Establishing Renal Proximal Tubule Epithelial-Derived Cell Lines Expressing Human Telomerase Reverse Transcriptase for Studying BK Polyomavirus

We previously established an infection model for BK polyomavirus (BKPyV) in primary human renal proximal tubule epithelial (RPTE) cells. Use of these cells is limited by their inability to be passaged extensively. Here, we describe RPTE cells immortalized with human telomerase reverse transcriptase (hTERT), which can serve as a model system for acute or persistent BKPyV infection.

48 and 72 h posttransfection and filtered with a 0.45-m polyethersulfone filter (MilliporeSigma). Filtered medium was overlaid on 20% sucrose in 1ϫ phosphatebuffered saline, and lentivirus was concentrated by centrifuging at 24,000 rpm for 2 h (AH-629 rotor). Pelleted lentiviruses were resuspended in complete REGM/REBM. RPTE cells at passage 3 were grown in REGM/REBM medium in a 10-cm dish. hTERT-expressing lentivirus at a multiplicity of infection (MOI) of 0.3 was directly added to the cells and inoculated at 37°C overnight. Cells were passaged 3 days postransduction and further passaged at 70% confluence until passage 20 to select against nontransduced cells. Single RPTE-hTERT subclones were isolated by seeding hTERTtransduced cells at passage 20 in 96-well plates at a concentration of 0.2 cells per well. Subclones were subsequently expanded in 6-well plates and 10-cm dishes before freezing down aliquots in REBM/REGM with 10% dimethyl sulfoxide (DMSO) and 10% fetal bovine serum (FBS) in liquid nitrogen. hTERT integration was confirmed by amplifying hTERT-WPRE fragment from cellular genomic DNA and Sanger sequencing (data not shown). Images of RPTE-hTERT cells are shown in Fig. 1B.
We believe that the RPTE-hTERT cell line will be a useful tool for studying biological aspects of BKPyV infection that cannot be performed in primary cells with limited life spans in culture.
Data availability. RPTE-hTERT cells are provided upon request by contacting the corresponding author.

ACKNOWLEDGMENTS
We thank Eric Campeau and Paul Kaufman for gifting the pLenti CMV GFP Puro plasmid through Addgene.
This report was supported in part by a Comprehensive Cancer Center Core grant from the National Cancer Institute of the National Institutes of Health (NIH) under award number P30 CA046592 and by NIH grant R01 AI060584 awarded to M.J.I.