VIROLOGY
190, 716-723 (1992)
Interaction of the Human Polyomavirus, JCV, with Human B-Lymphocytes WALTER J . ATWOOD, KEI AMEMIYA, RENEE TRAUB, JURGEN HARMS',
AND
EUGENE O . MAJOR'
Section on Molecular Virology and Genetics, Laboratory of Viral and Molecular Pathogenesis, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 Received April 20, 1992 ; accepted June 18. 1992
The human polyomavirus, JCV, is the causative agent of the central nervous system demyelinating disease progressive multifocal leukoencephalopathy (PML) . The principal target of JCV infection in the central nervous system (CNS) is the myelinating oligodendrocyte . However, the site of JCV multiplication outside of the CNS and the mechanism by which virus gains access to the brain are not known . Recently, JCV infected B-lymphocytes have been demonstrated in PML patients in several lymphoid organs, in circulating peripheral lymphocytes, and in brain, suggesting a possible role of B-lymphocytes in the dissemination of virus to the brain . The experiments reported here were undertaken to understand more about the interactions of JCV with human B-lymphocytes . The data show that JCV is able to multiply in either Epstein-Barr virus transformed (EBV) or EBV negative human B cell lines resulting in production of infectious, progeny virions. In addition, nuclear proteins extracted from these B cells bind to similar nucleotides within the JCV regulatory region that are bound by nuclear proteins extracted from human fetal glial cells, the most susceptible host and principal target cell for JCV infection in vitro . It is not known, however, whether these DNA binding proteins from susceptible B cells and glial cells are similar . 9 1992 Academic Press, Inc .
INTRODUCTION
JCV latency and the mechanism that underlies its reactivation are currently unknown . In a previous report, JCV DNA was found to be widely disseminated in many
Seroepidemiological surveys of the human population indicate that infection with the human polyomavirus, 1CV, is very common and occurs worldwide (Brown et al., 1975 ; Rziha et al ., 1978 ; Padgett and Walker, 1983 ; Walker and Padgett, 1983) . Conversion
different organs including lymphoid tissue (Grinnell at al_ 1983) . The cell type harboring JCV DNA was not identified in that report . We have examined tissues of PML patients with or without AIDS for the detection of the JCV genome and
to seropositive status usually takes place during childhood, ages 5-8, with antibody levels remaining at de-
have described B-lymphocytes in the bone marrow, spleen, and brain as possible sites of JCV infection (Houff etal., 1988 ; Major etal., 1990) . Using PCR analy-
tectable levels throughout adulthood . Despite the prevalence of JCV in the human population the only known manifestation of JCV infection is the relatively rare central nervous system demyelinating disease, progressive multifocal leukoencephalopathy, or PML(Richardson, 1961 ; Padgett at al ., 1971) . PML occurs most
sis, JCV DNA was found associated with peripheral blood lymphocytes in 89% of PML patients (Tornatore
et al., 1992) . The histopathology of demyelinated lesions in the brain of PML patients suggest a hematogenous spread of virus to the brain . It has been hypothesized thatJCV gains entry to the brain in B-lymphocytes of immune-suppressed individuals (Major etat, 1992) . In this report the ability of JCV to infect human B-lymphocyte cell lines was investigated . Introduction of the
frequently in individuals with an underlying immunosuppressive disorder . Based largely upon studies of AIDS patients, it appears that there is a direct correlation between the extent of immune deficiency and the incidence and severity of PML (Ho at al., 1984 ; Krupp et al., 1985 ; Stoner et al., 1986 ; Berger at al., 1987 ; Chaisson and Griffin, 1990) . The observations of the
1CV genome into B-lymphocytes either by infection with JCV virions or by transfection with JCV DNA results in de novo replication of viral DNA and the multiplication of infectious virions . In addition, nuclear proteins extracted from human B cell lines are shown by competitive gel shift analyses and DNase I footprinting
common nature of 1CV infection with a low incidence of PML and its association with immunocompromised individuals indicate that JCV probably establishes a latent infection which is activated at times of immune suppression (Brooks and Walker, 1984) . The site of
assays to interact with the regulatory region of the JCV genome at a site previously identified as a binding site for nuclear factor-1 (NF-1) . The nucleotide sequence in
' Present address : Deutsches Primatenzentrum ABT . Immunologic, Gottingen, Germany. ' To whom reprint requests should be addressed . 0042-6822/92 $0 .00 Ccp9'ight O 1992 by Academic Pmas . Inc . Ai rghIe of -eproduction in any form reserved .
the JCV regulatory region bound by the B cell nuclear proteins map to the same location as the nucleotides 716
1C VIRUS IN 3 CELLS
bound by proteins from human fetal glial cells . These results strengthen the suggestion that B-lymphocytes can serve as a susceptible cell for JCV infection and allow a more detailed investigation of their role in the pathogenesis of PML .
MATERIALS AND METHODS Cells and virus Preparation of glial cell cultures from human fetal tissue has been described in detail (Elder and Major, 1988 ; Major and Vacante, 1989) . The human Epstein Barr virus (EBV) negative B cell line, BJA-B, was obtained from L . Staudt . EBV-positive Namalwa cells were obtained from American Type Culture Collection (ATCC) . The LyT3 B cell line was produced by longterm culture of lymphocytes from human tonsil of a presumably healthy child and was a generous gift of A . DeGrassi . The EBV status of this cell line has not been determined (A . DeGrassi, personal communication) . BJA-B and Namalwa cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum . LyT3 cells were grown in RPMI 1640 supplemented with nonessential amino acids (Whittaker Bioproducts, Inc .) and
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Transfection of B cells with JCV DNA BJA-B and Namalwa cells were suspended in PBS at a density of 1 X 10' cells per milliliter and held on ice for 10 min . JCV DNA (6 µg/10 6 cells), prepared by EcoRl digestion of the plasmid pM1-TC (Frisque etal., 1984), was introduced into the B cell lines by electroporation (BTX Transfector 300) at a capacitance of 1000 A F and a voltage of 350V for BJA-B cells and 300V for Namalwa cells . The transfected B cells were lysed by freeze-thawing and virus was released from membranes by incubation with 0 .25% sodium deoxycholic acid . The lysates were then used to infect human fetal glial cells growing on glass coverslips . JCV-infected glial cells were detected by indirect immunofluorescent staining of T antigen with the monoclonal antibody PAB 416 (Oncogene Sciences ; Majoretak, 1992) and a secondary goat anti-mouse IgG conjugated to FITC . The T-antigen-positive cells were visualized with a Zeiss ICM 405 epifluorescent microscope .
Nuclear extract preparation Nuclear extracts were prepared from 1-10 X 10 8 cells either growing on plates (HFGC, HeLa) or in spin-
10% fetal bovine serum . All cell lines were grown in the presence of 250 µg/ml gentamicin (Sigma) .
ner flasks (Namalwa, LyT3) by a modification of the procedure of Dignam etal . (1983) . Briefly, washed cells were swollen for 10-15 min on ice in four packed cell
Infection of B cells with JC virus
volumes (PCV) of buffer A [10 mM Tris-HCL (pH 7 .9), 1 .5 mM MgCl 2 , 10 mM KCL, 0 .5 mM dithiothreitol (DTT), and 0 .2 mM phenylmethylsulfonyl fluoride
BJA-B and Namalwa cells were washed three times in phosphate-buffered saline (PBS) and incubated for 2 hr at 37° in PBS with JCV at 1 X 10 4 hemagglutination (HA) units of virus per 1 x 106 cells . At 13 days postinfection (p .i .) cells were harvested and JCV DNA replica-
(PMSF)] . The cells were collected by centrifugation at 2000 rpm for 10 min and lysed with at least 10 strokes in a Wheaton homogenizer with a type B pestle . Nuclei were extracted in 4 PCV of buffer C, [20 mM Tris-HCL (pH 7 .9), 420 mM NaCl, 1 .5 mM MgCI 2 , 0 .2 mM EDTA, 25% glycerol (v/v), 0 .5 mM DTT, and 0 .5 mM PMSF],
tion was assayed by in situ DNA hybridization with a full-length genomic JCV-specific biotinylated probe (ENZO Diagnostics, Inc ., New York, NY) . Briefly, cells
The extracted nuclear proteins were precipitated with ammonium sulfate and collected by centrifugation for 30 min at 10,000 rpm . The pellet was resuspended in
were dried onto glass coverslips, fixed in 4%% paraformaldehyde and dehydrated in graded ethanol (60-
buffer BC [20 mM Tris-HCL (pH 7 .9), 100 mM KCI, 0 .2 mM EDTA, 20% glycerol (v/v), and 0 .5 mM DTT], and dialyzed overnight against the same buffer . The dia-
100%) . The cells were then rehydrated in PBS, treated for 30 min with methanol and H 2 0 2 before preparation for in situ hybridization to eliminate endogenous peroxidase activity (Aksamit et al ., 1985, 1986) . For Southern blot hybridization, nitrocellulose filters were hybrid-
lyzed nuclear proteins were cleared of insoluble material by centrifugation and the protein content of the samples estimated by the method of Bradford (1976) . Buffers A and C contained the protease inhibitors anti-
ized to a 32 P-labeled JCV DNA probe . Hybridization was done at 42° for 48 hr in 6x SSC, 50% formamide, 5X Denhardt's . Kodak X-AR film was exposed for 24 hr . Quantitation of JCV was made by hemagglutination (HA) of human type 0 erythrocytes (Padgett et al., 1971) . The virus titer is expressed as the reciprocal of
pain, leupeptin, pepstatin A, chymostatin, and aprotinin at 5 µg/ml, buffer BC contained these same protease inhibitors at 1 ug/ml .
the highest dilution which results in hemagglutination (HA unit) .
