orf21  (Addgene inc)

Journal: bioRxiv

Article Title: The Kaposi’s sarcoma-associated herpesvirus viral genome packaging accessory factor ORF68 forms cytoplasmic puncta dependent on the viral tyrosine kinase

doi: 10.64898/2026.02.23.707506

Figure Lengend Snippet: A . Recombinant mNG-ORF68, mNG-ORF68/HA-ORF21, and mNG-ORF68/ORF21.stop BACs were digested with RsrII to assess that no large-scale rearrangements occurred during cloning. B . Western blot of whole cell lysate (25 μg) from unreactivated or reactivated WT iSLK cells or iSLK cells harboring the ORF21.stop BAC, representative of 4 biological replicates.

Article Snippet: The vector for transient expression of ORF21 was previously described (pcDNA4/TO-ORF21-2xStrep, Addgene #136181) .

Techniques: Recombinant, Cloning, Western Blot

Journal: bioRxiv

Article Title: The Kaposi’s sarcoma-associated herpesvirus viral genome packaging accessory factor ORF68 forms cytoplasmic puncta dependent on the viral tyrosine kinase

doi: 10.64898/2026.02.23.707506

Figure Lengend Snippet: A . Z-slices of iSLK mNG-ORF68/ORF21-HA cells. Cells were fixed 72 hours post-reactivation, with or without PAA treatment, imaged for mNG-ORF68 (green), and stained for phalloidin (blue dashed lines), lamins A and C (cyan dashed lines), and HA-tag (magenta). Representative of at least 10 cells from two biological replicates. B . Intensity profile plots showing intensity of Lamin (blue), mNG (green), or HA-tag (magenta) signal at each point along lines (yellow or orange dashes). C . Whole-cell maximum Z-projections of iSLK mNG-ORF68/ORF21.stop cells. Cells were imaged for mNG-ORF68 (green) and stained for phalloidin (blue dashed lines), lamins A and C (cyan dashed lines), and HA-tag (magenta), which should be undetectable in cells lacking an HA epitope tag. Representative of at least 10 cells from two biological replicates. Scale bars are 10 μm; color bars represent the modified minimum and maximum pixel intensity values. D . Quantification of nuclear to cytosolic ratio of mNG-ORF68 in the presence or absence of viral-expressed ORF21. Error bars indicate the mean, S.D. (***) indicates a p-value < 0.0001 calculated using Welch’s T-test.

Article Snippet: The vector for transient expression of ORF21 was previously described (pcDNA4/TO-ORF21-2xStrep, Addgene #136181) .

Techniques: Staining, Modification

Journal: bioRxiv

Article Title: The Kaposi’s sarcoma-associated herpesvirus viral genome packaging accessory factor ORF68 forms cytoplasmic puncta dependent on the viral tyrosine kinase

doi: 10.64898/2026.02.23.707506

Figure Lengend Snippet: A . Z-slices of transfected HEK293T cells expressing HA-ORF68 and/or ORF21-Strep constructs, including ORF21 full-length, N-terminal region (residues 1-248), and kinase domain (residues 248-580). Stained for Hoechst (cyan dashed lines), HA-tag (green), and Strep-tag (magenta), representative of at least 10 cells from 2 biological replicates. Color bars represent the modified minimum and maximum pixel values. Scale bar is 10 μm. B . Schematic of ORF21 domain organization, showing disordered region and kinase domain. C . Western blot of whole cell lysate (25 μg) from HEK293T cells transfected with ORF21 constructs (Strep) and ORF68 (HA). Vinculin serves as the loading control. D . ORF68 participates in viral genome packaging in nuclear viral replication compartments, but is drawn to the cytoplasm through an interaction with the disordered N-terminal tail of ORF21.

Article Snippet: The vector for transient expression of ORF21 was previously described (pcDNA4/TO-ORF21-2xStrep, Addgene #136181) .

Techniques: Transfection, Expressing, Construct, Staining, Strep-tag, Modification, Western Blot, Control

Journal: bioRxiv

Article Title: The Kaposi’s sarcoma-associated herpesvirus viral genome packaging accessory factor ORF68 forms cytoplasmic puncta dependent on the viral tyrosine kinase

doi: 10.64898/2026.02.23.707506

Figure Lengend Snippet: Z-slices of transfected HEK293T cells expressing HA-ORF68 and full-length ORF21-strep with mutations G260V and Y120A. Stained for Hoechst (cyan dashed lines), HA-tag (green), and Strep-tag (magenta), representative of at least 5 cells from 1 biological replicate. Scale bars are 10 μm.

