A system for the analysis of BKV non-coding control regions: application to clinical isolates from an HIV/AIDS patient

Abstract

The human polyomavirus BK virus (BKV) is an important opportunistic pathogen whose disease prevalence continues to increase with the growing immunocompromised population. To date, the major determinant of replication in cell culture has not been formally proven. BKV exists as archetype virus and rearranged variants, which are classified based on the DNA sequence of their non-coding control regions (NCCRs). The archetype BKV NCCR is divided into five blocks of sequence and rearranged variants contain deletions and duplications of these blocks. In this study, a genetic system was developed and used to identify the major determinant of replication ability in primary renal proximal tubule epithelial cells, the natural host cell of BKV. This system was also used to analyze NCCR variants isolated from an immunocompromised patient which contain assorted rearrangement patterns and functional differences. This study solidifies the NCCR as the major genetic determinant of BKV replication ability in vitro.

Fig. 1

The NCCR determines replication efficiency of BKV. (A) Schematic of the swap genome with the archetype virus (Dik) NCCR. SpeI and SacII sites were inserted into the pBR322-Dunlop or –Dik vectors flanking the majority of the NCCR and a PmlI site was inserted between the early and late regions. (B) RPTE cells were transfected with recircularized viral genome and low molecular weight DNA was harvested 5 dpt. Samples were linearized, digested with DpnI, and analyzed by Southern blotting. The left panel shows Dik and Dunlop wt replication compared to the swap vectors containing the three inserted restriction enzyme sites, Dik3 and Dun3 respectively. The right panel shows all possible swap combinations. Each construct is designated by a three letter abbreviation. A=archetype and R=rearranged. The first letter denotes the NCCR; second letter, early region; third letter, late region. Marker, HindIII digest of pGEM-TU; Mock, mock transfection.

Fig. 2

(A) Schematic of the NCCRs of viruses isolated from a single clinical specimen. The NCCR is divided into blocks of sequence O, P, Q, R, and S. The block structures of the archetype virus (Dik), the rearranged variant (Dun), and the variants isolated from the patient are shown. The * represents the O block polymorphism and the † represents other mutations relative to the archetype P block sequence. The P′ and P″ blocks of Dunlop are variations of the P block. (B) A schematic of the Dik C65T and TCH2 T65C mutants created by site directed mutagenesis.

Fig. 3

Clones isolated from a clinical specimen display variable replication abilities. RPTE cells were transfected with recircularized viral genomes. (A, B) Low molecular weight DNA was harvested 4 dpt and analyzed as in Fig. 1. (A). Representative Southern blot. The two panels are different exposures of the same blot. Arrow indicates the DpnI-digested band used for normalization among samples. Mock, mock transfection. (B) Quantification of replication data from three independent experiments, error bars represent standard error of the mean.. (C) Viral lysates were prepared from parallel transfections at 7 dpt and assayed for infectious progeny as described in the Materials and Methods. Results are from three independent experiments, error bars represent standard error of the mean. The * represents a measurement below the limit of detection.

Fig 4

The O block polymorphism does not affect replication ability. The C65T mutation was created and reversed in the O blocks of Dik3 and TCH2, respectively, and the genomes were assayed as in Fig. 3A. The blot is representative of two independent repeats, and the two panels are different exposures of the same blot. Marker: HindIII digest of pGEM7-TU (size of bands in kb); Mock, mock transfection.