The plasmid, pJC188 °r „ contains one 98-bp repeat unit of the JC virus enhancer/promoter region and its
DNA probe and competitor preparation
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ATWOOD ET AL .
construction has been previously described (Amemiya et at, 1989) . The plasmid was linearized with Hindlll, the 5' phosphate removed using calf intestine alkaline phosphatase (Boehringer-Mannheim) and replaced with a y-32 P-labeled ATP (DuPont-NEN) . The 188-bp fragment containing the JCV regulatory region was separated from the rest of the plasmid by Pvull digestion, electrophoresis on a 15% polyacrylamide gel, and electroelution with an Elutrap (Schleicher and Schuell) . The labeled DNA was precipitated in ethanol, suspended in 100 pl dH 2 O, and an aliquot removed to determine the specific activity of the probe . Oligonucleotides used as competitors were synthesized on an Applied Biosystems 380A DNA synthesizer . Three double-stranded oligonucleotides were used as competitors ; one contained an intact binding site for NF-1, another contained a mutated NF-1 binding site (NF-1 m), and a third contained the TATA sequence . NF-1 : 5'-GGTATGAGCTCATGCTTGGC TGGCAGCCATCCC-3' NF-1 m : 5'-GGTATGAGCTCATGCT TTAC TGGCAG CTGTCCC-3' TATA : 5'-GGCGGAGGCGGCCTCGGCCTCCTGTATATATAAAAAAAAG-3' . Gel mobility shift DNA binding assay The DNA binding assay (20 µl) contained 10 mM Tris--HCL (pH 7 .9), 50 mM NaCl, 5 mM MgCl 2 , 0 .5 mM EDTA, 1 mM DTT, 10% glycerol (v/v), 4 µg of poly(dldC) (Pharmacia), 32 P-labeled DNA, and 5 Ag of nuclear extracts . The reactions were carried out at room temperature for 15 min and electrophoresed on a 6% polyacrylamide Tris--glycine gel . The gel was dried and samples visualized by autoradiography with Kodak XAR-5 film with an intensifying screen . DNase I protection assays DNase I protection assays have been described in detail elsewhere (Amemiya et al., 1989) . Briefly, 40-50 mg of nuclear extracts from various cells were incubated with the 32 P-labeled JC188 od probe for 15 min at room temperature . The samples were placed on ice for 2 min before the addition of an equal volume of a solution containing 5 MM CaCl 2 , and 10 MM MgC1 2 . After a 2-min incubation at room temperature DNase I (1Northington), freshly diluted to 10 pg/ml, was added and digestion was allowed to proceed for 30-60 sec at room temperature . The digestion was stopped and the samples were extracted with phenol-chloroform . The samples were dried under vacuum for 30 min before loading onto an 8% sequencing gel . The Maxam-Gilbert (Maxam and Gilbert, 1980) sequencing reaction
FIG . 1 . In situ DNA hybridization of JCV infected BJA-B cells . B1A-B cells were infected with 1CV at a multiplicity of 0 .01 HA units/cell . At 13 days postinfection cells were harvested, dried on glass coverslips, and fixed in 4% paraformaldehyde . JCV-positive cells are detected by incubating the fixed cells with a biotinylated JCV probe and detection with a peroxidase conjugated avidin-biotin complex reacted with diaminobenzidene and H 20,, resulting in a brown precipitate . Cells were visualized on a Zeiss ICM 405 equipped with Nomarski optics ; magnification was 200x .
was performed on the 32 P-labeled JC188 or, probe used in the DNase I protection assays for size markers . RESULTS Multiplication of JC virus in human B-lymphocytes Infection of BJA-B and Namalwa cells was initiated by incubation of the cells with 0 .01 HA units/cell of the Mad-1 strain of JCV at 37° for 2 hr . By the 13th day postinfection JCV DNA was detected in both cell lines by in situ DNA hybridization with a biotinylated JCV probe (Fig . 1) . Detection of JCV by this method is indicative of viral replication as 200-500 copies of the DNA must be present in a cell to yield a positive signal (Aksamit et al., 1985, 1986) . Southern blots of DNA extracted from these infected B cell cultures contain pre-
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FIG. 2 . Southern blot hybridization of BJA-B and Namalwa cells 4 weeks following 1CV infection . Cellular DNA 20 pg was digested with EcoRl, electrophoresed in a 1 .4% agarose gel, and transferred to a nitrocellulose filter by capillary blotting . 32 P-labelled JCV DNA probe was used for hybridization . Lanes marked JCV BJA-B (two experiments) and JCV NAM contained DNA extracted from infected cells and lane marked Control contained DNA extracted from uninfected control cells . EcoRl digestion of 1 .0 ug of circular 1CV DNA results in linear form of the JCV genome indicated as 5 .1-kb marker .
dominantly full-length copies of JCV DNA shown in Fig . 2 as a linearviral genome after digestion with the endonuclease EcoRl . JCV-positive BJA-B and Namalwa cells could routinely be scored using in situ hybridization demonstrating approximately 1% infected cells . Infection of BJA-B and Namalwa cells could also be initiated
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by electroporation of JCV DNA into the cells . Two weeks following electroporation DNA was extracted from the cultures and digested with the methylation sensitive restriction enzyme Mbol . Following transfer to nitrocellulose JCV-specific DNA was detected by hybridization with a 32 P-labeled JCV probe (data not shown) . Progeny JCV virions could not be detected in infected B cell cultures by HA assay of culture fluids which may indicate that few virions are produced per cell . Because the sensitivity of HA assays is low, it is possible that virions were being produced at a level that was undetectable by this assay . To test this possibility, lysates from the infected B cell cultures were used as an inoculum on susceptible cultures of human fetal glial cells . JCV T antigen could be detected in these cultures several weeks after treatment with the B cell lysates . This experiment was duplicated by electroporation of the B cells with the pV1-TC molecular clone of JCV . Two weeks following infection of the glial cells with the transfected B cell lysates approximately 15% of the glial cells stained positive for JCV T antigen (Fig . 3) . Nuclear proteins present in B cell extracts bind specifically to JCV regulatory sequences : DNase I footprint assay DNase I protection experiments were performed using nuclear proteins prepared from two B cell lines (LyT3 and Namalwa), human fetal glial cells, and HeLa
FIG . 3 . BJA-B and Narnalwa cells were transfected with JCV DNA as described under Materials and Methods . 1- he B cells were lysed and the lysates used in a blind passage assay to infect susceptible cultures of human fetal glial cells . Infected glial cells are detected by Irnmunofluorescent staining with the anti-T antigen antibody PAB 416 and visualized with a secondary goat anti-mouse FITC-conjugated antibody .
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FIG . 4 . DNase I footprint analysis of the interaction of nuclear proteins extracted from HFG cells (lane 2), Namalwa cells (lane 3), LyT3 cells (lane 4), and HeLa cells (lane 5), with the JCV regulatory region (JC188,,;) . Lanes designated G and G + A are Maxam-Gilbert sequencing reactions of the 1CV regulatory region . Lane 1 is the probe alone . The regulatory region probe used is illustrated diagrammatically at the left . Footprints A and C have previously been identified as a binding site for the nuclear factor-1 . The panel below is an expansion of the rightmost part of the upper panel .
cells . Equivalent amounts of nuclear proteins from glial, Namalwa, LyT3 and HeLa cells were allowed to bind to a JCV regulatory probe (JC188 o ,, ; see Materials and Methods) . Nuclear proteins from each of the extracts protected two regions of the probe from digestion with DNase 1 (Fig . 4, lanes 2-5, sites A and C) . The region designated site A is contained within the 98-bp repeat of the JCV regulatory region and spans a region between nucleotides 32 and 71 (Frisque et al ., 1984) . A second region, designated site C, is outside of the 98-bp repeat and spans a region between nucleotides 207 and 231 . The region within the 98-bp repeat protected from nuclease digestion (site A) was previously identified as containing potential binding sites for both the nuclear factor 1 (NF-1) (Amemiya et al., 1989) and c-Jun (Amemiya et al., J . Biol . Chem . 1992, in press) . In this report nuclear proteins present in two B-lymphocyte cell lines, LyT3 and Namalwa, are shown to footprint to the same region of the JCV probe as proteins present in glial cells (Fig . 4, lanes 2, 3, and 4) . Consistent with our earlier finding, nuclear extracts prepared from HeLa cells also protect this region from digestion with DNase I (Fig . 4, lane 5) .