Article Snippet: The vector for transient expression of ORF21 was previously described (pcDNA4/TO-ORF21-2xStrep, Addgene #136181) .

Techniques: Transfection, Expressing, Staining, Strep-tag

Journal: International Journal of Molecular Sciences

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

doi: 10.3390/ijms24021238

Figure Lengend Snippet: Construction of ORF21-kinase dead (21KD) and ORF21-deleted (21del) KSHV BAC16. ( a ) Schematic diagrams of the KSHV genome, including the ORF21-coding region. Two types of mutations (i.e., ORF21-kinase dead and ORF21-deleted) were generated in the ORF21 coding region (nucleotides (nt)35202 to nt36943) of the wildtype (WT) KSHV BAC16 clone (WT-BAC16) (GenBank accession number: GQ994935) by a two-step red recombination method. ORF21-kinase dead BAC16 (21KD-BAC16) had a kinase activity deficient mutation through the substitution of three Gly residues (G260, G263, and G265) within the ORF21 coding region in WT-BAC16. ORF21-deleted BAC16 (21del-BAC16) had a three stop codons insertion located after the fourth Met123-codon within the ORF21 coding region in WT-BAC16. a.a., amino acids. ( b ) Agarose gel electrophoresis of mutated BAC16 clones digested with Hind III. The asterisks indicate the insertion and deletion of the kanamycin resistance cassette in each BAC clone. The asterisks (*) indicate the insertion and deletion of a kanamycin-resistance cassette in each BAC clone. Original images are shown in . ( c , d ) DNA sequencing results for ORF21 mutagenesis sites in 21KD-BAC16 and 21del-BAC16. ( e , g ) Establishment of mutated BAC16-harboring cell lines. WT-BAC16, 21KD-BAC16, and 21del-BAC16 were stably transfected into iVero cells (or iSLK cells), and the established BAC16-harboring cell lines were designated as iVero-WT (or iSLK-WT), iVero-21KD (or iSLK-21KD), and iVero-21del (or iSLK-21del), respectively. Each picture shows the fluorescence signal derived from the GFP gene in the BAC16 of the established cell lines. ( f , h ) Western blotting data showing the elimination of ORF21 expression in the lytic-induced iVero-21del and iSLK-21del cells. Cells were treated for 48 h with 1.5 mM NaB and 8 μg/mL Dox to induce lytic replication and were subjected to Western blotting using the anti-ORF21 polyclonal antibody. ( f , h ) Original images are shown in .

Article Snippet: To construct the 3×Flag-ORF21-wildtype plasmid (p3F-21WT), the KSHV ORF21 coding DNA fragment was obtained by PCR from HBL6 PEL cells [ ] and was subcloned into a 3×Flag-tagged pCI-neo vector, which was generated by inserting oligonucleotides encoding three repeats of the Flag-tag sequence into the pCI-neo mammalian expression vector (Promega).

Techniques: Generated, Activity Assay, Mutagenesis, Agarose Gel Electrophoresis, Clone Assay, DNA Sequencing, Stable Transfection, Transfection, Fluorescence, Derivative Assay, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

doi: 10.3390/ijms24021238

Figure Lengend Snippet: ORF21 is localized in the cytoplasm and is involved in cell contraction. ( a ) The endogenous expression of ORF21 protein in lytic-induced cells. iSLK-WT cells were treated for 0–60 h with NaB and Dox for lytic reactivation, and lytic-induced cells were analyzed by Western blotting using the anti-ORF21 polyclonal antibody. Original images of the blotting are shown in . ( b ) Localization of endogenously expressed ORF21 protein in lytic-induced cells. iSLK-WT cells were treated with 1.5 mM NaB and 8 μg/mL Dox for 48 h and analyzed with IFA. ORF21 protein (orange) and DNA (blue) were stained with the anti-ORF21 polyclonal antibody and DAPI, respectively. Scale bars represent 5 µm. ( c ) Effect of endogenous ORF21 expression on cell contraction in iSLK-WT during lytic replication. iSLK-WT cells were treated with (or without) Dox and NaB for 48 h and analyzed with a fluorescence microscope. Scale bars represent 20 µm. ( d ) Effect of exogenous ORF21 expression on cell contraction in iSLK-21KD cells during lytic replication. iSLK-21KD cells were transiently transfected with either 3×Flag-ORF21 wildtype (p3F-21WT) or an empty plasmid and cultured with Dox and NaB for 48 h. The left images show the GFP signal (green) derived from KSHV-BAC16, ORF21 (red), and nuclei (white). The right images show phalloidin signal (violet), GFP signal (green), and nuclei (white). The cell areas of the GFP- and phalloidin-positive cells were measured. Scale bars represent 20 µm. ( c , d ) p < 0.001 and p < 0.005 indicate a statistically significant difference.

