Appendix 1—key resources table.
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Kaposi’s sarcoma-associated herpesvirus) | ORF68 | Human gammaherpesvirus 8 clone BAC16, complete genome | GenBank: MK733609.1; Uniprot: D2XQF2 | |
| Gene (Epstein–Barr virus) | BFLF1 | Human gammaherpesvirus 4 isolate NPCT090, complete genome | GenBank: MK540447.1 | |
| Gene (MHV68) | mu68 | Murid herpesvirus 4 strain g2.4, complete genome | GenBank: AF105037.1 Uniprot: O41969 |
|
| Gene (human cytomegalovirus) | UL52 | Human herpesvirus 5 strain Towne, complete genome | GenBank: FJ616285.1; Uniprot: O56765 | |
| Gene (Herpes simplex virus 1) | UL32 | Human herpesvirus 1 isolate KOS, complete genome | GenBank: KT899744.1; Uniprot: H9E939 | |
| Strain, strain background (Escherichia coli) | GS1783 | Brulois et al., 2012 | PMID: 22740391 | WT BAC16-containing E. coli used for construction of BAC16 mutants |
| Genetic reagent (Kaposi’s sarcoma-associated herpesvirus) | KSHV BAC16 WT | Brulois et al., 2012 | PMID: 22740391 | WT BAC16 containing KSHV genome |
| Genetic reagent (Kaposi’s sarcoma-associated herpesvirus) | KSHV BAC16 ORF68.stop (68S) | Gardner and Glaunsinger, 2018 | PMID: 29875246 | BAC16 mutant containing a premature stop codon in ORF68 |
| Genetic reagent (Kaposi’s sarcoma-associated herpesvirus) | KSHV BAC16 ORF68.stop-MR (68S-MR) | Gardner and Glaunsinger, 2018 | PMID: 29875246 | Mutant rescue of BAC16 ORF68.stop |
| Genetic reagent (Kaposi’s sarcoma-associated herpesvirus) | KSHV BAC16 ORF68-K435A | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | BAC16 mutant containing ORF68-K435A | |
| Genetic reagent (Kaposi’s sarcoma-associated herpesvirus) | KSHV BAC16 ORF68-K435A-MR | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | Mutant rescue of BAC16 ORF68-K435A | |
| Genetic reagent (Kaposi’s sarcoma-associated herpesvirus) | KSHV BAC16 ORF68-K174A/R179A/K182A (3+) | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | BAC16 mutant containing ORF68-K174A/R179A/K182A | |
| Genetic reagent (Kaposi’s sarcoma-associated herpesvirus) | KSHV BAC16 K174A/R179A/K182A-MR (3+-MR) | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | Mutant rescue of BAC16 ORF68-K174A/R179A/K182A | |
| Genetic reagent (Kaposi’s sarcoma-associated herpesvirus) | KSHV BAC16 ORF68-TS | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | BAC16 mutant containing ORF68-TS | |
| Genetic reagent (Kaposi’s sarcoma-associated herpesvirus) | KSHV BAC16 ORF68-TS-MR | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | BAC16 mutant containing ORF68-TS-MR | |
| Cell line (Homo sapiens) | HEK-293T | ATCC | CRL-3216 | This cell line is commercially available from ATCC |
| Cell line (Homo sapiens) | iSLK-puro | Myoung and Ganem, 2011; Stürzl et al., 2013 | PMID: 21419799; PMID: 22987579 | Renal-cell carcinoma cell line containing doxycycline-inducible KSHV RTA |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 WT | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | iSLK-puro latently infected with KSHV derived from BAC16 WT | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68.stop | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | iSLK-puro latently infected with KSHV derived from BAC16 ORF68.stop | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68.stop-MR | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | iSLK-puro latently infected with KSHV derived from BAC16 ORF68.stop-MR | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68-K435A | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | iSLK-puro latently infected with KSHV derived from BAC16 ORF68-K435A | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68-K435A-MR | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | iSLK-puro latently infected with KSHV derived from BAC16 ORF68-K435A-MR | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68-K174A/R179A/K182A | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | iSLK-puro latently infected with KSHV derived from BAC16 ORF68- K174A/R179A/K182A | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68- K174A/R179A/K182A -MR | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | iSLK-puro latently infected with KSHV derived from BAC16 ORF68- K174A/R179A/K182A -MR | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68-TS | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | iSLK-puro latently infected with KSHV derived from BAC16 ORF68-TS | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68-TS-MR | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | iSLK-puro latently infected with KSHV derived from BAC16 ORF68-TS-MR | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68.stop + pLJM1-zeo-empty | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | Lentivirally transduced iSLK-BAC16 ORF68.stop | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68.stop + pLJM1-zeo-2xStrep-ORF68 | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | Lentivirally transduced iSLK-BAC16 ORF68.stop | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68.stop + pLJM1-zeo-2xStrep-ORF68-C52A | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | Lentivirally transduced iSLK-BAC16 ORF68.stop | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68.stop + pLJM1-zeo-2xStrep-mu68 | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | Lentivirally transduced iSLK-BAC16 ORF68.stop | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68.stop + pLJM1-zeo-2xStrep-BFLF1 | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | Lentivirally transduced iSLK-BAC16 ORF68.stop | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68.stop + pLJM1-zeo-2xStrep-UL52 | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | Lentivirally transduced iSLK-BAC16 ORF68.stop | |