Gel shift assay The specificity of the B cell nuclear proteins which were binding to this site in the JCV genome was determined using competitive gel shift experiments . When equivalent amounts of B cell nuclear extracts from either Namalwa or LyT3 cells were reacted with the JCV regulatory region probe (JC188 or), five gel-shifted bands were evident (Fig . 5, lanes 2 and 6) . The gel shift patterns in both B cell lines were similarto one another but were different from the pattern seen with the HeLa cell extract (Fig . 5, lane 10) . Competitions with an oligonucleotide containing a binding site for the nuclearfactor-1 (NF-1) specifically eliminated a single gel-shifted band present in both B cell extracts (Fig . 5, lanes 3 and 7, bold arrow) . This oligonucleotide competed for different bands in the HeLa cell extract (Fig . 5, lane 11) . An oligonucleotide containing a mutated NF-1 binding site failed to compete for any of the gel-shifted bands using B cell extracts (Fig . 5, lanes 4 and 8) but did compete for one of the larger complexes using the HeLa cell extract (Fig . 5, lane 12, dashed arrow) . As an additional control an oligonucletide containing a TATA
JC VIRUS IN B CELLS
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Ww~urr FIG . 5 . Competitive gel retention assay of a JCV regulatory region probe (JC188 o „) incubated with nuclear extracts from Namalwa, LyT3 or HeLa cells . Lane 1 is the probe alone . Lanes 2, 6, and 10 are the probe without competitor . Lanes 3, 7, and 11 are the probe incubated with a 100-fold molar excess of NF-1 competitor . Lanes 4, 8, and 12 are the probe incubated with a 100-fold molar excess of an oligonucleotide containing a mutated NF-l binding site (NF-1m) . Lanes 5, 9, and 13 are the probe incubated with a 100-fold molar excess of an oligonucleotide containing a TATA sequence as competitor. The bold arrow indicates the position of a band present in Namalwa and LyT3 lanes but not in the HeLa cell lane that is specifically competed for by an NF-1 oligonucleotide but not by a mutated NF-1 oligonucleotide . The light arrow indicates a band competed for by the TATA-containing oligonucleotide . The dashed arrow indicates the position of a higher molecular weight band that is competed for by both the NF-1 and MF-1 m but not by the TATA oligonucleotide in HeLa cell extracts .
sequence was used which competed for a different band, representing the A/T rich sequences present in the probe . (Fig . 5, lanes 5, 9, 13, thin arrow) . DISCUSSION JCV-infected B cells have been identified in brain, spleen, bone marrow, and peripheral lymphocytes of PML patients (Houff at al ., 1988 ; Major et al., 1990, 1992 ; Tornatore et al., 1992) . In order to study the biology and understand the molecular control of JCV in human B lymphocytes in more detail, attempts were made to infect cell cultures of human B cell lines . Several B cell lines were chosen for this study ; EB virus Namalwa and BJA-B lymphoma cell lines, and a B cell line developed from tonsils, LyT3 . The data here show that approximately 1% of the cells from each of the three cell lines becomes infected with JCV when adsorbed with 0 .01 HA units of virus per cell . The JCV DNA that replicated in these B cells is full-length, infectious viral DNA . This differs from pre-
721
vious observations that had demonstrated JCV multiplication in other non-brain-derived cells (Miyamura at al., 1980) . The JCV DNA that was isolated from these cultures had multiple genomic deletions and rearrangements (Yoshiike et at, 1982 ; Miyamura et al., 1985) . Similar altered or rearranged viral DNAs have not been identified from infected B cells in these cultures . Also, recent nucleotide sequencing of the regulatory region of JCV-amplified DNA from peripheral lymphocytes from two PML patients demonstrated the prototype Mad-1 and Mad-4 genotypes (Tornatore et al., 1992) . The level of infectivity of JCV in B cells is consistent with what has been observed for other lymphotropic viruses including lymphotropic simian polyomavirus, LPV, which specifically targets B lymphocytes (Mosthaf at al., 1985 ; McChesney and Oldstone, 1987 ; Erselius at al., 1990) . Currently we are examining the effects of different B cell mitogens on the multiplication of JCV in B cell lines representing various differentiative states of B cells as well as in primary human B cells . JCV has usually been considered a neurotropic virus, Its presence in peripheral lymphocytes of PML patients and its ability to infect B lymphocyte lines in culture suggest an expanded host range . Cell type specificity of JCV is determined at the level of transcription of the early viral genes (Kenney at at, 1984 ; Feigenbaum et al ., 1987 ; Tada et at, 1989) . These current observations suggest some common molecular control of viral gene expression between glial cells and B cells . The gel-shift and DNase footprint data show that nuclear proteins present in B cells bind to critical regulatory elements in the JCV genome that are also bound by nuclear proteins from human fetal glial cells . The most prominent protein-binding region on the JCV genome contains consensus binding sites for nuclear factor-1 (NF-1) (Amemiya at al., 1989) and c-Jun (Amemiya at al., 1992) . NF-1 and Jun binding to these regions have been confirmed using purified NF-1 and c-Jun protein preparations (Amemiya et al., 1992) . In gel-shift competition assays an oligonucleotide containing an NF-1 binding site but not an oligonucleotide containing a mutated NF-1 binding site specifically competes for a gel shifted band unique to B cells . A similar gel shifted band was seen with the human fetal glial extracts (not shown), The nuclear factor-1 is a ubiquitous nuclear protein that was originally identified as a stimulator of adenovirus DNA replication (Nagata et at, 1983 ; Rawlins et at, 1984) . NF-1 is part of a family of different polypeptides encoded by multiple genes whose products are differentially spliced (Gill etat, 1988 ; Paonessa etal., 1988 ;_ Santoro et al., 1988 ; Nermod et at, 1989) . It has been shown to stimulate replication of a number of viral
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ATWOOD ET AL .
DNAs including JCV DNA (Nagata etal., 1983 ; Rawlins et aL, 1984 ; Socket al., 1991) . NF-1 is identical to the cellular transcription factor (CTF) (CCAATtranscription factor) and has been found to play a dual role in both transcription and replication of DNA (Rosenfeld and Kelly, 1986 ; Jones at at, 1987 ; DePamphilis, 1988 ; Santoro et al., 1988) . Experiments designed to test whether NF-1 or NF-1-like proteins from B cells and glial cells function in the multiplication cycle of JCV in B cells and glial cells are underway . Future studies on the infectivity of JCV in additional B cell lines as well as in primary B cells should help to clarify the role that B cells may play in the life cycle of JCV and in the pathogenesis of PML . In addition, molecular analyses of transcription factor binding to the JCV regulatory region from these B cells should help to elucidate the signals that may be involved in regulating the expression of JCV in cells of both lymphoid and glial origin . ACKNOWLEDGMENTS We thank Linda Durham and Blanche Curfman for technical assistance and Peter Pares for preparation of the ol,gonucleotides . We also thank Carlo Tornatore for his many helpful suggestions .
REFERENCES AKSAMIT, A. J ., MOURRAIN, P ., SEVER, J . L., and MAJOR, E . 0 . (1985) . Progressive Multifocal Leukoencephalopathy: Investigation of Three Cases Using In Situ Hybridization with JC Virus Biotinylated DNA Probe . Ann . Neurol. 18, 490-496 . AKSAMIT, A . 1 ., SEVER, J . L., and MAJOR, E . O . (1986) . Progressive Multifocal Leukoencephalopathy : JO virus detection by in situ hybridization compared with immunocytochemistry . Neurology 36, 499-504 . AMEMIYA, K ., TRAUB, R ., DURHAM, L ., and MAJOR, E . O . (1989) . Interaction of nuclear factor-1-like protein with the regulatory region of the human polyomavirus JC virus . J. Biol. Chem. 264, 7025-7032 . AMEMIYA, K ., TRAUB, R ., DURHAM, L ., and MAJOR, F . O . (1992) . Adjacent nuclear factor-1 and activator protein binding sites in the enhancer of the neurotropic JC Virus . J . Biol. Chem . 267, 1420414211 . HERGER, J . R ., KASLOVnz, B ., PosT, M . J ., and DICKINSON, G . (1987) . Progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection . A review of the literature w with a report of sixteen cases . Ann . Intern . Med. 107, 78-87 . BRADFORD, M . M . (1976) . A rapid and quantitative method for quantitation of microgram quantities of proteins using the principle of protein-dye binding . Anal . Biochem . 72, 248-254 . BROOKS, B . R ., and WALKER, D . L . (1984) . Progressive multifocal leukoencephalopathy . Neurol. Clin. 2, 299-313 . BROWN, P ., TSAI, T ., and GAJDUSEK, D . C . (1975) . Seroepiderniology of human papovaviruses : Discovery of virgin populations and some unusual patterns of antibody prevalence among remote peoples of the World . Am, . J. Epidemiol. 102, 33t-340 . CHAISSON, R . E ., and GRIFFIN, D . E . (1990) . Progressive multifocal leukoencephalopathy in AIDS . ]AMA 264, 79-82 . DEPAMPHILIS, M . (1988) . Transcriptional elements as components of eukaryotic origins of DNA replication . Cell 62, 635-638 .
