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Article by m.l. chapagain and v.r. nerurkar - j. infect. dis. 2010

Human Polyomavirus JC (JCV) Infection of HumanB Lymphocytes: A Possible Mechanism for JCVTransmigration across the Blood-Brain Barrier Moti L. Chapagain and Vivek R. Nerurkar
Retrovirology Research Laboratory, Department of Tropical Medicine, Medical Microbiology, and Pharmacology, John A. Burns School of Medicine,University of Hawaii at Manoa, Honolulu, Hawaii (See the editorial commentary by Houff and Berger, on pages 181–183.)
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 11000-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 dem-
onstrated 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.
Progressive multifocal leukoencephalopathy (PML), a semination throughout the body and trafficking across subacute demyelinating disease of the central nervous the blood-brain barrier remains poorly understood.
system [1] results from the lytic infection of oligoden- A possible role of B cells in JCV transmigration across drocytes, the myelin-producing cells in the brain, with the blood-brain barrier has been suggested [8, 12, 14– human polyomavirus JC (JCV) [2–4]. JCV foci in a 18]. JCV-infected B cells were first detected in the spleen brain with PML are closely related to the blood vessels and bone marrow of 2 patients with PML [15] and [5], and JCV is presumably spread by the hematogenous were subsequently detected in the central nervous sys- route from the primary site of infection to secondary tem of a patient with PML [19]. Moreover, JCV DNA sites, such as kidneys, lymphoid tissues, and brain, to was reported to be associated with peripheral blood establish focal areas of infection or persistence [2, 6– lymphocytes in 89.5% and 38% of patients with PML 13]. However, the precise mechanism(s) of JCV dis- and patients with AIDS, respectively, with varying de-grees of immunodeficiency [7, 10]. Demonstration ofviral genome in the peripheral blood lymphocytes sug-gests the possibility of hematogenous spread of JCV.
Received 24 November 2009; accepted 14 January 2010; electronically published 15 June 2010.
Furthermore, JCV infection of human B cells in vitro Potential conflicts of interest: none reported.
was suggested [12, 18, 20], and it was argued that JCV Financial support: National Institute of Neurological Disorders and Stroke (grant R03NS060647), Research Centers in Minority Institutions Program (grants infection of Epstein-Barr virus (EBV)–transformed B G12RR003061 and P20RR011091), and Centers of Biomedical Research Excellence cells or B cell lines resulted in viral DNA replication (grant P20RR018727), National Center for Research Resources, National Institutesof Health, and Institutional Funds.
and production of infectious virions [18, 20]. However, Reprints or correspondence: Vivek R. Nerurkar, PhD, John A. Burns School of evidence for JCV replication in B cells was inconclusive, Medicine, University of Hawaii at Manoa, 651 Ilalo St, BSB 325AA, Honolulu, HI96813 ([email protected]).
because none of these studies quantitated the viral The Journal of Infectious Diseases
DNA, and messenger RNA (mRNA) transcripts were  2010 by the Infectious Diseases Society of America. All rights reserved.
rarely detected [18]. Moreover, infectious JCV virions in the B cells might be the residual virus inoculum used 184 • JID 2010:202 (15 July) • Chapagain and Nerurkar
to infect the B cells rather than de novo production of the 250, 1000, or 2500 HA units of JCV(Mad1) for 2 h at 37C, virus in the B cells.
were washed with phosphate-buffered saline (PBS) 3 times, and The possible role of B lymphocytes in JCV transmigration 2.5 ⫻ 10 cells were harvested at days 0 (2 h), 5, 10, across the blood-brain barrier came to light again in 2005 after 15, and 20 after infection, for DNA and RNA extraction.
the development of PML in patients with multiple sclerosis and Fluorescein isothiocyanate (FITC) labeling of JCV.
