Plasmids and expression vectors
Two short hairpin RNAs (shRNA) for vIRF-1 were cloned into pYNC352/puro (a derivative of pYTF 
) using Bam
HI and Mlu
I enzyme sites; target sequence of the shRNAs correspond to 5′- AGCCGGACACGACAACTAAGA -3′
(sh1) and 5′-ATCAAGGATTGGATAGTATGT-3′
(sh2). Sequences specifying wild-type or mutated forms of vIRF-1 were cloned into lentivirus vector pYNC352/SV40/puro using Mlu
I and Bam
HI cloning sites. BimEL
cDNA sequences linked to Flag were cloned between the Bam
HI and Eco
RI sites of pcDNA3.1 (Invitrogen; Carlesbad, CA), for expression in transfected cells. Coding sequence for the nuclear localization signal (NLS) of SV40 large T antigen was inserted between the Hind
III and Bam
HI sites of pcDNA3.1-flag-BimEL
to generate a eukaryotic expression vector encoding NLS-flag-BimEL
. Bacterial expression plasmids for BimEL
and vIRF-1 were generated by cloning of the respective coding sequences into pTYB4 (New England Biolabs; Ipswich, MA) and pGEX-4T-1 (GE Life Sciences; Piscataway, NJ), using Nco
I and Sma
I sites and Bam
HI and Eco
RI sites, respectively. The BimEL
and vIRF-1 proteins were fused to intein/chitin-binding-domain (CBD) and GST, respectively, used for precipitation and purification via chitin- and glutathione-bead capture. Fas ligand promoter sequences encompassing 1.2-kb upstream of the initiator codon 
were amplified from BCBL-1 cell DNA by PCR and cloned between the Xho
I and Hind
III sites of pGL3/basic to provide a reporter construction responsive to IRF-1. The NLS and BBD coding sequences were cloned between Kpn
I and Age
I and Bsr
GI and Xba
I sites, respectively, of pEGFP-N1 (Clontech Laboratories; Mountainview, CA) to generate nuclear-directed GFP and GFP-BBD proteins. Coding sequences for wild-type or mutated vIRF-1 proteins were cloned between the Bam
HI and Mlu
I sites of pRetroX-Tight-Pur (Clontech Laboratories) to construct viral vectors for the generation of TIME cultures conditionally expressing the proteins (+Dox).
Cell culture, transfection and viral transduction
TIME cells 
were maintained in EGM-2 MV medium (Lonza, Walkersville, MD) containing 5% fetal bovine serum (FBS) and cytokine supplements. HEK293 and HEK293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS and gentamicin. BCBL-1 cells 
were cultured in RPMI 1640 supplemented with 20% heat-inactivated FBS and gentamicin. For lentivirus production, HEK293T cells were transiently transfected with virus vector and gag/pol-encoding packaging plasmids using standard calcium-phosphate precipitation method and virus harvested after 48 h by centrifugation at 49,000×g, essentially as described previously 
. Other transfections were performed using Lipofectamine 2000 (Invitrogen). Stable transduction of shRNA or cDNA into TIME cells using lentivirus vectors was performed under puromycin selection.
Cell extracts, immunoblotting and immunofluorescence
For whole cell extracts, cells were lysed in lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1% IGEPAL CA-630, 0.25% sodium deoxycholate, and protease inhibitor cocktail). For nucleo-cytoplasmic fractionation, cells were homogenized in buffer A (10 mM HEPES [pH 8.0], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and protease inhibitor) using a Dounce homogenizer. After centrifugation of the homogenate at 1,500×g, the supernatant was used as the cytoplasmic fraction and the pellet, after resuspended in buffer B (20 mM HEPES [pH 8.0], 1.5 mM MgCl2, 420 mM NaCl, and 0.2 mM EDTA), was used as the nuclear fraction. For immunoblotting, proteins were size fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Immunoreactive bands were detected with enhanced chemiluminescence solution (GE Healthcare, Piscataway, NJ) and visualized on X-ray film. For immunofluorescence assay (IFA), cells on a 0.1% gelatin-coated coverglass were fixed and permeabilized in chilled methanol. Following incubation with superblock blocking buffer (Thermo Scientific Inc., Rockford, IL), coverslips were incubated with primary antibody, washed with phosphate-buffered saline (PBS), and then incubated with appropriate fluorescent dye-conjugated secondary antibody. Coverslips were mounted in 90% glycerol in PBS containing 10 mg/ml p-phenylenediamine. Nuclei were visualized by staining with Hoechst 33342.
