BK virus large T and VP-1 expression in infected human renal allografts

Abstract

Objective: We investigated the expression of early and late phase BK virus (BKV) proteins and their interactions with host cell proteins in renal allografts, with ongoing polyomavirus associated nephropathy (PVAN), and correlated this with the nuclear and cell morphology.

Methods: Frozen sections from three patients with renal allografts (two biopsies, one explant) with PVAN were analysed by indirect immunofluorescence using BKV specific anti-polyoma large T-antigen and anti-VP-1 antibodies, as well as anti-p53, anti-Ki67, anti-caspase-3, anti-bcl2 and anti-cytokeratin 22 antibodies. Nuclear morphology and size were estimated by DNA Hoechst staining.

Results: In infected tubular cells the early and late phases of infection could be distinguished according to expression of large T-antigen or VP-1. The early phase revealed almost normal nuclear proportions, whereas in later phases nuclear size increased about 2 to 3 fold. Expression of large T-antigen was strongly associated with accumulation of p53 in the nucleus, accompanied by the activation of the cell cycle associated cell protein Ki67. In contrast, expression of BKV VP1 correlated only weakly with p53. Virus dependent cell lysis was due to necrosis, since neither caspase 3 nor nuclear nor cytoskeleton changes indicated apoptosis.

Conclusion: In our selected patients with PVAN a clear distinction between early and late phases was possible, according to the protein expression patterns of BKV markers. Striking nuclear enlargement is only present in the late phase of infection. In the inflammatory setting of PVAN, BKV dependent effects appear to be mediated by the inhibition of p53, resulting in the activation of the cell cycle. We assume that in PVAN similar BKV mechanisms are operative as in certain in vitro systems.

Fig. 1

Histology of our three patients (A, B, C) with ongoing PVAN in the corresponding stages A, B and C. (A) (Patient A): cortico-medullary junction: tubules with cells showing typical inclusion bodies. In the corresponding frozen sections much more infected cells were present than in the paraffin sections. PAS stain, original magnification 400×. (B) (Patient B): cortex with focal inflammation surrounding tubules with infected cells. PAS stain, original magnification 200×. (C) (Patient C): cortex with severe interstitial fibrosis and tubular atrophy. In-between better preserved areas of cortical tissue. Trichrome stain, original magnification 100×.

Fig. 2

Investigation of the expression of large T-antigen and VP1 BKV proteins and their interaction with host cell proteins by immunfluorescence (IF) double labelling techniques in kidney transplanted patients with ongoing PVAN. The IF images are taken from patient B (Figure 1a and c), and the quantifications of the results includes all three patients (Figure 1b and d). (A) Double labelling of large T-antigen with p53, Ki67, caspase 3 and Bcl-2 in single, double or triple channels including the DNA staining of the nuclei by Hoechst (H) dye in blue. In a double channel, overlay of the red and green signal results in an orange-yellow colouration. (B) Quantification of the number of cells expressing large T-antigen and co-localizing p53, Ki67, caspase 3 and Bcl-2. The numbers of T-antigen expressing cells is adjusted to 1 (=100%). Bars indicate the standard deviation (SD) between the three patients. (C) Double labelling of VP1 with large T-antigen, Ki67, p53 and caspase 3 in single, double or triple channels. In addition, DNA is stained by Hoechst (H) dye in blue. Co-expression of the red and green signal causes an orange-yellow colouration. (D) Quantification of the number of cells expressing VP1 and co-localizing large T-antigen, Ki67, p53 and caspase 3. The numbers of VP1 expressing cells are adjusted to 1 (=100%). Bars indicate the SD between the three patients.

Fig. 2

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Fig. 3

Correlation of the nuclear size with expression of early and late phase BKV protein. On the Y-axis the numbers of pixels are given corresponding to the nuclear size, on the X-axis the different phases of the BKV cycle with respect to the expression of large T-antigen and VP1. Bars represent the mean and standard error (SE) of pooled nuclear measurement in all three patients (A, B and C). The nuclear size increases significantly with the course of the virus infection and reaches a maximum upon expression of VP1 in the late replication phase (Wilcoxon test: negative versus T-antigen, versus double versus VP1 = P < 0.02, P < 0.003, P < 0.0001, T-antigen versus double versus VP1 = P < 0.003, P < 0.003, double versus VP1 P < 0.05).

Fig. 4

Estimation of the duration of the BKV infectious cycle in vivo. Assuming that the action of BKV T-antigen of p53 binding is a direct effect, a minimal induction time of p53 of 3 h could be assumed (see the Discussion section). From the 70% overlay of p53 and T-antigen a T-antigen expression period of 10 h is assessed. The 20% overlay of VP1 expression and T-antigen would correspond to 2 h co-expression of the viral proteins. Consecutively, VP1 would be expressed for an additional 8 h resulting finally in the lysis of the infected cell. Taking into account that the time between virus reception and first viral transcriptional activity would last 24–36 h as described by Low et al. [11] we arrive at a virus life cycle of 42–54 h.