Crohn disease who were treated with a monoclonal antibody crose-purified JCV [31] was labeled with FITC using the Fluoro- natalizumab (Tysabri; Biogen Idec and Elan Pharmaceuticals) Tag FITC Conjugation Kit (Sigma) by modifying the published [21–23]. Natalizumab is a recombinant humanized monoclonal protocol [37, 38]. Briefly, 850 mL of the sucrose-purified virus antibody directed against the adhesion molecules a4b1 and containing 100 HAU/mL of JCV was dialyzed overnight with a4b7 integrins [24]. Because a4b1 integrin interacts with very labeling buffer (0.05 mol/L boric acid, 0.2 mol/L NaCl, pH 9.2), late antigen-4 (VLA-4) and this interaction is required for gen- and the volume was adjusted to 1 mL. The dialyzed virus (1 erating T and B cells from bone marrow progenitor cells in mL) was then transferred into a 2-mL tube containing a small adult mice [25], it is argued that a4-integrin blockade mobilizes stirrer, and 250 mL of FITC solution (1 mg/mL) in 0.1 mol/L JCV-infected pre-B cells from bone marrow into the circulation carbonate-bicarbonate buffer (pH 9.0) was added slowly drop and, thus, facilitates JCV dissemination and PML development by drop and incubated for 8 h at room temperature in the [26, 27]. Recently, 3 patients treated with efalizumab for pso- dark, with continuous gentle stirring. The FITC-labeled virus riasis also developed PML [28]. Efalizumab (Raptiva; Genen- was then dialyzed overnight with PBS (pH 7.2), was aliquoted, tech) is a humanized monoclonal antibody (IgG1) that binds and was stored at ⫺80C. As a control, 5 mg of bovine serum to the alpha chain (CD11a) of the leukocyte function associated albumin (BSA) in 1 mL of PBS was also conjugated with FITC, antigen (LFA-1) [29, 30]. LFA-1 is a member of the hetero- was aliquoted, and was stored at ⫺80C. Protein and FITC dimeric b2 integrin family, and it interacts with intercellular levels in the FITC-conjugated viral or BSA suspension were adhesion molecules (ICAM) expressed on antigen presenting determined according to the company's protocol (Sigma) by cells and endothelial cells and is necessary for T cell activation, measuring the absorbance at 280 nm and 495 nm, respectively.
T cell helper and B cell responses, natural killer cell cytotoxicity, The FITC-conjugated BSA was diluted to get the same absor- and antibody-dependent cytotoxicity [30]. Efalizumab can se- bance as FITC-conjugated JCV at 495 nm and was used as a lectively and reversibly block the activation, reactivation, and nonspecific fluorescence control.
trafficking of T cells [29, 30]. Natalizumab and efalizumab both Characterization of EBV-transformed B cell infection with
could create artificial cell-mediated immune deficiency in the One million B cells were incubated central nervous system by inhibiting the migration of lympho- with 1000 HAU of FITC-conjugated JCV (FITC-JCV) or FITC- cytes across the blood-brain barrier and, thus, may facilitate conjugated BSA (FITC-BSA) containing the same amount of the development of PML.
fluorescence in 500 mL of RPMI-1640 for 2 h. The B cells were To better understand the possible role of B cells in JCV washed 3 times with PBS, and the pellet was dissolved in 1 mL transmigration across the blood-brain barrier, we studied the of PBS. Forty microliters of cell suspension containing ∼40,000 kinetics of JCV(Mad1) infection of B cells in vitro. Our data B cells were transferred into each well of a multiwell slide, were confirm that JCV nonproductively infects B cells. However, JCV air dried, and were fixed with 4% paraformaldehyde for 10 virions persist in B cells, and B cells can transmit infectious min. Cells were then washed with PBS and permeabilized with virions to naive primary human fetal glial (PHFG) cells, sug- 0.4% Triton-X 100 for 5 min. The permeabilized cells were gesting that B cells can act as a vehicle for JCV transmigration blocked with 5% BSA for 1 h and were incubated with anti- across the blood-brain barrier.
rabbit protein disulphide isomerase (1:500), to visualize theplasma membrane, and were washed 3 times with PBS. The MATERIALS AND METHODS
cells were further incubated with anti-rabbit secondary anti- Virus and cell cultures.
