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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 991-999
Latent Infection and Reactivation of Human Herpesvirus 6 in Two
Novel Myeloid Cell Lines
By
Masaki Yasukawa,
Hideki Ohminami,
Eiji Sada,
Yoshihiro Yakushijin,
Masahiko Kaneko,
Kohsuke Yanagisawa,
Hidehisa Kohno,
Shiro Bando, and
Shigeru Fujita
From the First Department of Internal Medicine, Ehime University
School of Medicine, Ehime; the Department of Medical Technology, Ehime
Medical College of Health Science, Ehime; the Department of Internal
Medicine, Uwajima City Hospital, Ehime; and the Division of Clinical
Laboratory Medicine, Ehime University Hospital, Ehime, Japan.
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ABSTRACT |
It has been reported that reactivation of human herpesvirus-6
(HHV-6) causes a failure of hematopoiesis. To clarify the mechanisms of
bone marrow suppression induced by HHV-6 infection, it is necessary to
establish an in vitro model of HHV-6 infection in hematopoietic progenitor cells. We have established two novel Philadelphia
chromosome-positive myeloid cell lines, SAS413 and SAS527, which
possess different hematologic characteristics and show distinct
susceptibility to infection by HHV-6, from a patient with blast crisis
of chronic myelogenous leukemia (CML). HHV-6 subgroup A (HHV-6A) showed
marked replication in SAS413, forming syncytia and inducing cell lysis in short-term culture. On the other hand, HHV-6A-inoculated SAS527 continued to proliferate without cell lysis and only a few cells showed
HHV-6 antigen expression. In contrast to HHV-6A infection, inoculation
with HHV-6 subgroup B (HHV-6B) did not induce any cytopathic
effect (CPE) or viral antigen expression in either of the cell
lines. Although HHV-6B replication was undetectable, the presence of
the HHV-6 genome in both cell lines was shown by polymerase chain
reaction (PCR) during culture for more than 10 months, suggesting that
HHV-6B latently infected SAS413 and SAS527. Phorbol ester treatment of
SAS527 latently infected with HHV-6B resulted in reactivation of HHV-6,
as shown by the appearance of a CPE, positive reactivity for the HHV-6
antigen, and isolation of infectious HHV-6. These novel cell lines
should be useful for studying the mechanisms of HHV-6-induced
hematopoietic failure and HHV-6 latency and reactivation, as well as
differentiation, of the myeloid cell lineage.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN HERPESVIRUS-6 (HHV-6) was first
isolated from immunocompromised patients with lymphoproliferative
disorders.1 HHV-6 is divided into two subgroups, HHV-6A and
HHV-6B, on the basis of reactivity with monoclonal antibodies and
HHV-6-specific T-cell clones and restriction enzyme cleavage
patterns.2-4 Recent studies have shown that HHV-6 induces
biologic alterations of T lymphocytes5,6 and trans
activation of the human immunodeficiency virus type 1 (HIV-1) long
terminal repeat.7,8 HHV-6 is thought to infect most
individuals at an early age and to persistently infect throughout life,
as with other HHVs. It has been established that HHV-6 is a causative
agent of exanthem subitum,9 and various disorders are
caused by reactivation of HHV-6. It has been reported recently that
reactivation of HHV-6 occurs frequently in patients with
immunodeficient conditions such as acquired immune deficiency syndrome
and recipients of organ transplantation, and it causes bone marrow
suppression in some patients.10-15 To clarify the
mechanisms of hematopoietic failure mediated by HHV-6, it is necessary
to establish an in vitro experimental model of HHV-6 infection of hematopoietic precursor cells.
We have established two novel myeloid cell lines, designated SAS413 and
SAS527, from a patient with blast crisis of chronic myelogenous
leukemia (CML). Although their bcr gene rearrangement patterns
are identical, their growth patterns, cytochemistry, and surface
phenotypes are different. In the present study, to establish an in
vitro experimental system for HHV-6 infection of myeloid cells, we
examined the characteristics of these cell lines with a focus on their
susceptibility to infection with HHV-6A and HHV-6B. The results showed
that SAS413 and SAS527 possess distinct characteristics of
susceptibility to HHV-6A, and both support latent infection with
HHV-6B. In addition, phorbol ester treatment of SAS527 that was
latently infected with HHV-6B resulted in reactivation of HHV-6. These
cell lines should therefore be useful for studying the mechanisms of
HHV-6-induced hematopoietic failure and HHV-6 latency and reactivation
in myeloid cells.
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MATERIALS AND METHODS |
Case history.
A 41-year-old man was admitted to Uwajima City Hospital on May 20, 1989 because of leukocytosis. A cytogenetic study of his bone marrow cells
demonstrated the karyotype 46, XY, t(9;22)(q34;q11). The patient was
diagnosed as being in the chronic phase of CML and was treated with
interferon alfa, busulfan, and 6-mercaptopurine. He remained in stable
condition until January 1994, when the number of blasts in peripheral
blood and the size of the spleen increased rapidly. Karyotype analysis
of the bone marrow cells demonstrated karyotype 46, XY,
t(9;22)(q34;q11),  5, 17p+. The patient was diagnosed as having CML
blast crisis and was treated with combined chemotherapy. Despite
intensive chemotherapy, he died on May 31, 1994 due to intracranial hemorrhage.
Cell culture.