| Cell line (Homo sapiens, Kaposi’s sarcoma-associated herpesvirus) | iSLK-BAC16 ORF68.stop + pLJM1-zeo-UL32 | This paper; 'Materials and methods' (Cell line establishment and viral mutagenesis) | Lentivirally transduced iSLK-BAC16 ORF68.stop | |
| Antibody | Anti-vinculin (rabbit polyclonal) | Abcam | Cat. no. ab91459 | WB primary (1:1000) |
| Antibody | Strep-Tag II HRP conjugate | Novagen | Cat. no. 71591-3 | WB primary (1:2500) |
| Antibody | Anti-K8.1 (rabbit polyclonal) | Gardner and Glaunsinger, 2018 | PMID: 29875246 | WB primary (1:10,000) |
| Antibody | Anti-ORF68 (rabbit polyclonal) | Gardner and Glaunsinger, 2018 | PMID: 29875246 | WB primary (1:5000) |
| Antibody | Anti-ORF6 (Rabbit polyclonal) | Gardner and Glaunsinger, 2018 | PMID: 29875246 | WB primary (1:10,000) |
| Antibody | Anti-GAPDH (mouse monoclonal) | Abcam | Cat. no. ab8245 | WB primary (1:5000) |
| Antibody | Anti-rabbit IgG-HRP (goat polyclonal) | Southern Biotech | Cat. no. 4030-05 | WB secondary (1:5000) |
| Antibody | Anti-mouse IgG-HRP (goat polyclonal) | Southern Biotech | Cat. no. 1031-05 | WB secondary (1:5000) |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162625 | For transient expression of ORF68 |
| Recombinant DNA reagent | pcDNA4/TO-ORF68-2xStrep | Davis et al., 2015 | Addgene: 136229 | For transient expression of ORF68 |
| Recombinant DNA reagent | pcDNA4/TO-ORF68 (tagless) | This paper; 'Materials and methods' (Plasmids) | Addgene: 166025 | For transient expression of ORF68 |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-BFLF1 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162626 | For transient expression of BFLF1 |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-mu68 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162627 | For transient expression of mu68 |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-UL52 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162628 | For transient expression of UL52 |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-UL32 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162629 | For transient expression of UL32 |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 C52A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162630 | For transient expression of ORF68 C52A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 C373A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162631 | For transient expression of ORF68 C373A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 C191A/C192A | This paper; 'Materials and methods' (Plasmids) | Addgene:162632 | For transient expression of ORF68 C191A/C192A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 C415A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162633 | For transient expression of ORF68 C415A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 H452A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162634 | For transient expression of ORF68 H452A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 C79A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162635 | For transient expression of ORF68 C79A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 K435A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162636 | For transient expression of ORF68 K435A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 R443A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162637 | For transient expression of ORF68 R443A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 K450A/R451A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162638 | For transient expression of ORF68 K450A/R451A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 K174A/R179A/K182A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162639 | For transient expression of ORF68 K174A/R179A/K182A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 R14A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162640 | For transient expression of ORF68 R14A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 K310A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162641 | For transient expression of ORF68 K310A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 R14A/K310A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162642 | For transient expression of ORF68 R14A/K310A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-ORF68 K395A/K396A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162643 | For transient expression of ORF68 K395A/K396A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-UL32 C128A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162644 | For transient expression of UL32 C128A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-UL32 C502A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162645 | For transient expression of UL32 C502A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-UL32 C308A/C309A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162646 | For transient expression of UL32 C308A/C309A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-UL32 C544A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162647 | For transient expression of UL32 C544A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-UL32 H581A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162648 | For transient expression of UL32 H581A |