JCV(Mad1) was propagated in PHFG body conjugated with Alexa-594 and were mounted with Vec- cells and was purified and quantitated by the HA assay and tashield mounting medium with DAPI (Vector Laboratories).
real-time polymerase chain reaction (PCR) [31–34]. EBV-trans- Fluorescent cells were examined using a Axiocam MRm camera formed primary B cells were provided by Dr Allison Imrie, mounted on a Zeiss Axiovert 200 microscope, equipped with University of Hawaii, and were cultured in RPMI-1640 sup- appropriate fluorescence filters and objectives as described else- plemented with 10% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 mg/mL), and 2 mmol/L L-glutamate at 37C, One million EBV-transformed B cells were incubated with with 5% CO as described elsewhere [35, 36].
125, 250, 500, or 1000 HAU of FITC-JCV for 2 h and were JCV infection of EBV-transformed B cells.
washed with PBS. Cells were incubated with antibody against 2.5 ⫻ 10 cells) in suspension were infected with CD19 conjugated with PE (CD19-PE) and were subjected to JCV Infection of Human B Lymphocytes • JID 2010:202 (15 July) • 185

flow cytometry in Guava EasyCyte Plus platform. BSA labeledwith FITC containing the same amount of fluorescence wasused as a control.
DNA and RNA extraction.
Uninfected and JCV-infected B cells in T25 flasks were washed with PBS, were counted, andan aliquot of 0.25 million cells in duplicate were either frozenfor DNA extraction or lysed with 350 mL of RLT plus bufferfor RNA extraction (Qiagen) and stored at ⫺80C. RNA wasisolated using protocols reported elsewhere [32]. Additionally,100 mL of DNA was extracted using the Qiagen QIAprep SpinMiniprep Kit from each 0.25 million cells harvested at differenttime points, according to the manufacturer's protocol.
Quantitation of viral DNA and mRNA transcripts and re-
verse transcription PCR.
Two microliters of template DNA or complementary DNA were amplified and quantitated in theBio-Rad's iCycler iQ Multicolor Real-Time PCR Detection Sys-tem with use of primers, probes, and protocols described else-where [39]. Copies of JCV TAg or VP-1 genomes or mRNAtranscripts in experimental samples were calculated from thestandard curve and expressed as copies of viral genome per250,000 B cells or mRNA transcripts per microgram of totalRNA [32, 39].
JCV infection of B lymphocytes is nonproductive.
1 genome copies recovered from 2.5 ⫻ 10 infected B cells de- creased 11000-fold from day 0 to day 20 after infection (Figure1A) and neither early (TAg) nor late (VP-1) mRNA transcripts Figure 1.
JC virus (JCV) infection of B cells is nonproductive. Epstein- were detected (data not shown), suggesting that JCV infection Barr virus–transformed B cells ( 2.5 ⫻ 10 ) were infected with 250, 1000, or 2500 HA units of JCV(Mad1) for 2 h, and aliquots of of B cells is nonproductive. Our quantitative PCR and quan- were harvested at days 0 (2 h), 5, 10, 15, and 20 after infection. A, DNA titative reverse transcription PCR assays were sensitive and con- was extracted from 2.5 ⫻ 10 cells, and VP-1 DNA was amplified and sistently detected as low as 10–100 copies of JCV DNA and quantitated by quantitative polymerase chain reaction. VP-1 DNA genome mRNA transcripts, respectively [31]. Because JCV infection of copies decreased exponentially from days 0 to 20 after infection. B, Trypsin EBV-transformed B cells did not result in viral genome repli- treatment had no effect on further reducing the JCV genome copies at cation or transcripts expression, it is possible that JCV might any time point. JCV VP-1 genome copies recovered from 25, 100, or 250HAU of JCV equivalent used to infect 250,000 B lymphocytes was shown have remained associated with the B cells by attaching to the for comparison. ID, infection dose.