Article Snippet: To construct the 3×Flag-ORF21-wildtype plasmid (p3F-21WT), the KSHV ORF21 coding DNA fragment was obtained by PCR from HBL6 PEL cells [ ] and was subcloned into a 3×Flag-tagged pCI-neo vector, which was generated by inserting oligonucleotides encoding three repeats of the Flag-tag sequence into the pCI-neo mammalian expression vector (Promega).

Techniques: Expressing, Western Blot, Staining, Fluorescence, Microscopy, Transfection, Plasmid Preparation, Cell Culture, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

doi: 10.3390/ijms24021238

Figure Lengend Snippet: The effects of ORF21 and ORF21-kinase activity on the replication of intracellular viral DNA and the transcription of the lytic genes. Viral DNA replication ( a , b ) and viral gene transcription ( c – f ) in iVero (or iSLK) cells harboring WT-BAC16, 21KD-BAC16, and 21del-BAC16. The recombinant BAC16-transfected iVero cells ( a , c , e , g ) or iSLK cells ( b , d , f , h ) were treated for 48 h with Dox and NaB to induce lytic replication, and DNA genomes containing viral DNA were prepared from harvested cells. ( a , b ) The copies of intracellular viral DNA in the lytic-induced cells were measured using real-time PCR and normalized by the total amount of obtained DNA. ( c – h ) Quantities of mRNA expression levels of viral genes, immediate-early gene: ORF16 (vBcl-2); early gene: ORF59 (DNA processivity factor); late gene: K8.1 (glycoprotein) in the mutated KSHV-harboring cells. The total RNA was purified from lytic-induced cells and was subjected to RT real-time PCR. The values obtained from Dox- and NaB-untreated iSLK (or iVero)-WT cells were defined as 1.0. Statistical analysis was performed between Dox/NaB(+) groups in RT-qPCR. These results were not detected to have a statistically significant difference. ( a – h ) N.S., not significant.

Article Snippet: To construct the 3×Flag-ORF21-wildtype plasmid (p3F-21WT), the KSHV ORF21 coding DNA fragment was obtained by PCR from HBL6 PEL cells [ ] and was subcloned into a 3×Flag-tagged pCI-neo vector, which was generated by inserting oligonucleotides encoding three repeats of the Flag-tag sequence into the pCI-neo mammalian expression vector (Promega).

Techniques: Activity Assay, Recombinant, Transfection, Real-time Polymerase Chain Reaction, Expressing, Purification, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

doi: 10.3390/ijms24021238

Figure Lengend Snippet: The effects of ORF21 on the production of cell-free genome-encapsidated particles. ( a ) Recombinant BAC16-harboring iVero cells (iVero-WT, iVero-21KD, and iVero-21del) or ( b ) iSLK cells (iSLK-WT, iSLK-21KD, and iSLK-21del) were cultured for 48 h in a medium with Dox and NaB, and the culture supernatants were harvested. KSHV genomes were purified from the cell-free genome-encapsidated particles in culture supernatants, and viral DNA copies were determined by real-time PCR. N.S., not significant.

Article Snippet: To construct the 3×Flag-ORF21-wildtype plasmid (p3F-21WT), the KSHV ORF21 coding DNA fragment was obtained by PCR from HBL6 PEL cells [ ] and was subcloned into a 3×Flag-tagged pCI-neo vector, which was generated by inserting oligonucleotides encoding three repeats of the Flag-tag sequence into the pCI-neo mammalian expression vector (Promega).

