Cell lines, plasmids, and transfections.

TREx-BCBL-1-RTA cells (a kind gift from Jae Jung, University of Southern California) are a BCBL-1-based cell line that has been engineered to inducibly express exogenous Myc-tagged RTA by the addition of 1 μg/ml doxycycline hyclate, leading to a robust reactivation of the full KSHV lytic cycle (53); these were cultured in RPMI 1640 (Lonza) supplemented with 10% fetal bovine serum (FBS) (Life Technologies). Inducible SLK-BAC16 cells (iSLK-BAC16; also a gift from Jae Jung) were grown in Dulbecco modified Eagle medium (DMEM) (Lonza) supplemented with 10% FBS (Life Technologies). These maintain a latent infection with bacterial artificial chromosome 16 (BAC16)-derived KSHV (54). The parental SLK cell line, originally considered KSHV-negative endothelial cells derived from a KS patient, was subsequently found to be a contaminant of a renal carcinoma cell line, Caki1 (55). Nevertheless, these cells are widely used as a model for the study of KSHV biology. Initially, these cells were engineered to inducibly express RTA (iSLK) following the addition of doxycycline (56) and subsequently transfected with BAC16 and selected based on BAC-derived puromycin resistance (54). This cell line was demonstrated to support an authentic latent infection, and induction of the lytic cycle (with 1 μg/ml doxycycline hyclate) leads to the release of infectious virus (54).

HEK293T cells were used for reinfection assays as previously described (57). HEK293 rKSHV.219 cells (58) maintain KSHV as a latent infection and were generated by infecting HEK293T cells with a recombinant KSHV that contains a constitutively active puromycin resistance and green fluorescent protein (GFP) gene and a red fluorescent protein (RFP) gene that is fused to an RTA-responsive lytic cycle (PAN) promoter (not utilized in this study). Both HEK293T-based cell lines were maintained in DMEM (Lonza) supplemented with 10% FBS (Life Technologies).

To generate cells with inducible N-terminal FLAG-His-tagged RTA expression (iRTA-293), RTA was amplified from pRTS-ORF50 (14) (forward primer, 5′-ATCTTAAGGCCACCATGGATTATAAAGATGACGATGACAAGCATCATCATCATCATCATGCGCAAGATGACAAGGGTAAG-3′; reverse primer, 5′-ATCTCGAGTCAGTCTCGGAAGTAATTACG-3′) and ligated into the AflI-XhoI sites of pcDNA5-FRT-TO (Life Technologies) to generate pcDNA5-FH-RTA. Functionality of this vector was demonstrated due to its ability to reactivate the KSHV lytic cycle following its transfection. Flp-In-293 cells (Life Technologies) were transfected along with pOG44 (Flp recombinase) and subsequently selected using hygromycin B according to the manufacturer's protocol (Life Technologies). Clonal populations were generated by limiting dilution under hygromycin B selection, and clones with tightly regulated expression and normal growth properties (compared to parental cells) were selected. RTA expression was induced following treatment with 1 μg/ml doxycycline hyclate for the indicated times. iRTA-293 cells were maintained in DMEM (Lonza) supplemented with 10% FBS (Life Technologies).

The vector expressing C-terminally FLAG-tagged ARID3B was generated by PCR amplification using an ARID3B-containing plasmid (kind gift from Karen Cowden Dahl, Indiana University) as the template (forward primer, 5′-CGCGGATCCAAGCGATGGAGCCACTTCAGCAGCAGCAGCA-3′; reverse primer, 5′-CGCGAATTCTCACTTATCGTCGTCATCCTTGTAATCGAGGGACCAGCTGGTGGAGGGCTC-3′). Products were digested and ligated into the BamHI and EcoRI sites of pcDNA3 (pcDNA3-ARID3B-FLAG). FLAG-tagged SRAG was a kind gift from Stuart Wilson (University of Sheffield). Expression constructs were verified by DNA sequencing.

For transfections, cells were plated into 6-well plates, and transfections routinely used 1 μg plasmid DNA and Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions.

To determine the effects of overexpressed proteins on virus reactivation efficiencies, iSLK-BAC16 cells were transfected for 24 h and the lytic cycle was induced by doxycycline treatment for a further 24 h. Samples were processed for immunoblot analysis (see below).