DIGNAM, J . D ., LEaovITZ, R . M ., and ROEDER, R . G . (1983) . Acurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei . Nucleic Acid Res. 11, 14751489 . ELDER, G . A ., and MAJOR, E . O . (1988) . Early appearance of type II astrocytes in developing human fetal brain . Dev. Brain Res . 42, 146-150 . ERSELIUS, J . R ., JosTES, B ., HATzoPOULOS, A. K ., MOSIHAF, L ., and Gauss, P . (1990). Cell type specific control elements of the lymphotropic papovavirus enhances . J. Virol. 64, 1657-1666 . FEIGENBAUM, L ., KHALIu, K ., MAJOR, E . 0 ., and KHOURY, G . (1987) . Regulation of the host range of human papovavirusJCV . PNAS84, 3695-3698 . FRISOUE, R ., BREAM, G ., and CANNELLA, M . (1984) . Human polyomavirus JC virus genome .1. Uirdl. 51, 458-469 . GILL, G ., SMITH, J . R ., GOLDSTEIN . J . L ., SLAUGHTER, C . A ., ORTH, K ., BROWN, M . S ., and OSBORNE, T. F. (1968) . Multiple genes encode nuclear factor 1 like proteins that bind to the promoter for 3-hydroxy-3-mrthylglutaryl-coenzyme-A-reductase . Prop. Nat!. Acad. Sci. USA 85,8963-8967 . GRINNELL, B . W ., PADGETT, B . L, and WALKER, D . L. (1983) . Distribution of non-integrated DNA from JC papovavirus in organs of patients with progressive multifocal leukoencephalopathy . J. lnf. Dis. 147,669-675 . Ho, J . L, POLDRE, P . A., McENIRY, D ., HOWLEY, P . M ., SNYDMAN, D . R ., RUDDERS, R . A ., and WORTHINGTON, M . (1984) . Aquired immunodeficiency syndrome with progressive multifocal leukoencephalopathy and monoclcnal B-cell proliferation . Ann . Intern . Med. 100, 693-696 . HOUFF, S . A ., MAJOR, E . O ., KATZ, D . A ., KuFTA, C . V ., SEVER, J . L., PITTALUGA, S ., ROBERTS, J . R ., Grit, J ., SAINI, N ., and Lux, W. (1988) . Involvement of IC virus infected mononuclear cells from the bone marrow and spleen in the pathogenesis of progressive multifocal leukoencephalopathy . N. Engl. J. Med. 318, 301-305 . JONES, K . A ., KADONAGA, J . T ., ROSENFELD, P . J ., KELLY, T . J ., and THAN, R . (1987) . A cellular DNA-binding protein that activates eukaryotic transcription and replication . Cell 48, 79-89 . KENNEY, S ., NATARAJAN, V., STRIFE, D ., KHOURY, G ., and SALZMAN, N . P . (1984) . JC virus enhancer-promoter active in human brain cells . Science 226, 1337-1339 . KRUPP, L . B ., LIPTON, R. B ., SWERDLOW, M . L ., LEEDS, N . E ., and LLENA, J . (1985) . Progressive multifocal leukoencephalopathy : Clinical and radiographic features . Ann . Neurol. 17, 344-349 . MAJOR, E . 0 ., AMEMIYA, K ., ELDER, G ., and HOUFF, S . A . (1990) . Glial cells of the human developing brain and B cells of the immune system share a common DNA binding factorfor recognition of the regulatory sequences of the human polyomavirus, JCV . J. Neurosci. Res. 27, 461-471 . MAJOR, E . 0 ., and VACANTE, D . A . (1989) . Human fetal astrocytes in culture support the growth of the neurotropic human polyomavirus, JCV . J. Neuroparhol. Exp . Neurol. 48, 425-436 . MAJOR, E . 0 ., AMEMIYA, K ., TORNATORE, C . S ., HOUFF, S . A ., and BERGER, J . R . (1992) . Pathogenesis and molecular biology of Progressive Multifocal Leukoencephalopathy, a JC Virus-induced demyelinating disease of the human brain . Clin . Microbiol . Rev. 5, 49-73 . MAxAM, A . M ., and GILBERT, W . (1980) . Sequencing and end-labelled DNA with base specific chemical cleavages . Methods Enzymol. 65,499-560 . McCHESNEY, M . B ., and OLDSTONF M . B . A. (1987) . Viruses perturb lymphocyte functions : Selected principles characterizing virus induced immunosuppression . Ann . Rev. lmmunol. 5, 279-304 . MIYAMuRA, T ., FURANO, A ., and YOSHIIKE, K . (1985) . DNA rearrangement in the control region for early transcription in a human poly-
JC VIRUS IN B CELLS
omavirus JC host range mutant capable of growing in human embryonic kidney cells . J. Virel. 54, 750-756 . MIYAMURA, T ., YOSHIIKE, K ., andTAKEMOTO, K . K . (1980) . Characterization of JC papovavirus adapted to grow in human embryonic kidney cells . J. Virol. 35, 498-504 . MOSTHAF, L ., PAWLITA, M ., and GROSS, P . (1985) . A viral enhancer element specifically active in human haematopoietic cells . Science 315, 587-600 . NAGATA, K ., GuGGENHEIMER, R . A,, and HURWITZ, J . (1983) . Specific binding of a cellular DNA binding protein to the origin of replication of adenovirus DNA . Proc. Nat/. Acad. Sol. USA 80, 6177-6181 . NERMOD, N ., O'NEILL, E . A ., KELLY, T . J ., and TJAN, R . (1989) . The proline rich transcriptional activator of CTF/NF-1 is distinct from the replication and DNA binding domain . Cell 58, 741-753 . PADGETT, B . L ., and WALKER, D . L . (1983) . Virologic and serologic studies of progressive multifocal leukoencephalopathuy . Prog. Olin . Bini. Res. 105, 107-117 . PADGETT, B . L ., ZURHEIN, G . M ., WALKER, D . L ., ECHROADE, R. J ., and DESSEL, B . H . (1971) . Cultivation of papova-like virus from human brain Mth progressive multifocal leukoencephalopathy . Lancet 1, 1257-1260 . PAONESSA, G ., GOUNARI, F ., FRANK, R ., and CORTESE, R . (1988) . Purification of a NF-1 like DNA binding protein from rat liverand cloning of the corresponding cDNA . EMBO J. 7, 3115-3123 . RAWLINS, D . R ., ROSENFELD, P . J ., WIDES, J . R ., CHALLBERG, M . D ., and KELLY, T . J ., JR . (1984) . Structure and function of the adenovirus origin of replication . Cell 37, 309-319 . RICHARDSON, E . P ., JR . (1961) . Progressive multifocal leukoencephalopathy . N. Eng/. J. Med. 265, 815-823 . ROSENFELD, P . J ., and KELLY, T . J . (1986) . Purification of nuclearfactor
723
1 by DNA recognition affinity chromatography . J.. Bioi . Chem . 261, 1398-1408 . RZIHA, H . J ., BoRNKAMM, G . M ., and ZuRHAUSEN, H . (1978) . Seroepidemiological studies and serologic response to viral infection . Med. Microbiol. lmmunol . 165, 73-92 . SANTORO, C ., NERMOD, N ., ANDREWS, P . C ., and TJIAN, R . (1988) . A family of human COAT-box-binding proteins active in transcription and replication : Cloning and expression of multiple cDNAs . Nature 334,218-224 . SOCK, E ., WEGNER, M ., and GRUMMT, F . (1991) . DNA replication of human polyomavirus JC is stimulated by NF-1 in vivo . Virology 182,298-308 . STONER, G . L ., RYSCHKEWITSCH, C . F ., WALKER, 0 . I_ ., and WEBSTER, H . D . F . (1986) . JO papovavirus large tumor (T)-antigen expression in brain tissue of squired immune deficiency syndrome (AIDS) and non-AIDS patients with progressive multifocal leukoencephalopathy. Proc. Nail, Aced. Sci. USA 83, 2271-2275 . TADA, H ., LASHGARI, M ., RAPPAPORT, J ., and KHAUU, K . (1989) . Cell type-specific expression of JC virus early promoter is determined by positive and negative regulation . J. Virol. 63, 463-466 . TORNATORE, C ., BERGER, J . R ., HOUFF, S . A., CURFMAN, B ., MEYERS, K ., WINFIELD, D ., and MAJOR, F . 0 . (1992) . Detection of JC Virus DNA in peripheral lymphocytes from patients with and without progressive multifocal leukoencephalopathy . Ann . Neurot 31, 454-462 . WALKER, D . L ., and PADGETT, B . L . (1983) . The epidemiology of human polyornaviruses . In "Polyomaviruses and Human Neurological Diseases" (J . L. Sever and D . L. Madden, Eds .), pp . 99-106 . A. R . Liss, New York . YOSHIIKE, K ., MIYAMURA, T., CHAN, H . W ., and TAKEMOTD, K . K . (1982) . Two defective DNAs of human polyomavirus JC adapted to growth in human embryonic kidney cells . J Virel . 42, 395-401 .
