SPAG9 in HUVECs treated with PBS ( PBS ) or infected by KSHV wild-type virus ( KSHV ) (MOI of 3). (C). Western-blotting analysis of SPAG9 expression in HUVECs treated with PBS ( PBS ) or infected with wild-type KSHV ( KSHV_WT ) (MOI of 3) or vIRF1 mutant virus ( K9_mut ) (MOI of 3) for 30 h. (D). Hematoxylin and eosin (H&E) staining and immunohistochemical staining (IHC) of KSHV LANA, SPAG9 in normal skin, skin KS of patient #1 (Skin KS1), patient #2 (Skin KS2), and patient #3 (Skin KS3). Magnification, ×200, ×400. (E). Results were quantified in ( D ). Data were shown as mean ± SD. *** P < 0.001, Student's t-test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibodies were kindly provided by Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine.

Techniques: Western Blot, Transduction, Control, Infection, Virus, Expressing, Mutagenesis, Staining, Immunohistochemical staining

Journal: PLoS Pathogens

Article Title: Sperm associated antigen 9 promotes oncogenic KSHV-encoded interferon regulatory factor-induced cellular transformation and angiogenesis by activating the JNK/VEGFA pathway

doi: 10.1371/journal.ppat.1008730

Figure Lengend Snippet: (A). RT-qPCR analysis of mRNA level of SPAG9 in HUVECs transduced with lentiviral-vIRF1 or its control lentiviral-pHAGE. (B). RT-qPCR analysis of mRNA level of SPAG9 in treated with PBS ( PBS ) or infected by KSHV wild-type virus ( KSHV ) (MOI of 3). (C). Luciferase reporter assay of the activity of SPAG9 promoter in HUVECs transduced with lentiviral-vIRF1 or its control lentiviral-pHAGE. (D). Luciferase reporter assay of the activity of SPAG9 promoter in vIRF1-expressing HUVECs transduced with a mixture of lentivirus-mediated shRNAs targeting Lef1 ( shLef1 ). (E). Putative binding sites of Lef1 in the promoter region of SPAG9 gene. (F). ChIP assays of SPAG9 promoter. Immunoprecipitation was performed in vIRF1- or pHAGE-transduced HUVECs with anti-Lef1 antibody. Both SPAG9 primers (1) and (2) were used to amplify the sequences of the above two putative binding sites of Lef1 in the region of SPAG9 promoter as described in ( E ). (G) and (H). ChIP assays of SPAG9 promoter. Immunoprecipitation was performed in vIRF1-transduced HUVECs with anti-Flag antibody. Isotype IgG, anti-RNA polymerase II antibody and amplification of GAPDH promoter were used to examined the work of system. (I). RT-qPCR analysis of mRNA expression level of SPAG9 in vIRF1-expressing HUVECs transduced with a mixture of lentivirus-mediated shRNAs targeting Lef1 ( shLef1 ). (J). Western-blotting analysis of SPAG9 expression in vIRF1-expressing HUVECs transduced with a mixture of lentivirus-mediated shRNAs targeting Lef1 ( shLef1 ). Data were shown as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, Student's t-test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibodies were kindly provided by Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine.

Techniques: Quantitative RT-PCR, Transduction, Control, Infection, Virus, Luciferase, Reporter Assay, Activity Assay, Expressing, Binding Assay, Immunoprecipitation, Amplification, Western Blot

Journal: PLoS Pathogens

Article Title: Sperm associated antigen 9 promotes oncogenic KSHV-encoded interferon regulatory factor-induced cellular transformation and angiogenesis by activating the JNK/VEGFA pathway

doi: 10.1371/journal.ppat.1008730

Figure Lengend Snippet: (A). Western-blotting analysis of SPAG9 expression in vIRF1-expressing HUVECs transduced with lentivirus-mediated No.1 ( shSPAG9-1 ) and No. 2 ( shSPAG9-2 ) shRNAs targeting SPAG9. (B). Lentiviral vIRF1- or its control pHAGE-infected endothelial cell line were transduced with lentivirus-mediated No.1 ( shSPAG9-1 ) and No. 2 ( shSPAG9-2 ) shRNAs targeting SPAG9, and then were subjected to chicken chorioallantoic membranes (CAMs) assay. Representative images are shown. Magnification, ×100. Scar bars, 40 μm. (C). Quantification of CAMs assay described in ( B ). (D). Cells treated as in ( B ) were mixed with the high concentration Matrigel, and then were injected into the right flanks of nude mice. Plugs were harvested 10 days after the injection and photographed using stereomicroscope. Representative images of Matrigel plug assay in mice are displayed. (E). Quantification of Matrigel plug assay in mice described in ( D ). (F). CCK-8 assay of HUVECs treated as in ( A ). (G). Transwell migration analysis of HUVECs treated as in ( A ). The migrated and invaded HUVECs were counted at 6 h and 12 h post seeding. (H). Quantification of Transwell migration assay described in ( G ). (I). Western-blotting analysis of SPAG9 expression in KSHV-infected HUVECs transduced with lentivirus-mediated No.1 ( shSPAG9-1 ) and No. 2 ( shSPAG9-2 ) shRNAs targeting SPAG9. (J). CCK-8 assay of HUVECs treated as in ( I ). (K). Transwell migration analysis of HUVECs treated as in ( I ). The migrated and invaded HUVECs were counted at 6 h and 12 h post seeding. Data were shown as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, Student's t-test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibodies were kindly provided by Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine.

