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. 2008 Apr 14;205(4):841-52.
doi: 10.1084/jem.20072097. Epub 2008 Mar 17.

Polyomavirus BK with rearranged noncoding control region emerge in vivo in renal transplant patients and increase viral replication and cytopathology

Rainer Gosert  1 , Christine H Rinaldo, Georg A Funk, Adrian Egli, Emilio Ramos, Cinthia B Drachenberg, Hans H Hirsch
Affiliations

Polyomavirus BK with rearranged noncoding control region emerge in vivo in renal transplant patients and increase viral replication and cytopathology

Rainer Gosert et al. J Exp Med. .

Abstract

Immunosuppression is required for BK viremia and polyomavirus BK-associated nephropathy (PVAN) in kidney transplants (KTs), but the role of viral determinants is unclear. We examined BKV noncoding control regions (NCCR), which coordinate viral gene expression and replication. In 286 day-matched plasma and urine samples from 129 KT patients with BKV viremia, including 70 with PVAN, the majority of viruses contained archetypal (ww-) NCCRs. However, rearranged (rr-) NCCRs were more frequent in plasma than in urine samples (22 vs. 4%; P < 0.001), and were associated with 20-fold higher plasma BKV loads (2.0 x 10(4)/ml vs. 4.4 x 10(5)/ml; P < 0.001). Emergence of rr-NCCR in plasma correlated with duration and peak BKV load (R(2) = 0.64; P < 0.001). This was confirmed in a prospective cohort of 733 plasma samples from 227 patients. For 39 PVAN patients with available biopsies, rr-NCCRs were associated with more extensive viral replication and inflammation. Cloning of 10 rr-NCCRs revealed diverse duplications or deletions in different NCCR subregions, but all were sufficient to increase early gene expression, replication capacity, and cytopathology of recombinant BKV in vitro. Thus, rr-NCCR BKV emergence in plasma is linked to increased replication capacity and disease in KTs.