190, 716-723 (1992)
Interaction of the Human Polyomavirus, JCV, with Human B-Lymphocytes WALTER J . ATWOOD, KEI AMEMIYA, RENEE TRAUB, JURGEN HARMS',
AND
EUGENE O . MAJOR'
Section on Molecular Virology and Genetics, Laboratory of Viral and Molecular Pathogenesis, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 Received April 20, 1992 ; accepted June 18. 1992
The human polyomavirus, JCV, is the causative agent of the central nervous system demyelinating disease progressive multifocal leukoencephalopathy (PML) . The principal target of JCV infection in the central nervous system (CNS) is the myelinating oligodendrocyte . However, the site of JCV multiplication outside of the CNS and the mechanism by which virus gains access to the brain are not known . Recently, JCV infected B-lymphocytes have been demonstrated in PML patients in several lymphoid organs, in circulating peripheral lymphocytes, and in brain, suggesting a possible role of B-lymphocytes in the dissemination of virus to the brain . The experiments reported here were undertaken to understand more about the interactions of JCV with human B-lymphocytes . The data show that JCV is able to multiply in either Epstein-Barr virus transformed (EBV) or EBV negative human B cell lines resulting in production of infectious, progeny virions. In addition, nuclear proteins extracted from these B cells bind to similar nucleotides within the JCV regulatory region that are bound by nuclear proteins extracted from human fetal glial cells, the most susceptible host and principal target cell for JCV infection in vitro . It is not known, however, whether these DNA binding proteins from susceptible B cells and glial cells are similar . 9 1992 Academic Press, Inc .
INTRODUCTION
JCV latency and the mechanism that underlies its reactivation are currently unknown . In a previous report, JCV DNA was found to be widely disseminated in many
Seroepidemiological surveys of the human population indicate that infection with the human polyomavirus, 1CV, is very common and occurs worldwide (Brown et al., 1975 ; Rziha et al ., 1978 ; Padgett and Walker, 1983 ; Walker and Padgett, 1983) . Conversion
different organs including lymphoid tissue (Grinnell at al_ 1983) . The cell type harboring JCV DNA was not identified in that report . We have examined tissues of PML patients with or without AIDS for the detection of the JCV genome and
to seropositive status usually takes place during childhood, ages 5-8, with antibody levels remaining at de-
have described B-lymphocytes in the bone marrow, spleen, and brain as possible sites of JCV infection (Houff etal., 1988 ; Major etal., 1990) . Using PCR analy-
tectable levels throughout adulthood . Despite the prevalence of JCV in the human population the only known manifestation of JCV infection is the relatively rare central nervous system demyelinating disease, progressive multifocal leukoencephalopathy, or PML(Richardson, 1961 ; Padgett at al ., 1971) . PML occurs most
sis, JCV DNA was found associated with peripheral blood lymphocytes in 89% of PML patients (Tornatore
et al., 1992) . The histopathology of demyelinated lesions in the brain of PML patients suggest a hematogenous spread of virus to the brain . It has been hypothesized thatJCV gains entry to the brain in B-lymphocytes of immune-suppressed individuals (Major etat, 1992) . In this report the ability of JCV to infect human B-lymphocyte cell lines was investigated . Introduction of the
frequently in individuals with an underlying immunosuppressive disorder . Based largely upon studies of AIDS patients, it appears that there is a direct correlation between the extent of immune deficiency and the incidence and severity of PML (Ho at al., 1984 ; Krupp et al., 1985 ; Stoner et al., 1986 ; Berger at al., 1987 ; Chaisson and Griffin, 1990) . The observations of the
1CV genome into B-lymphocytes either by infection with JCV virions or by transfection with JCV DNA results in de novo replication of viral DNA and the multiplication of infectious virions . In addition, nuclear proteins extracted from human B cell lines are shown by competitive gel shift analyses and DNase I footprinting
common nature of 1CV infection with a low incidence of PML and its association with immunocompromised individuals indicate that JCV probably establishes a latent infection which is activated at times of immune suppression (Brooks and Walker, 1984) . The site of
assays to interact with the regulatory region of the JCV genome at a site previously identified as a binding site for nuclear factor-1 (NF-1) . The nucleotide sequence in
' Present address : Deutsches Primatenzentrum ABT . Immunologic, Gottingen, Germany. ' To whom reprint requests should be addressed . 0042-6822/92 $0 .00 Ccp9'ight O 1992 by Academic Pmas . Inc . Ai rghIe of -eproduction in any form reserved .
the JCV regulatory region bound by the B cell nuclear proteins map to the same location as the nucleotides 716
1C VIRUS IN 3 CELLS
bound by proteins from human fetal glial cells . These results strengthen the suggestion that B-lymphocytes can serve as a susceptible cell for JCV infection and allow a more detailed investigation of their role in the pathogenesis of PML .
MATERIALS AND METHODS Cells and virus Preparation of glial cell cultures from human fetal tissue has been described in detail (Elder and Major, 1988 ; Major and Vacante, 1989) . The human Epstein Barr virus (EBV) negative B cell line, BJA-B, was obtained from L . Staudt . EBV-positive Namalwa cells were obtained from American Type Culture Collection (ATCC) . The LyT3 B cell line was produced by longterm culture of lymphocytes from human tonsil of a presumably healthy child and was a generous gift of A . DeGrassi . The EBV status of this cell line has not been determined (A . DeGrassi, personal communication) . BJA-B and Namalwa cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum . LyT3 cells were grown in RPMI 1640 supplemented with nonessential amino acids (Whittaker Bioproducts, Inc .) and
71 7
Transfection of B cells with JCV DNA BJA-B and Namalwa cells were suspended in PBS at a density of 1 X 10' cells per milliliter and held on ice for 10 min . JCV DNA (6 µg/10 6 cells), prepared by EcoRl digestion of the plasmid pM1-TC (Frisque etal., 1984), was introduced into the B cell lines by electroporation (BTX Transfector 300) at a capacitance of 1000 A F and a voltage of 350V for BJA-B cells and 300V for Namalwa cells . The transfected B cells were lysed by freeze-thawing and virus was released from membranes by incubation with 0 .25% sodium deoxycholic acid . The lysates were then used to infect human fetal glial cells growing on glass coverslips . JCV-infected glial cells were detected by indirect immunofluorescent staining of T antigen with the monoclonal antibody PAB 416 (Oncogene Sciences ; Majoretak, 1992) and a secondary goat anti-mouse IgG conjugated to FITC . The T-antigen-positive cells were visualized with a Zeiss ICM 405 epifluorescent microscope .
Nuclear extract preparation Nuclear extracts were prepared from 1-10 X 10 8 cells either growing on plates (HFGC, HeLa) or in spin-
10% fetal bovine serum . All cell lines were grown in the presence of 250 µg/ml gentamicin (Sigma) .
ner flasks (Namalwa, LyT3) by a modification of the procedure of Dignam etal . (1983) . Briefly, washed cells were swollen for 10-15 min on ice in four packed cell
Infection of B cells with JC virus
volumes (PCV) of buffer A [10 mM Tris-HCL (pH 7 .9), 1 .5 mM MgCl 2 , 10 mM KCL, 0 .5 mM dithiothreitol (DTT), and 0 .2 mM phenylmethylsulfonyl fluoride
BJA-B and Namalwa cells were washed three times in phosphate-buffered saline (PBS) and incubated for 2 hr at 37° in PBS with JCV at 1 X 10 4 hemagglutination (HA) units of virus per 1 x 106 cells . At 13 days postinfection (p .i .) cells were harvested and JCV DNA replica-
(PMSF)] . The cells were collected by centrifugation at 2000 rpm for 10 min and lysed with at least 10 strokes in a Wheaton homogenizer with a type B pestle . Nuclei were extracted in 4 PCV of buffer C, [20 mM Tris-HCL (pH 7 .9), 420 mM NaCl, 1 .5 mM MgCI 2 , 0 .2 mM EDTA, 25% glycerol (v/v), 0 .5 mM DTT, and 0 .5 mM PMSF],
tion was assayed by in situ DNA hybridization with a full-length genomic JCV-specific biotinylated probe (ENZO Diagnostics, Inc ., New York, NY) . Briefly, cells
The extracted nuclear proteins were precipitated with ammonium sulfate and collected by centrifugation for 30 min at 10,000 rpm . The pellet was resuspended in
were dried onto glass coverslips, fixed in 4%% paraformaldehyde and dehydrated in graded ethanol (60-
buffer BC [20 mM Tris-HCL (pH 7 .9), 100 mM KCI, 0 .2 mM EDTA, 20% glycerol (v/v), and 0 .5 mM DTT], and dialyzed overnight against the same buffer . The dia-
100%) . The cells were then rehydrated in PBS, treated for 30 min with methanol and H 2 0 2 before preparation for in situ hybridization to eliminate endogenous peroxidase activity (Aksamit et al ., 1985, 1986) . For Southern blot hybridization, nitrocellulose filters were hybrid-
lyzed nuclear proteins were cleared of insoluble material by centrifugation and the protein content of the samples estimated by the method of Bradford (1976) . Buffers A and C contained the protease inhibitors anti-
ized to a 32 P-labeled JCV DNA probe . Hybridization was done at 42° for 48 hr in 6x SSC, 50% formamide, 5X Denhardt's . Kodak X-AR film was exposed for 24 hr . Quantitation of JCV was made by hemagglutination (HA) of human type 0 erythrocytes (Padgett et al., 1971) . The virus titer is expressed as the reciprocal of
pain, leupeptin, pepstatin A, chymostatin, and aprotinin at 5 µg/ml, buffer BC contained these same protease inhibitors at 1 ug/ml .
the highest dilution which results in hemagglutination (HA unit) .
