Enterovirus receptors and virus replication in human leukocytes
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Journal of General Virology (1999), 80, 921–927. Printed in Great Britain
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Enterovirus receptors and virus replication in human
leukocytes
Tytti Vuorinen,1 Raija Vainionpa$ a$ ,1 Jyrki Heino2 and Timo Hyypia$ 2, 3
1, 2
Department of Virology1 and MediCity Research Laboratory2, University of Turku, FIN-20520 Turku, Finland
3
Haartman Institute, Department of Virology, POB 21, University of Helsinki, FIN-00014 Helsinki, Finland
Although enteroviruses cause a great variety of diseases including meningitis, paralysis and
myocarditis, the life-cycle of these viruses in man is still quite poorly understood. The role of human
leukocytes as a target for enterovirus infections was studied in this report. Despite great similarity
in the structure and replication of coxsackievirus B3 (CBV3), echovirus 1 (EV1), and poliovirus 1
(PV1), the ability of these viruses to infect human peripheral blood mononuclear cells (PBMC), and
B (Raji), T (Molt-4) and monocytic (U-937) cell lines differed markedly. CBV3 attached both to
PBMC and the three haematopoietic cell lines studied, whereas EV1 bound only to PBMC.
Generally, the attachment of PV1 was very poor. The binding assays mostly correlated with the
expression of CD55 (decay accelerating factor, DAF) and α2β1-integrin (VLA-2), which are known to
act as receptors for CBV3 and EV1, respectively, as well as with the expression of the PV receptor
on the cell surface. To analyse virus replication in the cells, viral protein and nucleic acid syntheses
were studied by immunoprecipitation and RT–PCR. CBV3 was able to replicate in Raji and Molt-4
cells, even though no expression of DAF was detected, whereas in the monocytic cell line, viral
protein synthesis was detected only after transfection of virus RNA. Neither CBV3 nor EV1
replicated in PBMC and all haematopoietic cells studied seemed to be poorly permissive for PV1
replication.
Introduction and the fact that some enteroviruses are able to replicate in
Enteroviruses (polio-, coxsackie-, echo- and enteroviruses human peripheral blood mononuclear cells (PBMC) in vitro
68–71) are small RNA viruses in the family Picornaviridae. (Dagan & Menegus, 1992 ; Freistadt et al., 1993) indicate that
They are important human pathogens often causing mild infected cells may have a role in transporting the virus in the
febrile illness, but clinical manifestations of enterovirus body.
infections also include meningitis, encephalitis, paralysis and The initial step in virus infection is binding of the viruses to
myocarditis. In infants, the course of the disease can be severe specific receptors on the cell surface. In recent years, several
due to multisystem involvement. enterovirus receptors have been identified and characterized at
Primary replication of enteroviruses takes place in tissues of the molecular level. The poliovirus receptor (PVR) belongs to
the respiratory and gastrointestinal tracts. This may be the immunoglobulin superfamily. Although its structure has
followed by the occurrence of infectious virus in blood and been described in detail (Mendelsohn et al., 1989), its cellular
lead to spreading of the pathogens to secondary target organs. function is still unclear. Integrins serve as receptors for some
enteroviruses. Echovirus 1 (EV1) binds to α β -integrin (VLA-
During this viraemic phase, enteroviruses have been cultured # "
from the serum of patients (Dagan et al., 1985 ; Prather et al., 2 ; Bergelson et al., 1992, 1993), coxsackievirus A9 (CAV9) can
utilize αvβ -integrin in target cell recognition (Roivainen &
1984) indicating haematogenous spread of the viruses in free $
form. The finding that enteroviruses can also be isolated from Hovi, 1989), while CAV21 and some other coxsackie A viruses
blood cells of patients (Dagan et al., 1985 ; Prather et al., 1984) share a receptor (ICAM-1) with the major group of rhino-
viruses (Colonno, 1987 ; Pulli et al., 1995). Decay accelerating
factor (DAF ; CD55) has been reported to be a receptor for
Author for correspondence : Tytti Vuorinen. many echoviruses (Bergelson et al., 1994 ; Ward et al., 1994)
Fax j358 2 2513303. e-mail tytti.vuorinen!utu.fi and it also plays a role in the attachment of coxsackievirus B1
0001-5887 # 1999 SGM JCB
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(CBV1), CBV3 and CBV5 to host cells (Bergelson et al., 1995 ; by low-speed centrifugation, the virus was concentrated from the
Shafren et al., 1995). In addition, a molecule belonging to the supernatant by polyethylene glycol–NaCl precipitation and further
nucleolin family has been reported to recognize CBV3 on the purified in linear 5–20 % sucrose gradients (Abraham & Colonno, 1984).