Techniques: Western Blot, Expressing, Transduction, Control, Infection, Concentration Assay, Injection, Matrigel Assay, CCK-8 Assay, Migration, Transwell Migration Assay

Journal: PLoS Pathogens

Article Title: Sperm associated antigen 9 promotes oncogenic KSHV-encoded interferon regulatory factor-induced cellular transformation and angiogenesis by activating the JNK/VEGFA pathway

doi: 10.1371/journal.ppat.1008730

Figure Lengend Snippet: (A). Western-blotting analysis of the expression levels of SPAG9, phosphorylated JNK1/2 and total JNK1/2 in vIRF1-expressing HUVECs. (B). Western-blotting analysis of phosphorylated JNK1/2 expression in vIRF1-expressing HUVECs transduced with lentivirus-mediated No.1 (shSPAG9-1) and No. 2 (shSPAG9-2) shRNAs targeting SPAG9. (C). Western-blotting analysis of MKK4 expression in HUVECs transduced with lentiviral-vIRF1 or its control lentiviral-pHAGE. (D) and (E). Immunoprecipitation analyses of the interaction of JNK1/2-SPAG9-MKK4 complex in vIRF1-transduced or its control pHAGE-transduced HUVECs.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibodies were kindly provided by Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine.

Techniques: Western Blot, Expressing, Transduction, Control, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Sperm associated antigen 9 promotes oncogenic KSHV-encoded interferon regulatory factor-induced cellular transformation and angiogenesis by activating the JNK/VEGFA pathway

doi: 10.1371/journal.ppat.1008730

Figure Lengend Snippet: (A). Western-blotting analysis of phosphorylated JNK1/2 and total JNK1/2 in vIRF1-transduced HUVECs treated with the JNK inhibitor, SP600125 (50 μM) for 48 h. (B). CCK-8 assay of HUVECs treated as in ( A ). (C). Transwell migration analysis of HUVECs treated as in ( A ). The migrated and invaded HUVECs were counted at 6 h and 12 h post seeding. (D). Western-blotting analysis of phosphorylated JNK1/2 and total JNK1/2 expression in KSHV-infected HUVECs transduced with lentivirus-mediated No.1 (shSPAG9-1) and No. 2 (shSPAG9-2) shRNAs targeting SPAG9. (E). Western-blotting analysis of phosphorylated JNK1/2 and total JNK1/2 in KSHV-infected HUVECs treated with the JNK inhibitor, SP600125 (50 μM) for 48 h. (F). CCK-8 assay of HUVECs treated as in ( E ). (G). Transwell migration analysis of HUVECs treated as in ( E ). The migrated and invaded HUVECs were counted at 6 h and 12 h post seeding. (H). Lentiviral vIRF1- or its control pHAGE-infected endothelial cell line were treated with the JNK inhibitor, SP600125 (50 μM) for 48 h, and then were subjected to chicken chorioallantoic membranes (CAMs) assay. The quantified results represent mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, Student's t-test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibodies were kindly provided by Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine.

Techniques: Western Blot, CCK-8 Assay, Migration, Expressing, Infection, Transduction, Control

Journal: PLoS Pathogens

Article Title: Sperm associated antigen 9 promotes oncogenic KSHV-encoded interferon regulatory factor-induced cellular transformation and angiogenesis by activating the JNK/VEGFA pathway

