JCV Viral Genome Replication Assay

Replication assays were carried out as previously described [10 (link)]. Briefly, PHFG cells or SVG-A cells (2 × 10⁶ cells) grown in 75 cm² flasks were transfected either with JCV Mad-1 WT or JCV Mad-1 Pt mutant viral genome (8 μg/2 × 10⁶ cells/75 cm² flask) by lipofectin-2000 transfection method. Of note, The pBluescript (back bone) vector from both JCV and SV40 plasmids was digested with BamH I before using the plasmids in transfections. Lipofectin-DNA mixture was incubated with cells for 5 h and washed with PBS. Transfected SVG-A cells were fed with complete D-MEM media with 3% FBS. Transfected PHFG cells were fed with a special growth media D-MEM+F12 media containing 10% FBS, L-Glutamine (2 mM), gentamycin (50 mg/l) and insulin (2.5 mg/l). At indicated time points posttransfection, low-molecular-weight DNA containing both input and replicated viral DNA was isolated by the Hirt method [41 (link)], digested with BamH I and Dpn I enzymes, resolved on a 1% agarose gel and analyzed by Southern blotting.