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