The plasmid, pJC188 °r „ contains one 98-bp repeat unit of the JC virus enhancer/promoter region and its
DNA probe and competitor preparation
71 8
ATWOOD ET AL .
construction has been previously described (Amemiya et at, 1989) . The plasmid was linearized with Hindlll, the 5' phosphate removed using calf intestine alkaline phosphatase (Boehringer-Mannheim) and replaced with a y-32 P-labeled ATP (DuPont-NEN) . The 188-bp fragment containing the JCV regulatory region was separated from the rest of the plasmid by Pvull digestion, electrophoresis on a 15% polyacrylamide gel, and electroelution with an Elutrap (Schleicher and Schuell) . The labeled DNA was precipitated in ethanol, suspended in 100 pl dH 2 O, and an aliquot removed to determine the specific activity of the probe . Oligonucleotides used as competitors were synthesized on an Applied Biosystems 380A DNA synthesizer . Three double-stranded oligonucleotides were used as competitors ; one contained an intact binding site for NF-1, another contained a mutated NF-1 binding site (NF-1 m), and a third contained the TATA sequence . NF-1 : 5'-GGTATGAGCTCATGCTTGGC TGGCAGCCATCCC-3' NF-1 m : 5'-GGTATGAGCTCATGCT TTAC TGGCAG CTGTCCC-3' TATA : 5'-GGCGGAGGCGGCCTCGGCCTCCTGTATATATAAAAAAAAG-3' . Gel mobility shift DNA binding assay The DNA binding assay (20 µl) contained 10 mM Tris--HCL (pH 7 .9), 50 mM NaCl, 5 mM MgCl 2 , 0 .5 mM EDTA, 1 mM DTT, 10% glycerol (v/v), 4 µg of poly(dldC) (Pharmacia), 32 P-labeled DNA, and 5 Ag of nuclear extracts . The reactions were carried out at room temperature for 15 min and electrophoresed on a 6% polyacrylamide Tris--glycine gel . The gel was dried and samples visualized by autoradiography with Kodak XAR-5 film with an intensifying screen . DNase I protection assays DNase I protection assays have been described in detail elsewhere (Amemiya et al., 1989) . Briefly, 40-50 mg of nuclear extracts from various cells were incubated with the 32 P-labeled JC188 od probe for 15 min at room temperature . The samples were placed on ice for 2 min before the addition of an equal volume of a solution containing 5 MM CaCl 2 , and 10 MM MgC1 2 . After a 2-min incubation at room temperature DNase I (1Northington), freshly diluted to 10 pg/ml, was added and digestion was allowed to proceed for 30-60 sec at room temperature . The digestion was stopped and the samples were extracted with phenol-chloroform . The samples were dried under vacuum for 30 min before loading onto an 8% sequencing gel . The Maxam-Gilbert (Maxam and Gilbert, 1980) sequencing reaction
FIG . 1 . In situ DNA hybridization of JCV infected BJA-B cells . B1A-B cells were infected with 1CV at a multiplicity of 0 .01 HA units/cell . At 13 days postinfection cells were harvested, dried on glass coverslips, and fixed in 4% paraformaldehyde . JCV-positive cells are detected by incubating the fixed cells with a biotinylated JCV probe and detection with a peroxidase conjugated avidin-biotin complex reacted with diaminobenzidene and H 20,, resulting in a brown precipitate . Cells were visualized on a Zeiss ICM 405 equipped with Nomarski optics ; magnification was 200x .
was performed on the 32 P-labeled JC188 or, probe used in the DNase I protection assays for size markers . RESULTS Multiplication of JC virus in human B-lymphocytes Infection of BJA-B and Namalwa cells was initiated by incubation of the cells with 0 .01 HA units/cell of the Mad-1 strain of JCV at 37° for 2 hr . By the 13th day postinfection JCV DNA was detected in both cell lines by in situ DNA hybridization with a biotinylated JCV probe (Fig . 1) . Detection of JCV by this method is indicative of viral replication as 200-500 copies of the DNA must be present in a cell to yield a positive signal (Aksamit et al., 1985, 1986) . Southern blots of DNA extracted from these infected B cell cultures contain pre-
JC VIRUS IN B CELLS BJA-B
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FIG. 2 . Southern blot hybridization of BJA-B and Namalwa cells 4 weeks following 1CV infection . Cellular DNA 20 pg was digested with EcoRl, electrophoresed in a 1 .4% agarose gel, and transferred to a nitrocellulose filter by capillary blotting . 32 P-labelled JCV DNA probe was used for hybridization . Lanes marked JCV BJA-B (two experiments) and JCV NAM contained DNA extracted from infected cells and lane marked Control contained DNA extracted from uninfected control cells . EcoRl digestion of 1 .0 ug of circular 1CV DNA results in linear form of the JCV genome indicated as 5 .1-kb marker .
dominantly full-length copies of JCV DNA shown in Fig . 2 as a linearviral genome after digestion with the endonuclease EcoRl . JCV-positive BJA-B and Namalwa cells could routinely be scored using in situ hybridization demonstrating approximately 1% infected cells . Infection of BJA-B and Namalwa cells could also be initiated
719
by electroporation of JCV DNA into the cells . Two weeks following electroporation DNA was extracted from the cultures and digested with the methylation sensitive restriction enzyme Mbol . Following transfer to nitrocellulose JCV-specific DNA was detected by hybridization with a 32 P-labeled JCV probe (data not shown) . Progeny JCV virions could not be detected in infected B cell cultures by HA assay of culture fluids which may indicate that few virions are produced per cell . Because the sensitivity of HA assays is low, it is possible that virions were being produced at a level that was undetectable by this assay . To test this possibility, lysates from the infected B cell cultures were used as an inoculum on susceptible cultures of human fetal glial cells . JCV T antigen could be detected in these cultures several weeks after treatment with the B cell lysates . This experiment was duplicated by electroporation of the B cells with the pV1-TC molecular clone of JCV . Two weeks following infection of the glial cells with the transfected B cell lysates approximately 15% of the glial cells stained positive for JCV T antigen (Fig . 3) . Nuclear proteins present in B cell extracts bind specifically to JCV regulatory sequences : DNase I footprint assay DNase I protection experiments were performed using nuclear proteins prepared from two B cell lines (LyT3 and Namalwa), human fetal glial cells, and HeLa
FIG . 3 . BJA-B and Narnalwa cells were transfected with JCV DNA as described under Materials and Methods . 1- he B cells were lysed and the lysates used in a blind passage assay to infect susceptible cultures of human fetal glial cells . Infected glial cells are detected by Irnmunofluorescent staining with the anti-T antigen antibody PAB 416 and visualized with a secondary goat anti-mouse FITC-conjugated antibody .
720
ATWOOD ET AL .
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FIG . 4 . DNase I footprint analysis of the interaction of nuclear proteins extracted from HFG cells (lane 2), Namalwa cells (lane 3), LyT3 cells (lane 4), and HeLa cells (lane 5), with the JCV regulatory region (JC188,,;) . Lanes designated G and G + A are Maxam-Gilbert sequencing reactions of the 1CV regulatory region . Lane 1 is the probe alone . The regulatory region probe used is illustrated diagrammatically at the left . Footprints A and C have previously been identified as a binding site for the nuclear factor-1 . The panel below is an expansion of the rightmost part of the upper panel .
cells . Equivalent amounts of nuclear proteins from glial, Namalwa, LyT3 and HeLa cells were allowed to bind to a JCV regulatory probe (JC188 o ,, ; see Materials and Methods) . Nuclear proteins from each of the extracts protected two regions of the probe from digestion with DNase 1 (Fig . 4, lanes 2-5, sites A and C) . The region designated site A is contained within the 98-bp repeat of the JCV regulatory region and spans a region between nucleotides 32 and 71 (Frisque et al ., 1984) . A second region, designated site C, is outside of the 98-bp repeat and spans a region between nucleotides 207 and 231 . The region within the 98-bp repeat protected from nuclease digestion (site A) was previously identified as containing potential binding sites for both the nuclear factor 1 (NF-1) (Amemiya et al., 1989) and c-Jun (Amemiya et al., J . Biol . Chem . 1992, in press) . In this report nuclear proteins present in two B-lymphocyte cell lines, LyT3 and Namalwa, are shown to footprint to the same region of the JCV probe as proteins present in glial cells (Fig . 4, lanes 2, 3, and 4) . Consistent with our earlier finding, nuclear extracts prepared from HeLa cells also protect this region from digestion with DNase I (Fig . 4, lane 5) .