cell surface (Raab de Verdugo et al., 1995). More recently, a Isolation of RNA. Extraction of the RNA from the infected cells was
common receptor molecule (CAR) for CBVs and adenoviruses performed using the Ultraspec RNA isolation method with slight
modifications. In brief, 200 µl chloroform was added to the sample, which
has been identified (Bergelson et al., 1997 ; Tomko et al., 1997).
was mixed, incubated on ice for 5 min and centrifuged at 12 000 g and
Our previous results have shown that there are remarkable 4 mC for 15 min. RNA was precipitated by isopropanol in the presence of
differences in the susceptibility of haematopoietic cell lines to 0n04 µg glycogen. The samples were stored at k20 mC overnight,
CBV3 infection (Vuorinen et al., 1994, 1996). Both B and T cell centrifuged at 12 000 g at 4 mC for 20 min, and the precipitates were
lines support virus replication, whereas monocytic and granulo- washed twice with 75 % ethanol. Finally, the pellets were dried at room
cytic cell lines are non-permissive for infection. In spite of the temperature, dissolved in RNase-free water, incubated at 55 mC for
susceptibility of B and T cell lines to CBV3 infection, we have 15 min and stored at k70 mC until used in the RT reaction.
been unable to detect viral protein synthesis in in vitro-infected RT–PCR. An RT–PCR method was used to detect antigenomic
PBMC originating from different donors (Vuorinen et al., RNA. The oligonucleotide primers were from the conserved parts of the
1994). 5h-untranslated region of the enterovirus genome (Santti et al., 1997). The
cDNA reaction mixture (40 µl) contained 10 µl sample nucleic acid,
In light of the previously published work, enterovirus 50 mM Tris–HCl (pH 8n3), 75 mM KCl, 3 mM MgCl , 10 mM DTT,
infection in leukocytes and haematopoietic cell lines is rather #
0n5 mM dNTPs (Pharmacia), 20 U RNasin ribonuclease inhibitor
obscure. One explanation for the variability of the results could (Promega), 50 pmol genomic (j) primer and 20 U murine Moloney
be differences in the expression of enterovirus receptors on the leukaemia virus reverse transcriptase (Promega) incubated at 37 mC for
surfaces of blood cells. Moreover, intracellular factors essential 1 h.
for virus replication may play a role. In order to obtain a more The PCR reaction (100 µl) consisted of 10 µl cDNA mixture, 10 mM
Tris–HCl (pH 8n8), 50 mM KCl, 1n5 mM MgCl , 0n1 % Triton X-100,
detailed picture on the interactions of enteroviruses with blood #
0n2 mM dNTPs, 50 pmol primers (Arola et al., 1996) and 1 U DNA
cells, we have studied the attachment of radiolabelled CBV3, polymerase (Dynazyme). The mixture was amplified in a thermal cycler
EV1 and poliovirus 1 (PV1) to PBMC, as well as to monocytic, (Perkin-Elmer Cetus) through 40 cycles of denaturation, annealing and
B and T cell lines. Expression of the virus receptors on the cells DNA synthesis for 1 min at 95 mC, 1 min at 55 mC and 4 min at 72 mC.
correlated with findings concerning viral macromolecule The reaction products (18 µl) were analysed in 2n5 % agarose gels
synthesis in the infected cells. containing 1 µg\ml ethidium bromide.
Immunoprecipitation. The immunoprecipitation of viral capsid
proteins was performed according to a previously described procedure
Methods (Vuorinen et al., 1994). The cells were lysed in 50 mM Tris–HCl (pH 7n4),
Viruses, cells and cell lines. CBV3 (Nancy), EV1 (Farouk) and 0n15 M NaCl, 1 mM EDTA, containing 1 % Tween and 1 % sodium
PV1 (Mahoney) were obtained from the ATCC. Virus stocks were deoxycholate, sonicated and clarified by low-speed centrifugation. Rabbit
propagated in LLC-Mk cells (ATCC), stored at k70 mC and centrifuged antiserum to purified CBV3 virions, rabbit antiserum to poliovirus and
# horse antiserum to EV1 (ATCC) were used in the immunoprecipitation.
at low speed prior to use in order to remove cell debris. Human PBMC
were isolated from healthy adults by Ficoll–Isopaque (Ficoll–Paque ; Protein A–Sepharose CL-4B beads and 20 µl of the antiserum were
Pharmacia) gradients. Haematopoietic cell lines (Raji, Molt-4 and U-937) incubated at 37 mC for 60 min. The labelled cell lysate was added, the
were originally obtained from the ATCC. mixture was incubated overnight at 4 mC, washed four times with 0n1 M
Tris–HCl (pH 7n4), containing 0n01 % Tween, eluted with 0n1 M Tris–HCl
Infection and labelling of the cells, cell lines and viruses. (pH 7n4), 2 % SDS, 5 % mercaptoethanol, and analysed by electrophoresis
Cells (1i10' cells\ml) were infected at an m.o.i. of 5 in serum-free in 12 % SDS–polyacrylamide gels. The dried gels were exposed on an X-
Hanks’ balanced salt solution for 1 h at 37 mC, washed three times in ray film.