Techniques: Recombinant, Cell Culture, Purification, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

doi: 10.3390/ijms24021238

Figure Lengend Snippet: ORF21 is involved in infectious virus production. ( a – c ) Recombinant BAC16-harboring iVero cells (iVero-WT, iVero-21KD, and iVero-21del) or ( d – f ) iSLK cells (iSLK-WT, iSLK-21KD, and iSLK-21del) were treated for 96 h with Dox and NaB to produce recombinant KSHV, and the culture supernatants were harvested. The progeny viral particles were precipitated with ultracentrifugation, and partially purified viral particles (at approximately 10 5 viral genome copies/cell) were used to infect fresh Vero cells ( b , e ) and 293T cells ( c , f ). The GFP-positive cells (i.e., infected cells) were counted by flow cytometry 48 h post-infection to determine the infectivity of the produced recombinant viruses. N.S., not significant. p < 0.01 indicates a statistically significant difference compared with the iVero/iSLK-WT cells.

Article Snippet: To construct the 3×Flag-ORF21-wildtype plasmid (p3F-21WT), the KSHV ORF21 coding DNA fragment was obtained by PCR from HBL6 PEL cells [ ] and was subcloned into a 3×Flag-tagged pCI-neo vector, which was generated by inserting oligonucleotides encoding three repeats of the Flag-tag sequence into the pCI-neo mammalian expression vector (Promega).

Techniques: Virus, Recombinant, Purification, Infection, Flow Cytometry, Produced

Journal: International Journal of Molecular Sciences

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

doi: 10.3390/ijms24021238

Figure Lengend Snippet: Infectious virus production in iSLK-21del cells is rescued by ORF21 overexpression. To validate the rescue of infectious virus production in iSLK-21del cells by exogenous ORF21 expression, iSLK-21del cells were transiently transfected with the 3×Flag-ORF21 wildtype (p3F-21WT) plasmid (or empty plasmid) and treated for 96 h with Dox and NaB to produce recombinant KSHV. The culture supernatant was ultracentrifuged to precipitate the produced viruses, which were inoculated onto 293T cells. The infected cells (GFP-positive cells) were analyzed using a flow-cytometer, and the infectivity of the recombinant viruses is shown as a bar graph. p < 0.001 indicates a statistically significant difference compared with the empty plasmid-transfected cells. The endogenous or exogenenous ORF21 expression was analyzed by Western blotting using the anti-ORF21 polyclonal antibody. The original images are shown in .

Article Snippet: To construct the 3×Flag-ORF21-wildtype plasmid (p3F-21WT), the KSHV ORF21 coding DNA fragment was obtained by PCR from HBL6 PEL cells [ ] and was subcloned into a 3×Flag-tagged pCI-neo vector, which was generated by inserting oligonucleotides encoding three repeats of the Flag-tag sequence into the pCI-neo mammalian expression vector (Promega).

Techniques: Virus, Over Expression, Expressing, Transfection, Plasmid Preparation, Recombinant, Produced, Infection, Flow Cytometry, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

doi: 10.3390/ijms24021238

Figure Lengend Snippet: ORF21 upregulated the MEK phosphorylation and anchorage-independent cell growth. ( a – c ) Upregulation of MEK phosphorylation, anchorage-dependent proliferation, and anchorage-independent proliferation by ORF21. HeLa cells were transfected with empty, 3×Flag-ORF21 wildtype (p3F-21WT), or 3×Flag-ORF21-kinase dead (p3F-21KD) plasmid and cultured for 48 h, followed by ( a ) Western blotting, ( b ) cell proliferation assay, or ( c ) soft agar colony formation assay. ( a ) The band intensities of phospho-MEK were calculated using Fiji software. The values of phospho-MEK/total MEK are presented as a bar graph. The original images of phospho-MEK and Erk are shown in . ( b , c ) The value of 21WT plasmid-transfected cells is presented as 100. P -values indicate a significant difference. N.S., not significant. ( d – f ) KSHV lytic replication downregulated the MEK phosphorylation and the expression of the EGF receptor (EGFR). iSLK (without BAC16), iSLK-WT, iSLK-21KD, and iSLK-21del cells were treated for 48 h with Dox and NaB to express the lytic-related viral proteins, and EGFR and the phosphorylated MEK were analyzed. iSLK cells (without BAC16) were used as the uninfected control. The original images are shown in . ( e , f ) The values of EGFR/GAPDH, and phosphorylated MEK/MEK are presented as a bar graph. The values of Dox- and NaB-untreated iSLK cells were defined as 1.0. ( g ) Expression of EGFR mRNA in the lytic-induced iSLK-WT, iSLK-21KD, and iSLK-21del cells. Cells were treated with Dox and NaB for 48 h, and the expression of EGFR mRNA was determined by RT-qPCR and normalized by the expression of GAPDH mRNA. ( h ) The effect of the MEK-signaling inhibitor (U0126) on the new infection of the progeny virus with the recipient cells. The recipient (293T) cells were pretreated with 5 μM U0126 or control DMSO for 48 h, and WT-KSHV particles were infected into U0126-pretreated 293T cells. The number of infected cells (GFP-positive cells) was analyzed by flow cytometry. The inhibition of MEK signaling was validated by Western blotting. ( i ) The effect of U0126 on viral production in the lytic-induced WT-BAC16-harboring cells. The WT-BAC16-harboring cells were treated with Dox and NaB in the presence of 100 μM U0126 for 48 h, and the culture supernatant was harvested. The number of produced viruses in the culture supernatant was measured by qPCR. The inhibition of MEK signaling was validated by Western blotting. ( h , i ) N.S., not significant. p < 0.05 and p < 0.001 indicate a statistically significant difference. ( j ) Model of the KSHV ORF21-mediated upregulation of the MEK pathway and cell growth. The EGFR-MEK signaling pathway is known to be necessary for anchorage-independent cell growth in tumor cells. Unknown KSHV lytic-related proteins expressed by the lytic reactivation suppresse the expression of EGFR and MEK phosphorylation. ORF21 upregulates the MEK phosphorylation, resulting in enhancement of the infectivity of the progeny virus and anchorage-independent cell growth.