cell membrane and might not have entered the B cells. To testthis hypothesis, 2.5 ⫻ 10 B cells were inoculated with 250 HAU of JCV and incubated for 2 h at 37C. After incubation, the cells were incubated with 125, 250, 500, or 1000 HAU of JCV cells were washed twice with PBS and trypsinized for 10 min per million B cells, ∼1.5%, 1.7%, 3.9%, or 7.7% of B cells, with 0.5, 5, or 50 mg/mL of trypsin or with PBS alone, followed respectively, were infected with JCV (Figure 2A–2D).
by 2 washes of PBS. Cells were cultured for up to 10 days. On We further characterized JCV infection of EBV-transformed days 1, 5, and 10, 2.5 ⫻ 10 trypsin-treated and untreated B B cells with use of FITC-labeled JCV. B cells and human brain cells were harvested, and JCV VP-1 DNA was amplified and microvascular endothelial (HBMVE) cells were either infect- quantitated by quantitative PCR. The data demonstrate that ed with FITC-labeled JCV (Figure 3E–3P) or inoculated with trypsin treatment had no significant effect in further reducing FITC-conjugated BSA (FITC-BSA) containing the same amount the JCV genome copies (Figure 1B), suggesting that JCV indeed of fluorescence (Figure 3A–3D) and were examined by immu- entered into the B cells.
nofluorescence microscopy. Interestingly, all FITC-labeled JCV Moreover, by infecting EBV-transformed B cells with FITC- virions were seen inside the HBMVE cells (Figure 3O–3P), labeled JCV and by employing flow cytometry, we confirmed whereas FITC-labeled JCV virions appeared to be inside the B that JCV infects B cells in a dose-dependent manner. When B cells and on the B cells surfaces (Figure 3G–3H and 3K–3L).
186 • JID 2010:202 (15 July) • Chapagain and Nerurkar

Figure 2.
JC virus (JCV) infection of B cells is dose-dependent. One million Epstein-Barr virus–transformed B cells were incubated with 125 (A), 250 (B ), 500 (C ), or 1000 (D ) HAU of fluorescein isothiocyanate (FITC)-JCV or with equivalent fluorescence-containing bovine serum albumin (A–D ),similarly labeled with FITC (gray-filled histogram) as a control for 2 h, washed with phosphate-buffered saline, and subjected to flow cytometry.
These data confirm that JCV indeed infects the B cells. However, DNase-treated and DNase-untreated cells, DNA concentration in contrast to HBMVE cells where VP-1 and T antigen proteins was measured, and JCV VP-1 DNA was quantitated by quan- were observed predominantly in the cell nucleus [32], our re- titative PCR. Figure 4A demonstrates that the total cellular DNA peated attempts to demonstrate T antigen and VP-1 proteins recovered from the B cells increased ∼16-fold (mean  stan- expression in the B cells failed, and it is unlikely that JCV infection dard deviation, 10.7  1.5 to 170.9  21.1 mg/mL) from days of B cells is productive. Collectively, our data demonstrate that 0 to 15 after infection, suggesting that the B cells were actively JCV infects the B cells; however, JCV infection of B cells is replicating. However, mean JCV VP-1 DNA copies/well ( standard deviation) decreased ∼11-fold ( 18.0  8.6 ⫻ 10 to Infectious JCV virions persist in the B cells.