order to remove unabsorbed virus, and then maintained in RPMI-1640
medium supplemented with 10 % foetal calf serum (FCS) and 50 µg\ml Attachment of the viruses to the cells. Cells (1i10') were
gentamicin. The cells for antigenomic RT–PCR were collected after the washed once with Hanks’ balanced salt solution containing 10 mM
HEPES (pH 7n0) and 20 mM MgCl , incubated with 20 000 c.p.m. of
adsorption as well as at 1, 2, 3 and 4 days post-infection (p.i.). The cells #
were washed with PBS, 1 ml Ultraspec RNA (Biotecx Laboratories) labelled virus in 0n1 ml medium at room temperature for 30 min. They
solution was added and the mixture was stored at k70 mC until total were washed three times with the medium, and the bound radioactivity
RNA was isolated. was measured in a liquid scintillation counter. Monoclonal antibodies to
DAF (IF7), α -integrin (12F1 ; Pischel et al., 1987) and PVR (D171) were
The labelling of the infected cells with [$&S]methionine was performed #
at 3 h p.i. The cells were first cultured in methionine-free medium for 1 h used to confirm the specificity of the binding assays.
which was then replaced by medium containing [$&S]methionine Flow cytometric analysis. Cells (5i10&) were first incubated in
(50 µCi\ml ; Amersham). After 18 h, the cells were washed with PBS and PBS containing 1 % FCS at 4 mC for 20 min, then with the anti-receptor
stored at k70 mC. antibody for 30 min at 4 mC, and finally stained with FITC-conjugated
For labelling and purification of the viruses, infected LLC-Mk cells anti-mouse antibody (Zymed). Thereafter, the cells were washed twice
#
were preincubated in methionine-free medium for 30 min and then with PBS and 10 000 events were analysed by a FACScan cytometer
cultured in the medium containing [$&S]methionine. When complete (Becton Dickinson). In order to quantify the expression of virus receptors
cytopathic effect was observed, the cultures were freeze–thawed three on the cells, mean fluorescence intensity (MFI) for each staining was
times in order to release the intracellular virus. Cell debris was removed measured and the MFI ratio (MFIR) was calculated by dividing the MFI
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of the staining with specific antibody by the MFI of the staining with
FITC-conjugated anti-mouse antibody only. MFIR 1n5 was considered
positive. Lymphocytes and monocytes were gated according to their
light scattering characteristics.
Transfection experiments. CBV3 RNA (1 µg) in 100 µl -
MEM growth medium (Gibco) was added to 10 µl Lipofectin reagent
(Gibco) premixed with 90 µl -MEM. After 10 min incubation period
at room temperature, 800 µl -MEM was added and washed cells
(1i10') were overlaid with the mixture. Fresh -MEM (2 ml) was
added after 2 h incubation at 37 mC and incubation was continued
overnight. The synthesis of viral polypeptides was then revealed by
immunoperoxidase staining (Waris et al., 1990) using rabbit antiserum
against heat-treated echovirus 11. The antiserum is known to be broadly
reactive among enteroviruses (M. Waris and others, unpublished results).
Results
Expression of the virus receptors on the cell surface
Fig. 1 shows the flow cytometric histograms of the
expression of DAF (A), the α -integrin subunit (B) and PVR (C)
#
on the surface of the haematopoietic cell lines, human
Cell number
lymphocytes and monocytes. LLC-Mk cells were included as
#
a positive control. Expression of DAF and the α -integrin
#
subunit was detected both on monocytes (MFIR of 1n5 and 4n7,
respectively) and the monocytic cell line U-937 (MFIR of 2n2
and 2n7), while PVR was detected on T (Molt-4) and monocytic
cell lines, as well as on monocytes (MFIR of 2n1, 6n5 and 2n0).