Article Snippet: To construct the 3×Flag-ORF21-wildtype plasmid (p3F-21WT), the KSHV ORF21 coding DNA fragment was obtained by PCR from HBL6 PEL cells [ ] and was subcloned into a 3×Flag-tagged pCI-neo vector, which was generated by inserting oligonucleotides encoding three repeats of the Flag-tag sequence into the pCI-neo mammalian expression vector (Promega).

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Proliferation Assay, Soft Agar Assay, Software, Expressing, Control, Quantitative RT-PCR, Infection, Virus, Flow Cytometry, Inhibition, Produced

Journal: International Journal of Molecular Sciences

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

doi: 10.3390/ijms24021238

Figure Lengend Snippet: Primers for BAC mutagenesis, construction of expression plasmids, real-time PCR, and RT real-time PCR.

Article Snippet: To construct the 3×Flag-ORF21-wildtype plasmid (p3F-21WT), the KSHV ORF21 coding DNA fragment was obtained by PCR from HBL6 PEL cells [ ] and was subcloned into a 3×Flag-tagged pCI-neo vector, which was generated by inserting oligonucleotides encoding three repeats of the Flag-tag sequence into the pCI-neo mammalian expression vector (Promega).

Techniques: Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Sequencing, Plasmid Preparation

Journal: Journal of Virology

Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

doi: 10.1128/JVI.01555-19

Figure Lengend Snippet: Schematic of ORF21 luciferase (LUC) promoter constructs and results of activation of various promoter constructs by XBP-1u and XBP-1s. (A) ORF21 promoter contains 4 XBP-1 response element (XRE) core sequences (5ʹ-ACGT-3ʹ) (44), including 2 consensus (5ʹ-NNGNTGACGTGKNNNWT-3ʹ) XRE sequences (3 and 4) within 1,239 bp upstream of the ORF21 start codon (nucleotide positions 34144 to 35382 of KSHV-BAC36; GenBank accession number HQ404500). Consensus XREs are indicated as black squares and the other two (core only) XREs as gray squares. XRE1, −8 to −5; XRE2, −262 to −259; XRE3, −321 to −318; XRE4, −629 to −626. Direction of each XRE is indicated with an arrow. Consensus core HREs are shown as black triangles. Constructs of promoters pORF21-624, pORF21-316, and pORF21-256 were made by sequential deletions as shown. (B) Comparison of the activation of the ORF21-1239, vIL-6, and ORF36 promoter luciferase reporter constructs by XBP-1 unspliced (XBP-1u) or spliced (XBP-1s). HEK-293T cells were cotransfected with 300 ng of different promoter luciferase plasmids and 50 ng of an internal β-Gal control plasmid (pGL3 basic empty vector) in the presence of 100 ng of an expression plasmid encoding XBP-1u, XBP-1s, or pcDNA3.1 expression plasmid control. Values are expressed as fold increase over the respective control reporter plasmid transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values (*P ≤ 0.05, **P ≤ 0.01) for the comparison shown with the pGL3B control. (C) Comparison of the activation of ORF21 and truncated forms of the ORF21 luciferase reporter by XBP-1s or pcDNA3.1 plasmid control. 293T cells were cotransfected with 300 ng of each ORF21 promoter and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s or pcDNA3.1 control. Values are expressed as fold increase over pGL3basic transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values as in (B).

Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

Techniques: Luciferase, Construct, Activation Assay, Comparison, Control, Plasmid Preparation, Expressing, Transfection

Journal: Journal of Virology

Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

doi: 10.1128/JVI.01555-19

Figure Lengend Snippet: Effect of XRE3 and XRE4 mutations in the ORF21 promoter on the response to XBP1s. (A) Construct of the wild-type ORF21 and mutant reporter plasmids. Three different mutant reporters for each XRE were constructed in the pORF21-1239 full-length promoter, containing a 2- to 4-bp substitution within core XRE sequences. DNA sequences for the XRE3, XRE4 wild type, and mutant plasmids are shown. The underlined regions and bold letters indicate the mutations from wild type for each mutant construct. (B) Comparison of the activation of wild-type pORF21-1239 luciferase reporter with the XRE3 or XRE4 mutant luciferase reporters by XBP-1s. HEK-293T cells were cotransfected with 300 ng of pORF21-1239WT or X3 or X4 M1, 2, or 3 promoters and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s (black bars) or pcDNA3.1 expression plasmid control (gray bars). Values are expressed as fold increase over the pGL3basic reporter transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations. **P < 0.01 and ***P < 0.005 for the comparisons shown; none of the comparisons between the X4 mutations and wild type (WT) were significant (P > 0.05).

Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

Techniques: Construct, Mutagenesis, Comparison, Activation Assay, Luciferase, Control, Plasmid Preparation, Expressing, Transfection

Journal: Journal of Virology

Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

doi: 10.1128/JVI.01555-19

Figure Lengend Snippet: The ORF21 promoter contains potential HREs but does not respond to hypoxia in the absence of XBP-1s. (A). Comparison of the activation of wild-type pORF21 and pORF36 luciferase reporter in response to hypoxia. HEK-293T cells were cotransfected with 300 ng of pORF21 or 36 luciferase promoter, 100 ng of an expression plasmid encoding XBP-1s (black bars) or pcDNA3.1 control (gray bars), and 50 ng of an internal β-Gal control plasmid, cultured in normoxia for 32 hours, and then cells were treated in normoxic or hypoxic (1% oxygen) conditions for 16 hours. Values are expressed as the fold increase over the value for the pGL3 basic reporter transfected with an empty expression vector (pcDNA3.1) in normoxia and represent the mean of three independent experiments. Error bars denote the standard deviations. (*P ≤ 0.05; NS, not significant). (B). Comparison of the activation of the wild-type pORF21-1239 luciferase reporter or the HIF-responsive pORF36 reporter in response to degradation-resistant HIF-1 (dr-HIF-1). HEK-293T cells were cotransfected with 300 ng of the pORF21-1239 luciferase reporter or pORF36 promoter and 50 ng of an internal-β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding dr-HIF-1 or the pcDNA3.1 expression plasmid control. Values are expressed as the fold increase over the value of the pGL3basic reporter transfected with an empty expression vector pcDNA3.1 for each reporter construct and represent the mean of three independent experiments. Error bars denote standard deviation; ****P ≤ 0.001; NS, not significant. (C) Western blot showing XBP-1s (55 kDa) and RTA (85 kDa) expression in BCBL-1 cells 48 hours after treatment with 0.5 μg/ml to 2.5 μg/ml of tunicamycin. As seen, TM induces XBP-1s and RTA but does not induce HIF-1α production in BCBL-1 cells. However, HIF-1α protein expression is seen in BCBCl-1 cells 48 hours after treatment with CoCl2 at 75 μM. Actin was used as the loading control, and TPA was a control for KSHV activation. (D) Comparison of the activation of the pORF21-1239 luciferase reporter by RTA, XBP-1s, or both. HEK-293T cells were cotransfected with 300 ng of pGL3b control reporter plasmid DNA or the pORF21-1239 promoter luciferase reporter and 50 ng of an internal β-Gal control plasmid in the presence of 10 ng of a DNA expression plasmid encoding RTA and 100 ng pcDNA3.1, 100 ng of an expression plasmid for XBP-1s and 10 ng pcDNA3.1, or both RTA and XBP-1s expression plasmids. Values are expressed as the fold increase over the value for the pGL3b basic reporter transfected with pcDNA3.1. Shown are the mean ± standard deviation of triplicate determinations from one representative experiment expressed as the fold change compared with the level of nontreated cells.

Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

Techniques: Comparison, Activation Assay, Luciferase, Expressing, Plasmid Preparation, Control, Cell Culture, Transfection, Construct, Standard Deviation, Western Blot

Journal: Journal of Virology

Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

doi: 10.1128/JVI.01555-19

Figure Lengend Snippet: ChIP showing binding of XBP-1s to the ORF21 promoter in TM-treated cells. BCBL-1 cells were treated with DMSO (A) or TM treatment (0.5 μg/ml) (B) for 48 hours to induce XBP1s and then cross-linked. Chromatin IP of fragmented DNA was performed with anti-XBP-1 antibody, CHIP positive-control anti-histone H3 antibody, or control IgG. Precipitated DNA was assayed by qPCR with specific primers for amplification of XRE2, 3, or 4 of the ORF21 promoter and with primers for an ORF21, ORF36 non-XRE region as a negative control. The data were quantitated as described in the Materials and Methods. Results shown are the mean ± standard deviation of triplicate determinations from a typical experiment of three experiments performed. In controls performed at the same time, DNA immunoprecipitated with histone H3 antibody, but not XBP-1 antibody or control IgG, was enriched for RPL30 exon 3.

Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Positive Control, Control, Amplification, Negative Control, Standard Deviation, Immunoprecipitation

Journal: Journal of Virology

Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

doi: 10.1128/JVI.01555-19

Figure Lengend Snippet: XBP-1, ORF21, RTA ,and vIL-6 mRNA upregulation mediated by TM, a chemical inducer of XBP-1s, in the BCBL-1 PEL line. BCBL-1 cells were treated with increasing doses of TM to induce ER stress; cells were also treated with TPA as an inducer of RTA or a DMSO control. Real-time quantitative PCR showing expression of spliced XBP-1 (A) and total XBP-1 (B) in BCBL-1 cells treated with the compounds shown for 4 h, 8 h, and 24 h. (C, D, and E) Real-time quantitative PCR showing expression of ORF21, vIL-6, and RTA mRNA in BCBL-1 cells cultured in the same way and harvested at 4 hours (C), 8 hours (D), and 24 hours (E). Shown are the mean ± the standard deviation of triplicate determinations from one representative experiment out of three expressed as the fold change compared with the DMSO control.

Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

Techniques: Control, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Standard Deviation

Journal: Journal of Virology

Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

doi: 10.1128/JVI.01555-19

Figure Lengend Snippet: RNAscope analysis of ORF21 and XBP-1 in representative sections of a lymph node from a patient with KSHV-MCD. (A and B) A paraformaldehyde-fixed paraffin-embedded lymph node from a patient with KSHV-MCD was analyzed for ORF21 (green) and XBP-1 (red) mRNA as described in the Materials and Methods. The white arrows denote cells that express both ORF21 and XBP-1, while the pink arrow denotes a cell that expresses ORF21 only. In addition, CD20 protein expression is identified by immunohistofluorescence (blue), and nuclei identified by 4′,6-diamidino-2-phenylindole (DAPI) is shown in gray. (C) A probe-control section stained for CD20. It is worth noting that most KSHV plasmablasts do not express CD20. The scale bar is 100 μm.

Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

Techniques: RNAscope, Expressing, Immunohistofluorescence, Control, Staining

Journal: Journal of Virology

Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

doi: 10.1128/JVI.01555-19

Figure Lengend Snippet: Primers used in real-time PCR

Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

Techniques: Sequencing

Journal: Marine Drugs

Article Title: Diversity of Nonribosomal Peptide Synthetase Genes in the Microbial Metagenomes of Marine Sponges

doi: 10.3390/md10061192

Figure Lengend Snippet: Putative genes identified on the genomic fragment AANRPS (pANRPS19p18 and pANRPS32i21) from Aplysina aerophoba .

Article Snippet: ORF21 , 47982-48890 , Methyltransferase , MaviaA2_010100001311, (ZP_05214826), Mycobacterium avium ATCC 25291 , 40/49 , 302.

Techniques: Membrane