| Recombinant DNA reagent | pcDNA4/TO-2xStrep-UL32 C155A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162649 | For transient expression of UL32 C155A |
| Recombinant DNA reagent | pUE1-TSP-ORF68 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162650 | For transient overexpression of ORF68 |
| Recombinant DNA reagent | pUE1-TSP-ORF68 K435A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162651 | For transient overexpression of ORF68 K435A |
| Recombinant DNA reagent | pUE1-TSP-ORF68 R443A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162652 | For transient overexpression of ORF68 R443A |
| Recombinant DNA reagent | pUE1-TSP-ORF68 K450A/R451A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162653 | For transient overexpression of ORF68 K450A/R451A |
| Recombinant DNA reagent | pUE1-TSP-ORF68 K174A/R179A/K182A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162654 | For transient overexpression of ORF68 K174A/R179A/K182A |
| Recombinant DNA reagent | pUE1-TSP-ORF68 R14A/K310A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162655 | For transient overexpression of ORF68 R14A/K310A |
| Recombinant DNA reagent | pUE1-TSP-ORF68 K395A/K396A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162656 | For transient overexpression of ORF68 K395A/K396A |
| Recombinant DNA reagent | pUE1-TSP-BFLF1 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162657 | For transient overexpression of BFLF1 |
| Recombinant DNA reagent | pUE1-TSP-UL32 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162658 | For transient overexpression of UL32 |
| Recombinant DNA reagent | pLJM1-zeo-empty | Gardner and Glaunsinger, 2018 | PMID: 29875246 | Empty lentiviral vector |
| Recombinant DNA reagent | pLJM1-zeo-2xStrep-ORF68 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162659 | Lentiviral vector for stable expression of ORF68 |
| Recombinant DNA reagent | pLJM1-zeo-2xStrep-ORF68 C52A | This paper; 'Materials and methods' (Plasmids) | Addgene: 162660 | Lentiviral vector for stable expression of ORF68 C52A |
| Recombinant DNA reagent | pLJM1-zeo-2xStrep-BFLF1 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162661 | Lentiviral vector for stable expression of BFLF1 |
| Recombinant DNA reagent | pLJM1-zeo-2xStrep-mu68 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162662 | Lentiviral vector for stable expression of mu68 |
| Recombinant DNA reagent | pLJM1-zeo-2xStrep-UL52 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162663 | Lentiviral vector for stable expression of UL52 |
| Recombinant DNA reagent | pLJM1-zeo-UL32 | This paper; 'Materials and methods' (Plasmids) | Addgene: 162664 | Lentiviral vector for stable expression of UL32 |
| Recombinant DNA reagent | psPAX2 | A gift from Didier Trono | Addgene: 12260 | Lentiviral packaging plasmid |
| Recombinant DNA reagent | pMD2.G | A gift from Didier Trono | Addgene: 12259 | Lentiviral packaging plasmid |
| Peptide, recombinant protein | HRV 3C protease | Millipore Sigma | Cat. no: 71493 | For removal of Twin-Strep tag during protein purification |
| Commercial assay or kit | In-Fusion HD Cloning kit | Clontech | Cat. no. 639650 | For cloning |
| Commercial assay or kit | NucleoBond BAC 100 kit | Macherey Nagel | Cat. no. 740579 | For preparation of BAC DNA |
| Commercial assay or kit | NucleoSpin Blood kit | Macherey Nagel | Cat. no. 740951.50 | For isolation of DNA from iSLK BAC16 cell lines |
| Commercial assay or kit | iTaq Universal SYBR Green Supermix | Bio-Rad | Cat. no. 1725122 | For qPCR assays |
| Commercial assay or kit | DIG High Prime DNA Labeling and Detection Starter Kit II | Roche | Cat. no. 11585614910 | For probe labeling and detection of Southern blots |
| Software, algorithm | CTTFFIND4 | PMID: 26278980 | RRID: SCR_016732 | |
| Software, algorithm | Gautomatch | K. Zhang (MRC-LMB (https://www2.mrc-lmb.cam.ac.uk/research/locally-developed-software/zhang-software/)) | ||
| Software, algorithm | Relion | PMID: 23000701 | RRID: SCR_016274 | |
| Software, algorithm | SerialEM | PMID: 16182563 | RRID: SCR_017293 | |
| Software, algorithm | Motioncor2 | PMID: 28250466 | RRID: SCR_016499 | |
| Software, algorithm | Coot | PMID: 20383002 | RRID: SCR_014222 | |
| Software, algorithm | phenix.refine | PMID: 20124702, 22505256, 31588918 | RRID: SCR_016736 | |
| Software, algorithm | XDS | PMID: 20124692 | RRID: SCR_015652 | |
| Software, algorithm | POINTLESS | PMID: 21460446 | RRID: SCR_014218 | |
| Software, algorithm | STARANISO server | STARANISO (staraniso.globalphasing.org) | RRID: SCR_018362 | |
| Software, algorithm | PHASER | PMID: 19461840 | RRID: SCR_014219 | |
| Software, algorithm | PyMol | PyMol (pymol.org) | RRID: SCR_000305 | |
| Software, algorithm | APBS | PMID: 11517324 | RRID: SCR_008387 | |
| Software, algorithm | ConSurf Server | PMID: 27166375 | RRID: SCR_002320 | |
| Software, algorithm | SWISS-MODEL | PMID: 29788355 | RRID: SCR_018123 | |
| Software, algorithm | Clustal Omega | PMID: 30976793 | RRID: SCR_001591 | |
| Software, algorithm | GraphPad Prism | GraphPad | RRID: SCR_002798 | Version 8 |
| Other | Strep-Tactin Superflow high-capacity 50% suspension | IBA Lifesciences | Cat. no. 2-4030-025 |