16.0  7.5 ⫻ 10 copies/well) from days 0 to 15 after infection, data suggest that JCV infects EBV-transformed B cells but does further confirming our observation that JCV infection of B not replicate in these cells, intact virus must survive inside the cells is nonproductive. Although DNase treatment was very B cells long enough to be trafficked across the blood-brain effective and reduced the total cellular DNA recovered from barrier and must then have a mechanism to be released to JCV-infected B cells, up to 96% (mean  standard deviation, infect susceptible cells, particularly the oligodendrocytes. How- 170.9  21.1 to 6.9  1.7 mg/mL) on day 15 after infection ever, our data suggest that JCV genome copy numbers rapidly (Figure 4A), DNase treatment had very little effect on the JCV decrease in the EBV-transformed B cells after infection, and it VP-1 DNA copies recovered from the B cells (Figure 4B). These was unclear whether the decrease in viral genome was the result results suggest that JCV virions in the B cells remain intact for of degradation of viral DNA inside the B cells or merely a result at least 15 days after infection and are protected from DNase of a dilution effect of replicating B cells, because in vitro EBV- transformed B cells replicate rapidly. To address this question, To further verify that JCV virions inside the B cells were 2.5 ⫻ 10 EBV-transformed B cells with 250 HAU infectious, JCV-infected B cells or lysates after infection of B of JCV for 2 h. After a wash, 2.5 ⫻ 10 cells were seeded in cells with JCV for 24 h were further cocultured for an additional each well of a 24-well plate, were cultured, and all cells from 24 h with naive PHFG cells. After 24 h coculture, the PHFG each well were harvested at each time point. The cells were cells were washed; cells were harvested at days 5, 10, and 15 freeze-thawed 4 times to mechanically lyse the cells, and trip- and were analyzed for viral late gene (VP-1) expression. Al- licate samples at each time point were either left untreated or though no viral transcripts were recovered from PHFG cells treated with 125 U/mL of DNase for 1 h at 37C to digest cocultured with uninfected B cells (data not shown), JCV VP- genomic and free viral DNA. The DNase-treated cells were heat 1 mRNA transcripts increased 1100-fold in PHFG cells cocul- inactivated at 75C for 10 min, DNA was extracted from both tured with JCV-infected B cells or JCV-infected B cells lysate, JCV Infection of Human B Lymphocytes • JID 2010:202 (15 July) • 187

Figure 3.
Epifluorescence microscopy demonstrating infection of B cells by fluorescein isothiocyanate (FITC)–conjugated JC virus (JCV). One million B cells were inoculated with either FITC-labeled bovine serum albumin (A–D) as a negative control or 1000 HAU of FITC-labeled JCV (E–L). Membraneswere labeled with protein disulphide isomerase (PDI) and visualized with Alexa flour 594 (red ). Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei (blue). Merged images demonstrate FITC-labeled JCV on the B cell surface (arrowheads) and inside the Bcells (arrow ) (H and L). M–P, Human brain microvascular endothelial cells infected with FITC-labeled JCV (positive control). Scale bar, 5 mm.

infected monocytes (Trojan horse) [42]. Further studies havealso suggested the presence of JCV DNA in the peripheral bloodmononuclear cells of 17.4% of 69 HIV type 1–infected im-munocompetent patients, in 23.2% of 82 HIV type 1–infectedimmunocompromised patients, and in 60% of AIDS patientswith PML [43]. However, there was no expression of JCV earlyand late mRNA transcripts in the peripheral blood mononu-clear cells [43]. Recently, JCV DNA was demonstrated in theperipheral blood mononuclear cells and VP-1–stained CD138+positive plasma cells were visualized in the bone marrow of apatient with rheumatoid arthritis treated with methotrexate,who developed PML and had a rapid fatal outcome, furthersuggesting that B lymphocytes may play an important role inthe JCV latency and dissemination [14, 16]. Similarly, JCV DNAwas detected in the bone marrow of HIV-negative and HIV–positive patients with and without PML, and JCV large T an-tigen but not VP1 was detected by double immunostaining inCD138+ plasma cells in an archival bone marrow specimenfrom an HIV-infected patient without PML [41]. Moreover,Focosi et al [44] detected JCV DNA in serial bone marrowsamples from 4 hematological patients with histology-con-firmed PML. Importantly, JCV DNA was first detected in thebone marrow and later in CSF, peripheral blood, and brain.