Attachment of the viruses to the cells
The attachment of CBV3, EV1 and PV1 to the haemato-
poietic cell lines and PBMC differed noticeably (Fig. 2). CBV3
bound to all the haematopoietic cell lines and to the peripheral
blood leukocytes of the five donors. The amount of attached
virus was highest on B (Raji) and monocytic (U937) cell lines
but a great variety occurred in attachment to the PBMC of the
five donors. EV1 had highest affinity to PBMC, whereas the
haematopoietic cells lines, including the receptor (α -integrin
#
subunit)-containing U-937 cells, did not bind the virus in
detectable amounts. PV1 had a very low capacity to attach to
all the haematopoietic cells studied. LLC-Mk cells, which are
#
known to be susceptible for the replication of these viruses,
were analysed as control cells. Relative fluorescence intensity
Fig. 1. Cell surface expression of DAF (A), α2-integrin (B) and PVR (C)
analysed by fluorescence cytometry. Unstained cells, cells incubated with
Virus macromolecule synthesis in infected cells anti-mouse FITC–antibody alone, and cells incubated with DAF, α2-integrin
Viral protein synthesis was analysed by immuno- or PVR antibody before addition of FITC–antibody (shaded histograms) are
shown. The mean fluorescence intensity ratios are shown in the upper
precipitation using polyclonal antisera (Fig. 3). CBV3 poly- right corner. Values 1n5 were considered positive.
peptides were detected in Raji cells (CBV3 ; lane 1) and weak
signals were also observed in the Molt-4 cells (CBV3 ; lane 2),
whereas the other cells did not support virus protein synthesis replication suggesting that they may be virus-specific poly-
in detectable amounts. PV1 proteins were synthesized in both proteins. The interpretation of EV1 synthesis on the basis of
Molt-4 (PV1 ; lane 2) and U-937 (PV1 ; lane 3) cell lines. In immunoprecipitation was not possible due to the high non-
addition to the structural virus proteins, some other bands also specific background reactivity of the horse antiserum used.
co-precipitated. The appearance of the co-precipitated poly- Because the infectious virus contains positive-strand
peptides correlated to the susceptibility of the cells to virus genomic RNA, the synthesis of complementary (negative)
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Fig. 4. Detection of antigenomic RNA by RT–PCR in B (Raji), T (Molt-4)
Fig. 2. Attachment of the [35S]methionine-labelled CBV3, EV1 and PV1 to and monocytic (U-937) cell lines and PBMC infected with (A) CBV3, (B)
the haematopoietic cell lines and PBMC (hatched bars). The cells were EV1 or (C) PV1 at either 1 h or 2 days post-infection. Lanes : C,
incubated at room temperature for 30 min with radioactively labelled virus uninfected control cells ; MW, molecular mass markers. The arrows show
(" 20 000 c.p.m.) and the bound radioactivity was measured in a liquid the position of the specific amplification product.
scintillation counter. The black bars show the attachment of the labelled
viruses to the cells after treatment with monoclonal antibodies against
DAF, α2-integrin or PVR antibody. MeanpSD c.p.m. is shown. RT–PCR (Fig. 4), where the increase in the amount of
antigenomic RNA indicated productive RNA synthesis and
strands during the infection is a sensitive sign of de novo RNA susceptibility of the cells to infection. Active CBV3 RNA
replication (Vuorinen et al., 1996). Therefore, we analysed the synthesis was observed in both Raji and Molt-4 cell lines but
amount of antigenomic RNA in the haematopoietic cells by not in U-937 cells or in PBMC (Fig. 4 A), whereas only U-937
Fig. 3. Synthesis of CBV3 and PV1
proteins detected by
immunoprecipitation. Cells were infected
and subsequently labelled with
[35S]methionine for 22 h. The cell lysates
were immunoprecipitated with CBV3 or
PV1 antibodies and analysed by
SDS–PAGE. Lanes : 1, infected Raji cell
line ; 2, infected Molt-4 cell line ; 3,
infected U-937 cell line ; 4, 5, infected
PBMC from two donors ; 6, infected LLC-
Mk2 cells ; 7, uninfected LLC-Mk2 cells.
Virus polypeptides VP0, VP1, VP2 and
VP3 are indicated by the pointers.
Molecular mass markers are shown.
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Table 1. Summary of results
Receptor expression* Virus binding RNA synthesis
Cell DAF α2 PVR CBV3 EV1 PV1 CBV3 EV1 PV1
Raji k k k j k k j k k
Molt-4 k k j j j k j k k
U-937 j j j j k j k† j j
PBMC j j j j j j\k k k j
* A mean fluorescence intensity ratio (MFIR) 1n5 was considered to be positive.