Gel shift assay The specificity of the B cell nuclear proteins which were binding to this site in the JCV genome was determined using competitive gel shift experiments . When equivalent amounts of B cell nuclear extracts from either Namalwa or LyT3 cells were reacted with the JCV regulatory region probe (JC188 or), five gel-shifted bands were evident (Fig . 5, lanes 2 and 6) . The gel shift patterns in both B cell lines were similarto one another but were different from the pattern seen with the HeLa cell extract (Fig . 5, lane 10) . Competitions with an oligonucleotide containing a binding site for the nuclearfactor-1 (NF-1) specifically eliminated a single gel-shifted band present in both B cell extracts (Fig . 5, lanes 3 and 7, bold arrow) . This oligonucleotide competed for different bands in the HeLa cell extract (Fig . 5, lane 11) . An oligonucleotide containing a mutated NF-1 binding site failed to compete for any of the gel-shifted bands using B cell extracts (Fig . 5, lanes 4 and 8) but did compete for one of the larger complexes using the HeLa cell extract (Fig . 5, lane 12, dashed arrow) . As an additional control an oligonucletide containing a TATA
JC VIRUS IN B CELLS
Competitors
1
2
Namalwa 3 4 5
6
LyT3 7 8
9
Hells 10 11 12 13
NF1 NF1m TATA
~~
~iilfn
Ww~urr FIG . 5 . Competitive gel retention assay of a JCV regulatory region probe (JC188 o „) incubated with nuclear extracts from Namalwa, LyT3 or HeLa cells . Lane 1 is the probe alone . Lanes 2, 6, and 10 are the probe without competitor . Lanes 3, 7, and 11 are the probe incubated with a 100-fold molar excess of NF-1 competitor . Lanes 4, 8, and 12 are the probe incubated with a 100-fold molar excess of an oligonucleotide containing a mutated NF-l binding site (NF-1m) . Lanes 5, 9, and 13 are the probe incubated with a 100-fold molar excess of an oligonucleotide containing a TATA sequence as competitor. The bold arrow indicates the position of a band present in Namalwa and LyT3 lanes but not in the HeLa cell lane that is specifically competed for by an NF-1 oligonucleotide but not by a mutated NF-1 oligonucleotide . The light arrow indicates a band competed for by the TATA-containing oligonucleotide . The dashed arrow indicates the position of a higher molecular weight band that is competed for by both the NF-1 and MF-1 m but not by the TATA oligonucleotide in HeLa cell extracts .
sequence was used which competed for a different band, representing the A/T rich sequences present in the probe . (Fig . 5, lanes 5, 9, 13, thin arrow) . DISCUSSION JCV-infected B cells have been identified in brain, spleen, bone marrow, and peripheral lymphocytes of PML patients (Houff at al ., 1988 ; Major et al., 1990, 1992 ; Tornatore et al., 1992) . In order to study the biology and understand the molecular control of JCV in human B lymphocytes in more detail, attempts were made to infect cell cultures of human B cell lines . Several B cell lines were chosen for this study ; EB virus Namalwa and BJA-B lymphoma cell lines, and a B cell line developed from tonsils, LyT3 . The data here show that approximately 1% of the cells from each of the three cell lines becomes infected with JCV when adsorbed with 0 .01 HA units of virus per cell . The JCV DNA that replicated in these B cells is full-length, infectious viral DNA . This differs from pre-
721
vious observations that had demonstrated JCV multiplication in other non-brain-derived cells (Miyamura at al., 1980) . The JCV DNA that was isolated from these cultures had multiple genomic deletions and rearrangements (Yoshiike et at, 1982 ; Miyamura et al., 1985) . Similar altered or rearranged viral DNAs have not been identified from infected B cells in these cultures . Also, recent nucleotide sequencing of the regulatory region of JCV-amplified DNA from peripheral lymphocytes from two PML patients demonstrated the prototype Mad-1 and Mad-4 genotypes (Tornatore et al., 1992) . The level of infectivity of JCV in B cells is consistent with what has been observed for other lymphotropic viruses including lymphotropic simian polyomavirus, LPV, which specifically targets B lymphocytes (Mosthaf at al., 1985 ; McChesney and Oldstone, 1987 ; Erselius at al., 1990) . Currently we are examining the effects of different B cell mitogens on the multiplication of JCV in B cell lines representing various differentiative states of B cells as well as in primary human B cells . JCV has usually been considered a neurotropic virus, Its presence in peripheral lymphocytes of PML patients and its ability to infect B lymphocyte lines in culture suggest an expanded host range . Cell type specificity of JCV is determined at the level of transcription of the early viral genes (Kenney at at, 1984 ; Feigenbaum et al ., 1987 ; Tada et at, 1989) . These current observations suggest some common molecular control of viral gene expression between glial cells and B cells . The gel-shift and DNase footprint data show that nuclear proteins present in B cells bind to critical regulatory elements in the JCV genome that are also bound by nuclear proteins from human fetal glial cells . The most prominent protein-binding region on the JCV genome contains consensus binding sites for nuclear factor-1 (NF-1) (Amemiya at al., 1989) and c-Jun (Amemiya at al., 1992) . NF-1 and Jun binding to these regions have been confirmed using purified NF-1 and c-Jun protein preparations (Amemiya et al., 1992) . In gel-shift competition assays an oligonucleotide containing an NF-1 binding site but not an oligonucleotide containing a mutated NF-1 binding site specifically competes for a gel shifted band unique to B cells . A similar gel shifted band was seen with the human fetal glial extracts (not shown), The nuclear factor-1 is a ubiquitous nuclear protein that was originally identified as a stimulator of adenovirus DNA replication (Nagata et at, 1983 ; Rawlins et at, 1984) . NF-1 is part of a family of different polypeptides encoded by multiple genes whose products are differentially spliced (Gill etat, 1988 ; Paonessa etal., 1988 ;_ Santoro et al., 1988 ; Nermod et at, 1989) . It has been shown to stimulate replication of a number of viral
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ATWOOD ET AL .
DNAs including JCV DNA (Nagata etal., 1983 ; Rawlins et aL, 1984 ; Socket al., 1991) . NF-1 is identical to the cellular transcription factor (CTF) (CCAATtranscription factor) and has been found to play a dual role in both transcription and replication of DNA (Rosenfeld and Kelly, 1986 ; Jones at at, 1987 ; DePamphilis, 1988 ; Santoro et al., 1988) . Experiments designed to test whether NF-1 or NF-1-like proteins from B cells and glial cells function in the multiplication cycle of JCV in B cells and glial cells are underway . Future studies on the infectivity of JCV in additional B cell lines as well as in primary B cells should help to clarify the role that B cells may play in the life cycle of JCV and in the pathogenesis of PML . In addition, molecular analyses of transcription factor binding to the JCV regulatory region from these B cells should help to elucidate the signals that may be involved in regulating the expression of JCV in cells of both lymphoid and glial origin . ACKNOWLEDGMENTS We thank Linda Durham and Blanche Curfman for technical assistance and Peter Pares for preparation of the ol,gonucleotides . We also thank Carlo Tornatore for his many helpful suggestions .
REFERENCES AKSAMIT, A. J ., MOURRAIN, P ., SEVER, J . L., and MAJOR, E . 0 . (1985) . Progressive Multifocal Leukoencephalopathy: Investigation of Three Cases Using In Situ Hybridization with JC Virus Biotinylated DNA Probe . Ann . Neurol. 18, 490-496 . AKSAMIT, A . 1 ., SEVER, J . L., and MAJOR, E . O . (1986) . Progressive Multifocal Leukoencephalopathy : JO virus detection by in situ hybridization compared with immunocytochemistry . Neurology 36, 499-504 . AMEMIYA, K ., TRAUB, R ., DURHAM, L ., and MAJOR, E . O . (1989) . Interaction of nuclear factor-1-like protein with the regulatory region of the human polyomavirus JC virus . J. Biol. Chem. 264, 7025-7032 . AMEMIYA, K ., TRAUB, R ., DURHAM, L ., and MAJOR, F . O . (1992) . Adjacent nuclear factor-1 and activator protein binding sites in the enhancer of the neurotropic JC Virus . J . Biol. Chem . 267, 1420414211 . HERGER, J . R ., KASLOVnz, B ., PosT, M . J ., and DICKINSON, G . (1987) . Progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection . A review of the literature w with a report of sixteen cases . Ann . Intern . Med. 107, 78-87 . BRADFORD, M . M . (1976) . A rapid and quantitative method for quantitation of microgram quantities of proteins using the principle of protein-dye binding . Anal . Biochem . 72, 248-254 . BROOKS, B . R ., and WALKER, D . L . (1984) . Progressive multifocal leukoencephalopathy . Neurol. Clin. 2, 299-313 . BROWN, P ., TSAI, T ., and GAJDUSEK, D . C . (1975) . Seroepiderniology of human papovaviruses : Discovery of virgin populations and some unusual patterns of antibody prevalence among remote peoples of the World . Am, . J. Epidemiol. 102, 33t-340 . CHAISSON, R . E ., and GRIFFIN, D . E . (1990) . Progressive multifocal leukoencephalopathy in AIDS . ]AMA 264, 79-82 . DEPAMPHILIS, M . (1988) . Transcriptional elements as components of eukaryotic origins of DNA replication . Cell 62, 635-638 .
