Transfection experiments were carried out essentially as described previously (8). Briefly, viral DNA (1.5 μg/culture) was excised from recombinant plasmid and introduced into the cells using Lipofectamine (Invitrogen, Carlsbad, CA, USA). Thereafter, the transfected cells were transferred into 25-cm2 flasks containing culture medium and passaged at a split ratio of 1:3 or 1:4 every 3 or 4 days. Cells were harvested at 30, 43, and 50 days after transfection, and the HA titer was determined as described previously (8, 14). Experiments

were performed using four independent cultures. The transfected cells exhibited no obvious CPE and were able to be passaged serially for 3 weeks of incubation. Thirty days after transfection, obvious CPE selleck kinase inhibitor (rounding

of the cells) was observed in a small population of all COS-tat cell clones (data not shown). The cells were subjected to HA assay at 30, 43, and 50 learn more days after transfection. At 30 and 43 days after transfection, HA titers of COS-tat cell clones were significantly greater than those of parental COS-7 cells (Fig. 1a, b). In COS-tat7 cells, HA titer peaked at 43 days and remained unchanged up to 50 days after transfection (Fig. 1a–c). HA activity in COS-tat15 cells increased gradually from 30 to 50 days, with a peak at 50 days after transfection (640 ± 256 HA units) (Fig. 1a–c). HA activity in COS-tat 22 cells increased steeply up to 30 days compared to that in other COS-tat cell clones (Fig. 1a) and was similar to that in parental COS-7 cells at 50 days after transfection (Fig. 1c). These results indicate that stable expression of

HIV-1 Tat leads to increased production of PML-type JCV in COS-tat cells. The data also suggest that the kinetics of PML-type JCV propagation differ among COS-tat cell clones. To confirm HIV-1 Tat-mediated propagation of PML-type JCV, we examined the replication of viral genomic DNA in COS-tat cell clones. Total DNA was isolated from the above-mentioned HA samples using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) and subjected to real-time PCR analysis for quantification of JCV genomic DNA, essentially as described previously (8, 14). The detectable range of real-time PCR was more than 100 copies per reaction in this system (8, 14). The amount of viral DNA in COS-tat7, COS-tat15, and COS-tat22 cells was only significantly greater than that in parental COS7 cells at 30 days after transfection (Fig. 2a). In COS-tat7 cells, viral DNA level peaked at 43 days after transfection and declined at a later time point (Fig. 2b, c). The amount of viral DNA in COS-tat15 cells gradually increased from 30 to 50 days after transfection (Fig. 2a–c). In COS-tat22 cells, the amount of viral DNA increased steeply up to 30 days after transfection compared to other COS-tat cell clones. Although COS-tat22 cells exhibited a steep increase in the amount of viral DNA compared to other COS-tat cell clones on day 30, the amount decreased from 43 to 50 days (Fig. 2a–c).