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Figures

Figure 1.
Figure 1.
BKV NCCR in plasma and urine of KT patients. (A) Discordance of NCCR majority species in matched urine (U) and plasma (P) samples shown for 5 patients. Nested PCR targeting the BKV NCCR was resolved on 2% agarose-tris acetate, pH 8.0, electrophoresis gels and stained with ethidium bromide. NCCR ww, archetype; del, deletion, ins, insertion, bp, base pairs of marker. (B) Emergence of rr-NCCR in consecutive plasma shown for patient 2 (del-NCCR) and 5 (ins-NCCR) resolved as in A. (C) Time to rr-NCCR detection and peak plasma BKV load in consecutive plasma samples from 35 KT patients analyzed by linear and quadratic regression. Fine line, linear regression; bold line, quadratic regression; dashed lines, 90% confidence interval.
Figure 2.
Figure 2.
Histological patterns of PVAN in kidney biopsies. (A) Haematoxylin/eosin stain of a representative histological field showing PVAN pattern A consisting of focal viral cytopathic changes with little inflammation or tubular atrophy. Immunohistochemistry of LTag expression using the cross-reacting monoclonal anti–SV40 T-antigen Ab-2 and peroxidase-conjugated anti–mouse. Enlargement of the indicated area is shown. (B) Haematoxylin/eosin stain of a representative histological field showing PVAN pattern B with extensive viral cytopathic changes and inflammatory infiltrates. Immunohistochemistry of LTag expression using the cross-reacting monoclonal anti-SV40 T-antigen Ab-2 and peroxidase conjugated anti–mouse. Bars: (A and B) 200 μm; (A and B, insets) 100 μm.
Figure 3.
Figure 3.
BKV NCCR architecture and expression pattern in cell culture. (A) Architecture of archetype ww-NCCR in BKV genome, with arbitrarily denoted linear blocks (number of base pairs) O(142)-P(68)-Q(39)-R(63)-S(63) and position of primers (arrows). RFP, RFP for early genes; GFP, GFP for late genes; βG-pA, β-globin polyA; Hyg, hygromycin resistance; Amp, ampicillin resistance. (B) Architecture of rr-NCCR clones and gene expression pattern in HEK293 at 2 dat. ins, insertions corresponding to duplications of the numbered base pairs; del, deletions, denoted by gaps and nucleotides. VP1-Type, serotype; n.a., not applicable because generated by in vitro mutagenesis; Confluence, showing phase contrast; Early, red fluorescence; Late, green fluorescence; Expression level, percentage of green and red fluorescence–positive cells normalized to GFP-positive cells of phRG-ww(1.4) bearing the archetype ww-NCCR. (C) NCCR-driven early (E) and late (L) gene expression in different cell types. HEK293, monkey kidney cells (Vero), human umbilical vein endothelial cells (HUVECs), and primary RPTECs were transfected with phRG-bearing archetype ww(1.4)NCCR, del(5.3)NCCR, ins(7.3)NCCR, and del(5.3R)NCCR, which contained the del(5.3)NCCR in reverse orientation relative to RFP and GFP (results as above). Numbers indicate fold expression level for del(5.3)NCCR (green, late) and ww(1.4)NCCR (red, early), arbitrarily set to 1. (B and C) Mean of three independent experiments. Error bars represent the mean ± the SD.
Figure 4.
Figure 4.
Modulation of early and late gene expression. (A) Western blot detecting LTag in HEK293 cells stably expressing BKV LTag with monoclonal anti-SV40 and anti–mouse coupled to horseradish peroxidase at day 5 after transfection. Cells were transfected with phRG7.3 in the presence of Tetracycline (Tet). After 1 d, Tet was replaced by the indicated concentrations. Phase contrast (a–e), RFP (f–j), and GFP (k–o) expression in the presence of decreasing amounts of Tet (repression of LTag at 1,000 ng/ml Tet, maximum expression of LTag at 0 ng/ml Tet). Bar, 400 μm. (B) Effect of steroid treatment on early (RFP) and late (GFP) gene expression in Vero cells. -, absence; +, presence of 10−6 M dexamethasone. Cells were analyzed at 2 dat. (C) Effect of agnoprotein expression on early (RFP) and late (GFP) gene expression in Vero cells. Cells were analyzed at 2 dat. (A–C) Mean of three independent experiments. Error bars represent the mean ± the SD.
Figure 5.
Figure 5.
Replication of recombinant rr-NCCR BKV in cell culture. (A) Quantification of DNase-protected BKV load of rr-NCCR variants in Vero cell supernatants collected at indicated times after transfection. Viral load measurements for all 10 recombinant rr-NCCR BKV variants in Vero cells at 3 dat. Time course for archetype ww, ww(1.4)NCCR BKV; del, del(5.3)NCCR BKV). The data display a mean of three independent transfections determined in triplicate. Error bars represent the mean ± SD. (B) Cytopathology of the corresponding cell cultures at 21 dat by phase-contrast microscopy. (C) Western blot was performed with polyclonal anti-LTag, anti-VP1, and anti–rabbit conjugated to peroxidase, respectively. Tubulin was detected with monoclonal mouse anti-tubulin and peroxidase-conjugated anti–mouse. VP1, VP1 capsid protein; Tub, tubulin; at, after transfection. Samples were harvested at indicated times (hours) after transfection. Molecular weight of proteins is shown in kilodaltons. (D) Infection of primary RPTECs. Vero cell supernatants were harvested 7 dat and seeded onto RPTECs. Immunofluorescence for LTag (early gene, red) and agnoprotein (late gene, green) was performed on day 4 after infection using the monoclonal anti-SV40 LTag visualized with anti–mouse Alexa Fluor 568 and polyclonal rabbit anti-agno detected by anti–rabbit Alexa Fluor 488, respectively. Arrows indicate early LTag-positive staining cells without late agnoprotein staining. Cell nuclei are stained with DRAQ5 (blue). Bars: (B) 200 μm; (D, 10×) 200 μm; (D, 40×) 50 μm.
Figure 6.
Figure 6.
Emergence of rr-NCCR in the immunocompromised host. Naturally transmitted BKV with archetype ww-NCCR genome (black circles) undergo insertion and deletion errors during replication generating rr-NCCR genomes (red tagged circles). BKV rr-NCCR with increased early gene expression and corresponding higher replication capacity are eliminated in immunocompetent host, but outcompete ww-NCCR BKV in immunodeficient hosts. LT, LTag; VP1, capsid protein 1.
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