doi: 10.1371/journal.ppat.1008730

Figure Lengend Snippet: (A). Western-blotting analysis of SPAG9, phosphorylated JNK1/2, total JNK1/2 and VEGFA expression in HUVECs transduced with lentiviral-vIRF1or its control lentiviral-pHAGE. (B). Western-blotting analysis of phosphorylated JNK1/2, total JNK1/2 and VEGFA expression in vIRF1-expressing HUVECs transduced with lentivirus-mediated No.1 (shSPAG9-1) and No. 2 (shSPAG9-2) shRNAs targeting SPAG9. (C). Western-blotting analysis of phosphorylated JNK1/2, JNK1/2 and VEGFA in vIRF1-expressing HUVECs treated with the JNK inhibitor, SP600125 (50 μM) for 48 h. (D). Western-blotting analysis of phosphorylated JNK1/2, JNK1/2 and VEGFA in KSHV-infected HUVECs treated with the JNK inhibitor, SP600125 (50 μM) for 48 h. (E). Western-blotting analysis of SPAG9, phosphorylated JNK1/2, JNK1/2 and VEGFA expression in iSLK-RGB cells treated with doxycycline (Doxy) (1 μg/ml) for 48 h. (F). Western-blotting analysis of SPAG9, phosphorylated JNK1/2, JNK1/2 and VEGFA expression in Doxy-induced iSLK-RGB cells transduced with lentivirus-mediated No.1 (shSPAG9-1) and No. 2 (shSPAG9-2) shRNAs targeting SPAG9. (G). Luciferase reporter assay of the activity of VEGFA promoter in HUVECs transduced with lentiviral-vIRF1 or its control lentiviral-pHAGE. (H). Luciferase reporter assay of the activity of VEGFA promoter in vIRF1-expressing HUVECs treated with the JNK inhibitor, SP600125 (50 μM) for 48 h . (I). Western-blotting analysis of phosphorylated JNK1/2, JNK1/2 and VEGFA expression in vIRF1-expressing HUVECs transduced with lentivirus-mediated a mixture of shRNAs targeting Lef1 (shLef1). (J). Luciferase reporter assay of the activity of VEGFA promoter in vIRF1-expressing HUVECs transduced with lentivirus-mediated a mixture of shRNAs targeting Lef1 (shLef1). The quantified results represent mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, Student's t-test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibodies were kindly provided by Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine.

Techniques: Western Blot, Expressing, Transduction, Control, Infection, Luciferase, Reporter Assay, Activity Assay

Journal: PLoS Pathogens

Article Title: Sperm associated antigen 9 promotes oncogenic KSHV-encoded interferon regulatory factor-induced cellular transformation and angiogenesis by activating the JNK/VEGFA pathway

doi: 10.1371/journal.ppat.1008730

Figure Lengend Snippet: (A). Soft agar assay of MM cells, KSHV-infected and transformed MM cells (KMM) and a mutant with a deletion of KSHV ORF-K9 infected MM cells ( K9_Mut ) (MOI of 3). The representative images were captured at 2 weeks post seeding. Magnification, ×100. Scar bars, 40 μm. (B). CCK-8 assay of cells treated as in ( A ). (C). Western-blotting analysis of SPAG9, phosphorylated JNK1/2, JNK1/2 and VEGFA expression in cells treated as in ( A ). (D). Soft agar assay of MM and KMM cells treated with the JNK inhibitor, SP600125 (50 μM) for 48 h . The representative images were captured at 2 weeks post seeding. Magnification, ×100. Scar bars, 40 μm. (E). CCK-8 assay of cells treated as in ( D ). (F). Chicken chorioallantoic membranes (CAMs) assay of cells treated as in ( D ). (G). Matrigel plug assay in mice of cells treated as in ( D ). (H). Hematoxylin and eosin (H&E) staining analysis of histologic features (up; ×400) and immunohistochemical (IHC) staining analysis of the expression of SMA (down; ×400) in plugs in mice induced by cells treated as in ( D ). The newly formed blood vessels and the SMA expression were pointed out by black arrows, respectively. (I). Western-blotting analysis of SPAG9, phosphorylated JNK1/2, JNK1/2 and VEGFA expression in HUVECs treated with PBS ( PBS ) or infected with wild-type KSHV ( KSHV_WT ) (MOI of 3) or vIRF1 mutant virus ( K9_mut ) (MOI of 3) followed by transduction with lentiviral vIRF1 at MOI 2 at 6 hpi. (J). CCK-8 assay of cells treated as in ( I ). (K). A schematic working model of the mechanism by which vIRF1 facilitates angiogenesis and cell transformation. vIRF1 enhanced SPAG9 transcription by interacting with Lef1 to promote the transcriptional activity of Lef1. Increased SPAG9 expression enhanced the activation of JNK1/2 pathway and VEGFA transcription contributing to KSHV-induced angiogenesis and tumorigenesis.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibodies were kindly provided by Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine.

Techniques: Soft Agar Assay, Infection, Transformation Assay, Mutagenesis, CCK-8 Assay, Western Blot, Expressing, Matrigel Assay, Staining, Immunohistochemical staining, Immunohistochemistry, Virus, Transduction, Activity Assay, Activation Assay