DIGNAM, J . D ., LEaovITZ, R . M ., and ROEDER, R . G . (1983) . Acurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei . Nucleic Acid Res. 11, 14751489 . ELDER, G . A ., and MAJOR, E . O . (1988) . Early appearance of type II astrocytes in developing human fetal brain . Dev. Brain Res . 42, 146-150 . ERSELIUS, J . R ., JosTES, B ., HATzoPOULOS, A. K ., MOSIHAF, L ., and Gauss, P . (1990). Cell type specific control elements of the lymphotropic papovavirus enhances . J. Virol. 64, 1657-1666 . FEIGENBAUM, L ., KHALIu, K ., MAJOR, E . 0 ., and KHOURY, G . (1987) . Regulation of the host range of human papovavirusJCV . PNAS84, 3695-3698 . FRISOUE, R ., BREAM, G ., and CANNELLA, M . (1984) . Human polyomavirus JC virus genome .1. Uirdl. 51, 458-469 . GILL, G ., SMITH, J . R ., GOLDSTEIN . J . L ., SLAUGHTER, C . A ., ORTH, K ., BROWN, M . S ., and OSBORNE, T. F. (1968) . Multiple genes encode nuclear factor 1 like proteins that bind to the promoter for 3-hydroxy-3-mrthylglutaryl-coenzyme-A-reductase . Prop. Nat!. Acad. Sci. USA 85,8963-8967 . GRINNELL, B . W ., PADGETT, B . L, and WALKER, D . L. (1983) . Distribution of non-integrated DNA from JC papovavirus in organs of patients with progressive multifocal leukoencephalopathy . J. lnf. Dis. 147,669-675 . Ho, J . L, POLDRE, P . A., McENIRY, D ., HOWLEY, P . M ., SNYDMAN, D . R ., RUDDERS, R . A ., and WORTHINGTON, M . (1984) . Aquired immunodeficiency syndrome with progressive multifocal leukoencephalopathy and monoclcnal B-cell proliferation . Ann . Intern . Med. 100, 693-696 . HOUFF, S . A ., MAJOR, E . O ., KATZ, D . A ., KuFTA, C . V ., SEVER, J . L., PITTALUGA, S ., ROBERTS, J . R ., Grit, J ., SAINI, N ., and Lux, W. (1988) . Involvement of IC virus infected mononuclear cells from the bone marrow and spleen in the pathogenesis of progressive multifocal leukoencephalopathy . N. Engl. J. Med. 318, 301-305 . JONES, K . A ., KADONAGA, J . T ., ROSENFELD, P . J ., KELLY, T . J ., and THAN, R . (1987) . A cellular DNA-binding protein that activates eukaryotic transcription and replication . Cell 48, 79-89 . KENNEY, S ., NATARAJAN, V., STRIFE, D ., KHOURY, G ., and SALZMAN, N . P . (1984) . JC virus enhancer-promoter active in human brain cells . Science 226, 1337-1339 . KRUPP, L . B ., LIPTON, R. B ., SWERDLOW, M . L ., LEEDS, N . E ., and LLENA, J . (1985) . Progressive multifocal leukoencephalopathy : Clinical and radiographic features . Ann . Neurol. 17, 344-349 . MAJOR, E . 0 ., AMEMIYA, K ., ELDER, G ., and HOUFF, S . A . (1990) . Glial cells of the human developing brain and B cells of the immune system share a common DNA binding factorfor recognition of the regulatory sequences of the human polyomavirus, JCV . J. Neurosci. Res. 27, 461-471 . MAJOR, E . 0 ., and VACANTE, D . A . (1989) . Human fetal astrocytes in culture support the growth of the neurotropic human polyomavirus, JCV . J. Neuroparhol. Exp . Neurol. 48, 425-436 . MAJOR, E . 0 ., AMEMIYA, K ., TORNATORE, C . S ., HOUFF, S . A ., and BERGER, J . R . (1992) . Pathogenesis and molecular biology of Progressive Multifocal Leukoencephalopathy, a JC Virus-induced demyelinating disease of the human brain . Clin . Microbiol . Rev. 5, 49-73 . MAxAM, A . M ., and GILBERT, W . (1980) . Sequencing and end-labelled DNA with base specific chemical cleavages . Methods Enzymol. 65,499-560 . McCHESNEY, M . B ., and OLDSTONF M . B . A. (1987) . Viruses perturb lymphocyte functions : Selected principles characterizing virus induced immunosuppression . Ann . Rev. lmmunol. 5, 279-304 . MIYAMuRA, T ., FURANO, A ., and YOSHIIKE, K . (1985) . DNA rearrangement in the control region for early transcription in a human poly-
JC VIRUS IN B CELLS
omavirus JC host range mutant capable of growing in human embryonic kidney cells . J. Virel. 54, 750-756 . MIYAMURA, T ., YOSHIIKE, K ., andTAKEMOTO, K . K . (1980) . Characterization of JC papovavirus adapted to grow in human embryonic kidney cells . J. Virol. 35, 498-504 . MOSTHAF, L ., PAWLITA, M ., and GROSS, P . (1985) . A viral enhancer element specifically active in human haematopoietic cells . Science 315, 587-600 . NAGATA, K ., GuGGENHEIMER, R . A,, and HURWITZ, J . (1983) . Specific binding of a cellular DNA binding protein to the origin of replication of adenovirus DNA . Proc. Nat/. Acad. Sol. USA 80, 6177-6181 . NERMOD, N ., O'NEILL, E . A ., KELLY, T . J ., and TJAN, R . (1989) . The proline rich transcriptional activator of CTF/NF-1 is distinct from the replication and DNA binding domain . Cell 58, 741-753 . PADGETT, B . L ., and WALKER, D . L . (1983) . Virologic and serologic studies of progressive multifocal leukoencephalopathuy . Prog. Olin . Bini. Res. 105, 107-117 . PADGETT, B . L ., ZURHEIN, G . M ., WALKER, D . L ., ECHROADE, R. J ., and DESSEL, B . H . (1971) . Cultivation of papova-like virus from human brain Mth progressive multifocal leukoencephalopathy . Lancet 1, 1257-1260 . PAONESSA, G ., GOUNARI, F ., FRANK, R ., and CORTESE, R . (1988) . Purification of a NF-1 like DNA binding protein from rat liverand cloning of the corresponding cDNA . EMBO J. 7, 3115-3123 . RAWLINS, D . R ., ROSENFELD, P . J ., WIDES, J . R ., CHALLBERG, M . D ., and KELLY, T . J ., JR . (1984) . Structure and function of the adenovirus origin of replication . Cell 37, 309-319 . RICHARDSON, E . P ., JR . (1961) . Progressive multifocal leukoencephalopathy . N. Eng/. J. Med. 265, 815-823 . ROSENFELD, P . J ., and KELLY, T . J . (1986) . Purification of nuclearfactor
723
1 by DNA recognition affinity chromatography . J.. Bioi . Chem . 261, 1398-1408 . RZIHA, H . J ., BoRNKAMM, G . M ., and ZuRHAUSEN, H . (1978) . Seroepidemiological studies and serologic response to viral infection . Med. Microbiol. lmmunol . 165, 73-92 . SANTORO, C ., NERMOD, N ., ANDREWS, P . C ., and TJIAN, R . (1988) . A family of human COAT-box-binding proteins active in transcription and replication : Cloning and expression of multiple cDNAs . Nature 334,218-224 . SOCK, E ., WEGNER, M ., and GRUMMT, F . (1991) . DNA replication of human polyomavirus JC is stimulated by NF-1 in vivo . Virology 182,298-308 . STONER, G . L ., RYSCHKEWITSCH, C . F ., WALKER, 0 . I_ ., and WEBSTER, H . D . F . (1986) . JO papovavirus large tumor (T)-antigen expression in brain tissue of squired immune deficiency syndrome (AIDS) and non-AIDS patients with progressive multifocal leukoencephalopathy. Proc. Nail, Aced. Sci. USA 83, 2271-2275 . TADA, H ., LASHGARI, M ., RAPPAPORT, J ., and KHAUU, K . (1989) . Cell type-specific expression of JC virus early promoter is determined by positive and negative regulation . J. Virol. 63, 463-466 . TORNATORE, C ., BERGER, J . R ., HOUFF, S . A., CURFMAN, B ., MEYERS, K ., WINFIELD, D ., and MAJOR, F . 0 . (1992) . Detection of JC Virus DNA in peripheral lymphocytes from patients with and without progressive multifocal leukoencephalopathy . Ann . Neurot 31, 454-462 . WALKER, D . L ., and PADGETT, B . L . (1983) . The epidemiology of human polyornaviruses . In "Polyomaviruses and Human Neurological Diseases" (J . L. Sever and D . L. Madden, Eds .), pp . 99-106 . A. R . Liss, New York . YOSHIIKE, K ., MIYAMURA, T., CHAN, H . W ., and TAKEMOTD, K . K . (1982) . Two defective DNAs of human polyomavirus JC adapted to growth in human embryonic kidney cells . J Virel . 42, 395-401 .
