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