Efficient propagation of archetype BK and JC polyomaviruses

Abstract

BKPyV and JCPyV are closely related, ubiquitious human pathogens that cause disease in immunocompromised patients. The DNA sequence of the regulatory regions distinguishes two forms of these viruses, designated archetype and rearranged. Although cell culture systems exist for rearranged BKPyV and JCPyV, currently there is no robust cell culture system to study the archetype viruses. Large T antigen (TAg) is a virally encoded protein required to initiate viral DNA synthesis. Because archetype virus produces undetectable levels of TAg, we hypothesized that TAg overexpression would stimulate archetype virus replication. Efficient propagation of the archetype forms of BKPyV and JCPyV was observed in 293TT cells, human embryonic kidney cells overexpressing SV40 TAg. Importantly, the archetypal structure of the regulatory region was maintained during viral growth. Significant replication was not observed for Merkel cell, KI, or WU polyomaviruses. 293TT cells provide a means of propagating archetype BKPyV and JCPyV for detailed study.

miRNA regulation of BK polyomavirus replication during early infection

“…2A). The WT and miRNA mutant viruses were purified from 293TT cells, which allow both archetype virus and rearranged variants to replicate (8). RNA was harvested from infected 293TT cells and Northern blotting confirmed that the mutant virus does not produce its mature 5P and 3P miRNAs (Fig.…”

“…Archetype virus and rearranged variant miRNA mutated viruses were created using site-directed mutagenesis as described in SI Materials and Methods and a list of primers used is presented in Table S1. The WT and mutant viral genomes were transfected into 293TT cells to produce crude lysates (8). Viral stocks were expanded in 293TT cells and purified by density centrifugation on Cesium chloride gradients (30).…”

“…Viral stocks were expanded in 293TT cells and purified by density centrifugation on Cesium chloride gradients (30). Viral titers were determined by VP1 IU assay (8). We set the limit of detection for the IU assay as a minimum of 20 foci per field of view.…”

“…Rearranged variants, but not archetype virus, produce progeny in primary renal proximal tubule epithelial (RPTE) cells, a natural cell culture model of lytic replication (7). We have previously shown that archetype virus produces undetectable levels of TAg and very low, if any, viral DNA replication in RPTE cells (8). Therefore, there is currently no natural cell culture model of archetype virus replication.…”

Untitled

“…Therefore, BK polyomavirus may be reactivated when the immune system is suppressed. 7 Virus reactivation is a main complication after renal transplant, inducing BK virus-associated nephropathy (BKVAN). [4][5][6]8 BK virus-associated nephropathy is characterized by inflammatory interstitial nephropathy.…”