Human polyomavirus JC (JCV) infection of human B lymphocytes: a possible mechanism for JCV transmigration across the blood-brain barrier

Abstract

It has been suggested that JC virus (JCV) might travel to the central nervous system in infected B cells. Moreover, recent data suggest the presence of JCV in bone marrow plasma cells. However, the evidence for infection and replication of JCV in B cells is unclear. To address this question, we infected Epstein-Barr virus-transformed B cells with JCV and found that the viral genome decreased >1000-fold from days 0 to 20 after infection, which concurred with the absence of viral early and late messenger RNA transcripts and proteins. However, immunofluorescent images of B cells infected with fluorescein isothiocyanate-conjugated JCV demonstrated that JCV enters the B cells, and DNase protection assay confirmed the presence of intact JCV virions inside the B cells. Moreover, JCV-infected B cells were able to transmit infection to naive glial cells. These data confirm that JCV nonproductively infects B cells and possibly uses them as a vehicle for transmigration across the blood-brain barrier.

Figure 1

JCV infection of B cells is non-productive. 2.5 × 10⁶ EBV-transformed B cells were infected with 250, 1,000 or 2,500 HA units of JCV(Mad1) for 2 hr and aliquots of 2.5 × 10⁵ cells were harvested at days 0 (2 hr), 5, 10, 15 and 20 after infection. (A) DNA was extracted from 2.5 × 10⁵ cells and VP-1 DNA was amplified and quantitated by qPCR. VP-1 DNA genome copies decreased exponentially from days 0 to 20 after infection. (B) Trypsin treatment had no effect on further reducing the JCV genome copies at any time point. ID; infection dose - JCV VP-1 genome copies recovered from 25, 100 or 250 HAU of JCV equivalent used to infect 250,000 B lymphocytes was shown for comparison.

Figure 2

JCV infection of B cells is dose-dependent. One million EBV-transformed B cells were incubated with (A) 125, (B) 250, (C) 500 or (D) 1,000 HAU of FITC-JCV or with (A-D) equivalent fluorescence containing BSA similarly labeled with FITC (filled gray histogram) as control for 2 hr and washed with PBS and subjected to flow cytometry.

Figure 3

Epifluorescence microscopy demonstrating infection of B cells by FITC-conjugated JCV. One million B cells were inoculated with either (A-D) FITC-labeled BSA as negative control or (E-H and I-L) 1,000 HAU of FITC-labeled JCV. Membranes were labeled with PDI and visualized with Alexa flour 594 (red). Cells were counterstained with DAPI to visualize cell nuclei (blue). Merged images demonstrate FITC-labeled JCV on B cell surface (arrow heads) and inside the B cells (arrow) (H and L). HBMVE cells infected with FITC-labeled JCV (positive control) (M-P). DAPI; 4′,6-diamidino-2-phenylindole; PDI; protein disulphide isomerase; scale bar = 5 μm

Figure 4

JCV genome in B cells was virion protected. Infected B cells harvested at different time points after infection were mechanically lysed by repeated freeze-thaw, and untreated or treated with DNase. DNA was extracted, concentration was measured and JCV VP-1 DNA was quantitated by qPCR. (A) Total cellular DNA recovered increased from day 0 to day 15 suggesting B cells were actively replicating and DNase effectively reduced DNA concentration. (B) JCV VP-1 DNA copies decreased from days 0 to 15 after infection as expected but DNase treatment had very little effect on the viral DNA suggesting that JCV genome in the B cells was virion protected from DNase digestion.

Figure 5

JCV-infected B cells transmit JCV infection to naïve PHFG cells. PHFG cells were co-cultured for 24 hr with JCV-infected B cells or B cells lysate and JCV VP1 mRNA transcripts expression in PHFG cells was quantitated. JCV VP-1 mRNA transcripts increased several fold in PHFG cells co-cultured with JCV-infected B cells or B cells lysate. However, no viral transcripts were detected from PHFG cells co-cultured with uninfected B cells (data not shown).

On the cover: Epifluoresence images show human polyomavirus JC (JCV) infection of B lymphocytes. One million EBV-transformed B lymphocytes (left) were inoculated with 1000 HAU of FITC-labeled JCV (green). Cell membrane was labeled with anti-protein disulphide isomerase antibody and visualized using Alexa flour 594 (red), and nuclei were counterstained with DAPI (blue). Human brain microvascular endothelial cells (right) were used as a positive control.