HHV-8 infection, replication, and quantitative PCR
Infectious HHV-8 was obtained by inducing BCBL-1 cells with phorbol 12-myristate 13-acetate (PMA/TPA; 20 ng/ml) and calcium ionophore (A23187; 500 ng/ml). After 20 h, cells were pelleted and resuspended in fresh medium without TPA and A23187. After four days, virions were pelleted from culture media by centrifugation at 27,000×g for 2 h in an SW41 rotor and resuspended in basal EGM-2 MV medium. For HHV-8 infection, TIME cells were centrifuged at 1,000×g for 1 h in the presence of HHV-8 virions, and then cultured in fresh complete medium for 7 days to allow establishment of latency in the absence of ongoing lytic replication. Lytic replication of HHV-8 in TIME cells was induced by treatment with TPA. For determination of encapsidated HHV-8 genome copy number, viral DNA was extracted using guanidinium thiocyanate (6M) and silica gel following pre-treatment of virus suspensions with DNaseI for 20 min. at 37°C. Excess HHV-8 bacmid DNA was treated with DNaseI and processed identically to control for DNaseI efficacy. All qPCRs were performed in a 96-well microplate using an ABI Prism 7500 detection system (Applied Biosystems; Foster City, CA) with SYBR green/ROX master mix (SuperArray Bioscience Corp.; Frederick, MD). Reactions were performed in a total volume of 25 µl, containing viral DNA sample and 250 nM of each primer. To calculate copy number of viral DNA, BAC-cloned HHV-8 genomic DNA was used as a standard. PCR conditions included an initial incubation step of 2 min. at 50°C, and enzyme heat activation step of 10 min at 95°C, followed by 45 cycles of 15 seconds at 95°C for denaturing and 1 min at 60°C for annealing and extension.
HEK293T cells were transiently transfected with plasmids expressing vIRF-1 and p53, Smad3 or IRF-1 along with reporter plasmids, PG13-luc (Addgene; Cambridge, MA), SBEx4-luc (Addgene), or Fas ligand promoter-luc (see above), respectively, for 24 h and then lysed with passive lysis buffer (Promega, Madison, WI). Luciferase activity was measured by standard methods using D-luciferin and luminometry.
Cell viability assay
Bim-induced cell death of HEK293 cells was monitored by cotransfection of pEGFP-N1 (Clontech laboratories, Mountain view, CA) and measuring fluorescence by fluorometry. For terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL), cells were fixed in chilled methanol for 5 min and preincubated in TdT reaction buffer (25 mM Tris-HCl [pH 6.6], 200 mM sodium cacodylate, 0.25 mg/ml BSA, and 1 mM CoCl2) for 10 min. TUNEL reactions were carried out at 42°C for 2 h in TdT reaction buffer containing TdT and biotin-dUTP (Roche, Indianapolis, IN) and terminated with stop solution (300 mM NaCl and 30 mM sodium citrate). TUNEL-positive cells were visualized by staining with FITC-avidin. LANA IFA was performed after TUNEL reactions, using LANA monoclonal antibody (Advanced Biotechnologies Inc; Columbia, MD) and staining with Cy-3-conjugated secondary antibody.
Protein co-precipitation assay
Glutathione-S-transferase (GST)-fusion proteins were purified by standard methods. Proteins from BimEL-transfected HEK293T or BCBL-1 cells were incubated with purified GST or GST-vIRF-1 proteins immobilized on glutathione beads. After washing with lysis buffer, the bead-precipitated material was subjected to SDS-PAGE and analyzed by immunoblotting using Bim- or GST-specific antibodies. For binding-site mapping, BimEL (lacking the C-terminal 18 residues, affecting solubility) was fused to intein-chitin binding domain (CBD) in pTYB4 and purified according to the manufacturer's protocol. GST or a series of GST-vIRF-1 fusions were incubated with the purified Bim protein immobilized on chitin beads. After washing with lysis buffer, bead-associated proteins were size-fractionated by SDS-PAGE and analyzed by immunoblotting using GST- or CBD-specific antibodies. For peptide competition assays, peptides (35-fold molar excess) were pre-incubated with BimEL-intein-CBD for 1 h before addition of GST-vIRF-1. Peptide sequences (the first 11 residues comprising Tat basic region) were: BBD-WT, YGRKKRRQRRRGGGRTRGLQEIGKGISQDGHH; BBD-Mut, YGRKKRRQRRRGGGRTRGLQEIAAGISQDGHH (altered residues underlined). For immunoprecipitation, HEK293T cells transfected with plasmids expressing Flag-BimEL or vIRF-1 were lysed in lysis buffer and cell extracts were incubated with anti-Flag antibody (M2) and immune-complexes precipitated with protein A/G-agarose. After washing with lysis buffer, immune-complexes were subjected to SDS-PAGE and analyzed by immunoblotting using vIRF-1 antiserum, Bim antibody or biotinylated Flag antibody and secondary, detection reagents comprising HRP-conjugated anti-Ig antibody or sptreptavidin-HRP.
K8.1 and LANA antibodies were purchased from Advanced Biotechnologies Inc (Columbia, MD). Antisera directed to vIRF-1 and ORF59 were provided by Drs. Gary Hayward and Bala Chandran, respectively. Actin and Flag antibodies were purchased from Sigma (St. Louis, MO), HDAC1 and GST antibodies from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA), Bim antibody from Cell Signaling Technologies, Inc. (Beverly, MA), GFP antibody from Epitomics, Inc. (Burlingame, CA), and CBD antibody from New England Biolabs, Inc. (Ipswich, MA).