Furthermore, in 1 patient who survived, JCV was not detectedafter resolution of PML, suggesting that JCV indeed dissemi-nates by the hematogenous route [44].
It is often argued that JCV establishes low level of productive infection in B lymphocytes [8, 12, 18, 20]. Atwood et al [20] Figure 4.
JC virus (JCV) genome in B cells was virion protected. In- have demonstrated that 1% of B lymphocytes were JCV positive fected B cells harvested at different time points after infection were by in situ DNA hybridization on day 13 after infection, when mechanically lysed by repeated freeze-thaw and were untreated or treatedwith DNase. DNA was extracted, concentration was measured, and JCVVP-1 DNA was quantitated by quantitative polymerase chain reaction. A,Total cellular DNA recovered increased from day 0 to day 15, suggestingthat B cells were actively replicating and that DNase effectively reducedDNA concentration. B, JCV VP-1 DNA copies decreased from days 0 to15 after infection as expected, but DNase treatment had very little effecton the viral DNA, suggesting that JCV genome in the B cells was virionprotected from DNase digestion.
clearly indicating that the virions that remained inside the Bcell cytoplasm were infectious and replicated efficiently in naivePHFG cells (Figure 5).
The mechanism of JCV trafficking across the blood-brain bar-rier remains poorly understood. PCR analyses have demon- Figure 5.
JC virus (JCV)–infected B cells transmit JCV infection to strated that JCV may persist in the brain, tonsils, and lym- naive primary human fetal glial (PHFG) cells. PHFG cells were coculturedfor 24 h with JCV-infected B cells or B cells lysate, and JCV VP1 mes- phocytes of individuals with and without PML [2, 6–11, 13, senger RNA transcript expression in PHFG cells was quantitated. JCV 16, 40, 41], and it was proposed that JCV might employ B VP-1 messenger RNA transcripts increased several fold in PHFG cells lymphocytes to cross the blood-brain barrier [8, 12, 14–16], cocultured with JCV-infected B cells or B cells lysate. However, no viral similar to human immunodeficiency virus (HIV) and simian transcripts were detected from PHFG cells cocultured with uninfected B immunodeficiency virus, which gain entry into the brain via cells (data not shown).
JCV Infection of Human B Lymphocytes • JID 2010:202 (15 July) • 189
1 million B cells were infected with 10,000 HAU of JCV; they Although we always detected JCV TAg and VP-1 DNA, viral argued that it was indicative of DNA replication [20]. Monaco mRNA transcripts were never detected in JCV-infected B cells, et al [12] claimed that 3%–5% of hematopoietic cell line (KG- suggesting that JCV transcripts expression was either extremely 1a), primary hematopoietic progenitor cells (CD34+), and pri- low or completely shut off in the B cells. Further in vitro and mary B cells were positive for JCV on day 5 after infection in vivo studies using sensitive state-of-the-art real-time PCR when infected with 300 HAU/mL of JCV (Mad-4) [12]. Al- and quantitative reverse transcription PCR assays are warranted though TAg and VP-1 transcripts [18] and proteins [8] ex- to define the role of hematopetic precursor cells, primary B pression were demonstrated occasionally in JCV-infected B cell cells, and bone marrow–derived plasma cells in JCV replication, line or B lymphocytes in vitro, these studies were often limited latency, and dissemination.
to 1 time point after infection, or the transcripts were detectedonly after a second round of PCR (nested PCR) [18]. Taken together, in spite of several claims of replication of JCV genomeand production of infectious virions de novo in the B cells, We thank Dr Allison Imrie, University of Hawaii, for providing EBV- evidence for JCV replication in B lymphocytes is still lacking.
transformed primary B cells. We also thank Mr Nelson I. B. Lazaga, DrFrederic Mercier, Dr Pakieli Kaufusi, Ms Ulziijargal Gurjav, and Ms Al- In an attempt to better understand the mechanism(s) of JCV exandra Gurary for their technical assistance.
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