Journal: Scientific Reports

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF34 is essential for late gene expression and virus production

doi: 10.1038/s41598-017-00401-7

Figure Lengend Snippet: Construction of recombinant ΔORF34 KSHV BAC and its revertant. ( a ) Schematic illustration of the KSHV genome including the ORF34 coding region. Using a two-step Red recombination system, three stop codons were inserted into the ORF34 coding region of KSHV BAC16 (nt54653 – nt54654; Accession number: GQ994935) to construct ORF34-deficient BAC clone (ΔORF34-BAC16). The stop codons were replaced with the original wild-type sequence for revertant BAC clone (Revertant-BAC16). ( b ) Agarose-gel electrophoresis of the recombinant KSHV BACmids, digested with EcoRI. The asterisks (*) indicate Insertion and deletion of a kanamycin-resistance cassette in each BAC clone. Original data is shown in Supplementary Fig. . ( c ) DNA sequencing results of ORF34 mutagenesis sites in ΔORF34-BAC16 and Revertant-BAC16.

Article Snippet: To construct pENTR4-KSHV RTA/ORF50, KSHV RTA/ORF50 fragments were obtained by PCR from KSHV genome harboring PEL cells (BC3) and inserted into pENTR4 no ccdB (686-1), a gift from Dr. Eric Campeau (Addgene plasmid # 17424) .

Techniques: Recombinant, Construct, Sequencing, Agarose Gel Electrophoresis, DNA Sequencing, Mutagenesis

Journal: Scientific Reports

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF34 is essential for late gene expression and virus production

doi: 10.1038/s41598-017-00401-7

Figure Lengend Snippet: ORF34 is essential for virus production but not DNA replication of KSHV. ( a ) The effects of ORF34 deficiency on recombinant KSHV production. Each iVero cell line (iVero-WT, iVero-ΔORF34, or iVero-Revertant) were treated with Dox and NaB to induce the lytic-cycle and viral production, and cultured media was harvested after 96 h. Viral precipitates were inoculated onto cells (Vero and 293T) and infection with each KSHV was carried out for 48 h. GFP positive cells were analyzed by flow-cytometer to determine the infectivity of the recombinant viruses. The forward scatter/side scatter plots and gates of Fig. 2a are shown in Supplementary Fig. . ( b ) Virus production in iVero-WT, iVero-ΔORF34, iVero-Revertant. Each iVero cell line was cultured for 48 h with medium containing of NaB and Dox. KSHV DNA were purified from capsidated KSHV virions in culture supernatants, and KSHV genome copies were determined by real-time PCR. ( c ) KSHV DNA replication in iVero-WT, iVero-ΔORF34, and iVero-Revertant cells. The iVero cells were cultured for 48 h with medium containing of NaB and Dox. Cellular genome containing KSHV genome DNA were purified from each cell lines. KSHV genome copies were determined by real-time PCR and normalized by the total DNA amount. ( d ) Rescue of virus production in iVero-ΔORF34 cells by exogenous ORF34 expression. The iVero-WT or iVero-ΔORF34 cells were transfected with control plasmid or ORF34 plasmids. After 2 days, transfected cells were cultured with NaB and Dox-containing medium for 3 days, and culture supernatant containing virus was harvested. The KSHV genome was quantified by real-time PCR. ( b – d ) Three or four independent samples were evaluated by real-time PCR. The error bars indicate standard deviations.

Article Snippet: To construct pENTR4-KSHV RTA/ORF50, KSHV RTA/ORF50 fragments were obtained by PCR from KSHV genome harboring PEL cells (BC3) and inserted into pENTR4 no ccdB (686-1), a gift from Dr. Eric Campeau (Addgene plasmid # 17424) .

Techniques: Virus, Recombinant, Cell Culture, Infection, Flow Cytometry, Purification, Real-time Polymerase Chain Reaction, Expressing, Transfection, Control, Plasmid Preparation

Journal: Scientific Reports

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF34 is essential for late gene expression and virus production

doi: 10.1038/s41598-017-00401-7

Figure Lengend Snippet: ORF34 physically interacts with ORF24, ORF31, ORF18, ORF23, and ORF66. Cells (293T) were co-transfected with 2xS-tagged ORF34 and 6xMyc-ORF24 ( a ), 3xFLAG-ORF31 ( b ), 3xFLAG-ORF18 ( c ), 3xFLAG-ORF66 ( d ), 3xFLAG-ORF30 ( e ), or 6xMyc-ORF23 ( f ), upper panel) expression plasmids. Cell lysates were subjected to pull-down assays using S-protein-immobilized beads, which captures S-tagged ORF34. The precipitates were immunoblotted with anti-Myc or FLAG antibody to detect their interactions. ( e ) The pull-down sample was precipitated from cell extracts co-transfected with 2xS-ORF34 and 3xFLAG-ORF31, was applied in the left lane as the positive control. ( f ), lower panel) The pull-down sample was precipitated from cell extracts co-transfected with 2xS-ORF23 and 6xMyc-ORF34. ( a – f ) Original images of blotting data are shown in Supplementary Figs – . ( g ) Schematic depiction of the interactions between KSHV ORF34 and KSHV-PIC component candidates.

Article Snippet: To construct pENTR4-KSHV RTA/ORF50, KSHV RTA/ORF50 fragments were obtained by PCR from KSHV genome harboring PEL cells (BC3) and inserted into pENTR4 no ccdB (686-1), a gift from Dr. Eric Campeau (Addgene plasmid # 17424) .

Techniques: Transfection, Expressing, Positive Control

Journal: Scientific Reports

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF34 is essential for late gene expression and virus production

doi: 10.1038/s41598-017-00401-7

Figure Lengend Snippet: The interaction of the ORF34 C-terminal region and ORF24 is essential for virus production. The iVero-ΔORF34 cells were transfected with control, ORF34 or a ORF34-deletion mutant plasmid. After 2 days, transfected cells were stimulated with NaB and Dox for 3 days. Progeny KSHV was purified from harvested culture supernatant, and the KSHV genome was quantified by real-time PCR. Viral productivities were assessed using three independent samples, and error bars indicate standard deviations. Statistical significance between each group and the control was analyzed by two-tailed Student’s t -test (N.S.: not significant, P > 0.05).

Article Snippet: To construct pENTR4-KSHV RTA/ORF50, KSHV RTA/ORF50 fragments were obtained by PCR from KSHV genome harboring PEL cells (BC3) and inserted into pENTR4 no ccdB (686-1), a gift from Dr. Eric Campeau (Addgene plasmid # 17424) .

Techniques: Virus, Transfection, Control, Mutagenesis, Plasmid Preparation, Purification, Real-time Polymerase Chain Reaction, Two Tailed Test