† Viral protein synthesis was detected after transfection of genomic RNA.
used (data not shown). The unspecific staining was most
probably due to the known peroxidase activity of monocytes.
Discussion
Although enteroviruses have been isolated from blood cells
of patients (Dagan et al., 1985), detailed knowledge on the
interactions of enteroviruses with immune cells is still very
limited. Our previous studies have shown that B and T cell
lines support extensive CBV3 replication, but PBMC do not
produce significant amounts of virus (Vuorinen et al., 1994).
The purpose of the present study was to examine the blood cell
tropism of representatives of enteroviruses. The isolates
selected for study represent viruses with different molecular
and pathogenic characteristics. Polioviruses are the best known
members of the group and although they can cause epidemic
paralytic disease, they are more often responsible for asympto-
matic and mild infections. CBVs are associated with diverse
clinical manifestations including meningitis, encephalitis and
Fig. 5. Viral protein synthesis after transfection of CBV3 genomic RNA into myocarditis (Grist et al., 1978). Furthermore, increasing
the cells. The cells were stained by the immunoperoxidase method using a evidence supports association of CBV infections with type 1
broadly reacting echovirus 11 antibody. Raji cells were treated with diabetes (Hyo$ ty et al., 1995). The largest enterovirus group is
Lipofectin without (A) and with (B) CBV3 RNA. U-937 cells were
incubated with virus RNA in the absence (C) and presence (D) of the echoviruses, which comprises approximately 30 sero-
Lipofectin. logically distinct members. Echoviruses are a common cause of
meningoencephalitis and rashes.
In this study, PBMC were shown to contain specific
cells were able to support replication of EV1 RNA (Fig. 4 B). receptors (DAF, CD55) for CBV3, but neither viral macro-
PV1 RNA was produced in the U-937 cells and also in PBMC molecule nor RNA synthesis could be detected in PBMC. Also,
(Fig. 4 C). Table 1 summarizes the results of receptor ex- the monocytic cell line U-937 expressed CD55 and virus
pression, virus binding and RNA synthesis. binding occurred, but no virus replication was detected by
To test whether the block in the CBV3 replication cycle in RT–PCR or immunoblotting techniques. On the other hand,
the U-937 cells and PBMC is due to a lack of some intracellular immunoperoxidase staining experiments showed that trans-
factor, we carried out transfection experiments with RNA fected CBV3 RNA could be translated to proteins suggesting
isolated from CBV3. Viral protein synthesis was clearly incomplete uncoating or a defect in internalization during the
detected by immunoperoxidase staining after transfection in virus replication cycle. Recently, it has been shown that
Raji and U-937 cells (Fig. 5). However, the interpretation of interaction of CBV3 with two different receptor molecules,
immunoperoxidase staining of PBMC was not feasible because DAF and CAR, is needed for initiation of infection (Shafren et
unspecific staining of some cells was also detected even when al., 1997). The expression of CAR on PBMC is currently
only growth medium, Lipofectin reagent or RNA alone was unknown. In spite of the absence of DAF on B (Raji) and T
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(Molt-4) cells, CBV3 was able to infect cells probably through ness to infection. In general, there seem to be remarkable
another receptor, possibly CAR, since synthesis of virus RNA differences when the three enteroviruses, using different
was detected in the cells. Our results with the monkey kidney receptor systems for their entry into the blood cells, are
cell line LLC-Mk , commonly used for virus production, compared. Increasing knowledge on the molecular charac-
#
showed that CBV3 binding can only partially be blocked by teristics of the enteroviruses and further cell biological
the anti-DAF antibody, which also supports the idea of investigations will make it possible to understand the com-
alternative pathways for virus entry. Another explanation for plicated pathogenesis and clinical picture of enterovirus
the finding could be that the monoclonal antibody produced infections.
against human DAF is not equally effective in simian cells.
More efficient blocking was obtained in the blood cells and We thank Jeffrey Bergelson for the DAF antibody, Eckard Wimmer
CBV3 bound specifically to all three cell lines and to the for the PVR antibody, Virgil Woods for the 12F1 antibody, Tapani Hovi
and Aimo Salmi for critical comments, and Marita Maaronen and Marjut
PBMC. However, significant variation was observed in the
Sarjovaara for technical assistance. The study was supported by grants
binding to PBMC from different donors. In contrast to an from the Academy of Finland, the Sigrid Juselius Foundation and the
earlier report (Henke et al., 1992), we were unable to detect Turku University Foundation.
signs of virus replication (RNA and protein synthesis) in
PBMC cultures from different donors ; thus, our results were
similar to the work reported by Dagan & Menegus (1992). References
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JCH
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