to the linear TA cloning vector pCR2.1 (Invitrogen) and sequenced. The tAg cDNA contained one mutation, an artifact of the PCR amplification step, at nucleotide (nt) 4605. The resulting plasmid was digested with EcoNI and PflMI to isolate the tAg cDNA, which was ligated to pCMV:T+/t2/T9+ from which the corresponding sequence had been removed. The resulting mutant tAg expression vector, pCMV:T2/mut+/T’2, was sub- jected to PCR-mediated site-directed mutagenesis to correct the point mutation using primers WTtAgL (59-GCA ATC AAA GCA ATA GCA ATC TAT CCA CAC AAG TGG GC -39), and WTtAgE (59-GCC CAC TTG TGT GGA TAG ATT GCT ATT GCT TTG ATT GC-39), which span the coding sequence from nt 4628–4591. The tAg coding region of the PCR product was confirmed by DNA sequence analysis. The sequenced region, which contained the wild type tAg cDNA sequence, was isolated and ligated to the pCMV:T2/mut+/T’2 backbone with the corresponding region removed. This step ensured that any secondary mutations inadvertently introduced via PCR would not be present in the final construct. Next, the tAg cDNA region was isolated following BstXI and PstI digestion and ligated to pJR:T+/t+/T9+ with the corresponding sequence removed, to create pJR:T2/t+/T’2. The expected structure of the rDNA was confirmed by DNA sequence analysis. pJR:T+/t+/T9+ and pSR:T+/t+/T9+ contain wild-type JCV coding sequences linked to either the JCV or SV40 regulatory region, respectively. Each plasmid was used as a template for site- directed mutagenesis to generate constructs encoding P99A or C157A mutant tAgs. Complementary primers P99AR (59-CC CTT TAT TGC AAG GAA TGG GCT AAC TGT GCC ACT AAT CC-39) and P99AF( 59-GG ATT AGT GGC ACA GTT AGC CCA TTC CTT GCA ATA AAG GG-39), which span the coding sequence from nt 4739–4700, or C157AF (59-CCC AAG AAG CTC TTC ATT CCT GGG AGA AAG TTC TTG G-39) and C157AR (59-CCA AGA ACT TTC TCC CAG GAA TGA AGA GCT TCT TGG G -39), which span the coding sequence from nt 4562–4526, were used. Altered nucleotides are in bold and underlined. The PCR products were sequenced, and the regions of interest were removed by BstXI and PflMI digestion. The fragments containing the point mutations were ligated to pJR:T+/t+/T9+ or pSR:T+/t+/T9+ backbone vectors to create pJR:T+/P99At+/T9+, pJR:T+/C157At+/T9+, pSR:T+/P99At+/ T9+ and pSR:T+/C157At+/T9+. The structure of each construct was confirmed by DNA sequence analysis. To generate pJR:T2/H42Qt+/T92, PCR-mediated site-specific mutagenesis was performed on pSR:T+/t+/T9+ using primers H42Qf (59-CCCCACCTTTATCAGGTTGGAGTTCTTTGC- 39) and H42Qr (59-GCAAAGAACTCCAACCTGATAAAGGT- GGGG-39), which span the coding sequence from nt 4901–4872. The region spanning the H42Q mutation in the cloned PCR amplicon was subjected to DNA sequence analysis, isolated by restriction enzyme digestion, and ligated to the pJR:T2/t+/T92 backbone from which the corresponding fragment was removed via digestion. The rDNA structure was confirmed by DNA sequence analysis. Cell Culture Primary human fetal glial (PHFG), Rat2, mouse embryo fibroblast (MEF) and 3T3 (3T3-Swiss albino; American Type Culture Collection) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (PHFG) or 5% (R2, MEF, 3T3 cells) fetal bovine serum (FBS), 100 U/ml penicillin and 0.1 mg/ml streptomycin. U-87MG cells were cultured in minimum essential Eagle’s medium (MEM) with JCV Small t Protein Functions 10% FBS, 2% sodium pyruvate, 100 U/ml penicillin and 0.1 mg/ ml streptomycin. All cells were incubated at 37uC in 10% CO2. Generation of Cell Lines To create cell lines stably expressing all 5 JCV early proteins (T+/t+/T9+, T+/P99At+/T9+, T+/C157At+/T9+), or small tAg alone (T2/t+/T92), 3T3 cells in 100 mm dishes were co- transfected with 5 mg of DNA encoding JCV proteins driven by SV40 promoter-enhancer signals, and 0.5 mg of pCR3, which confers G418 [geneticin] resistance. Cells were selected for G418 resistance in the presence of 400 mg/ml geneticin in complete growth medium. Isolated colonies arising from single cells were transferred to new plates at 14 to 21 days post-transfection (p.t.). Protein extracts prepared from these cells were subjected to immunoprecipitation (IP) and Western blot (WB) analyses to confirm viral protein expression. Immunoprecipitation and Western Blot (IP/WB) Analysis Cell extracts were prepared with EBC lysis buffer (50 mM Tris, pH 8.0, 120 mM NaCl, 0.5% NP-40) supplemented with protease and phosphatase inhibitors (2 mg/ml leupeptin, 2 mg/ml E-64, 1 mg/ml aprotinin, 0.25 mM pefabloc, 1 mM sodium vanadate, 5 mM sodium fluoride, 25 mM b-glycerophosphate and 5 mM EDTA). Protein concentrations were determined by the Bradford assay using a micro protein assay (Bio-Rad Laboratories). Following initial IP experiments, the amounts of total protein in different cell lysates were adjusted to yield equivalent levels of tAg. These protein extracts were incubated with either the anti-JCV TAg monoclonal antibody PAb 962 [36] or anti-p107 antibody (C-18; Santa Cruz Biotechnology, Inc.) for one hour or with anti- PP2A-C antibody (clone ID6; Upstate Biotechnology) overnight on a rotary shaker at 4uC. Protein-antibody complexes were collected on Staph A (PANSORBIN; Calbiochem). After washing the pellets 3 times with EBC buffer, the protein complexes were disrupted by the addition of loading buffer and heating at 95uC for 4 minutes. Proteins were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated with a cocktail of anti-TAg monoclonal antibodies (PAb 962, 2000, 2001, 2003, 2024, 2030; [7]) to enhance detection, or with anti-p107 antibody. After exposing the membranes to BCIP/NBT (Sigma), secondary antibodies conju- gated to alkaline phosphatase permitted visualization of the proteins. DpnI DNA Replication Assay JCV DNA-containing vectors were digested with EcoRI to separate viral and plasmid DNAs. The linear viral DNA was self- ligated under dilute conditions to produce circular viral DNA. PHFG cells were seeded onto 60 mm plates and transfected 24 hours later with 100 ng of circular viral DNA using a modified DEAE-dextran protocol [37,38]. At various time points low molecular weight DNA was extracted from the cells using the Hirt protocol [39]. This DNA was cleaved with DpnI and EcoRI, and digestion products were electrophoresed on a 0.8% agarose gel. DNA was transferred to a nitrocellulose membrane using a Rapid Downward Transfer System and an alkaline transfer protocol (Schleicher and Schuell, Amersham). After exposure to UV light for 5 minutes to cross-link the DNA to the membrane, the DNA was hybridized with linear JCV DNA labeled with [a-32P] dCTP (Prime-a-Gene kit, Promega). Replication activity was determined by quantitating band intensities of linear DpnI- resistant viral DNA using ImageQuant 5.2 software (Molecular Dynamics). PLoS ONE | www.plosone.org 3 May 2010 | Volume 5 | Issue 5 | e10606