Peripheral blood mononuclear cells, of which greater than 90% were
leukemic blasts, were isolated by Ficoll-Conray gradient centrifugation
on April 13 and May 27, 1994. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) in 16-mm wells at
37°C in a 5% CO2 incubator. Half of the culture medium
was exchanged with fresh medium every 3 or 4 days. Rapid cell growth
was observed about 1 month after initiation of cell culture. The
growing cells were maintained in RPMI 1640 supplemented with 10% FCS
for more than 3 years, and were designated SAS413 and SAS527, respectively.
Cytochemical staining.
Cytocentrifuged cell preparations were stained with
May-Grünwald-Giemsa solution, myeloperoxidase, naphthol AS-D
chloroacetate esterase, and  -naphthyl butyrate esterase. Expression
of terminal deoxynucleotidyl transferase (TdT) was examined by immunofluorescence.
Chromosome analysis.
Chromosome analysis was performed on bone marrow cells and established
cell lines as described previously.16 Chromosomes were
banded by the trypsin-Giemsa method.
Southern blot analysis.
DNA samples prepared from peripheral blood mononuclear cells, of which
greater than 90% were leukemic cells, and established cell lines were
digested with restriction enzymes and size-fractionated on 0.8%
agarose gel. The DNA was then transferred to nylon filters, and
hybridized with a 32P-labeled 1.2-kb
HindIII-BglII bcr gene fragment.
Cell-surface molecule expression.
Expression of surface molecules on peripheral blood leukemic blasts and
cell lines was examined by direct and indirect immunofluorescence using
a flow cytometer. Control cells for background fluorescence were
stained with fluorescein isothiocyanate (FITC)-conjugated mouse Ig or
FITC-conjugated goat anti-mouse Ig. The analysis gate was set to
include 99% of the control cells, and the percentage of positive cells
was recorded.
Inoculation of cells with viruses.
The U1102 strain of HHV-6A and the Z29 strain of HHV-6B were grown in
cord blood mononuclear cells prestimulated with phytohemagglutinin (PHA).9 The cells were inoculated with HHV-6A or HHV-6B at a multiplicity of infection of approximately 1 50% tissue culture infective dose. Virus-inoculated cells were cultured continuously in
RPMI 1640 supplemented with 10% FCS for more than 10 months, and virus
replication and the presence of the virus genome were monitored
repeatedly during the culture. Effects of HHV-6 inoculation on cell
growth were examined by calculating viable cells periodically using the
trypan blue exclusion test.
Detection of virus replication.
The appearance of a cytopathic effect (CPE) of HHV-6 was examined with
an inverted microscope. The indirect immunofluorescence assay for
detection of HHV-6 antigen was performed using HHV-6-seropositive human serum as described previously.17 Briefly,
virus-infected cells were mounted on glass slides and fixed in cold
acetone. HHV-6-seropositive human serum diluted 20-fold was applied to the slide, followed by incubation for 30 minutes at 37°C. After washing, FITC-conjugated goat anti-human IgG (Organon Teknika, Durham,
NC) was added and incubated for 30 minutes at 37°C. After washing,
the slides were examined using a fluorescence microscope.
Transmission electron microscopy.
Transmission electron microscopy of SAS413 and SAS527 was performed as
described previously.18 Briefly, the cells were fixed with
2.0% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4), postfixed
with 1% osmium tetroxide, and gradually dehydrated. Samples were
embedded in Epon 812, sectioned, stained with uranyl acetate and lead
citrate, and examined with an H-800 electron microscope (Hitachi Co,
Ibaragi, Japan).
Detection of HHV-6 genome.
A polymerase chain reaction (PCR) assay for the HHV-6 genome was
performed as described previously.19 The primers used were as follows: 5'-GTGTTTCCATTGTACTGAAACCGGT-3' and
5'-TAAACATCAATGCGTTGCATACAGT-3'. The samples were amplified through 35 cycles. Annealing was performed at 60°C for 90 seconds, extension at
72°C for 120 seconds, and denaturation at 95°C for 60 seconds. The
expected product of HHV-6 DNA from the use of these primers is 776 bp.
We also used the following primers to examine the presence of the HHV-7
genome in the cell lines: 5'-TATCCCAGCTGTTTTCATATAGTAAC-3' and
5'-GCCTTGCGGTAGCACTAGATTTTTTG-3'. The expected product of HHV-7 DNA
from the use of these primers is 186 bp.
Detection of HHV-6 mRNA expression.
Expression of mRNA for the HHV-6 immediate-early gene was investigated
by reverse transcriptase (RT)-PCR. Total RNAs were extracted from
HHV-6-inoculated cell lines, and cDNA was synthesized by reverse
transcription with Moloney murine leukemia virus RT. cDNA amplification
by PCR was performed using the following primers: 5'-TTCTCCAGATGTGCCAGGGAAATCC-3' and
5'-CATCATTGTTATCGCTTTCACTCTC-3'. The expected length of the amplified
cDNA sequences for HHV-6A and HHV-6B was 325 and 553 bp,
respectively.20,21 Amplification of cDNA for the  -actin
gene was also performed as the control for RT-PCR using the following
primers: 5'-TCCTGTGGCATCCACGAAACT-3' and 5'-GAAGCATTTGCGGTGGACGAT-3'.
Treatment of cell lines with phorbol ester.
It is well known that some virus types can be reactivated from latency
by cell stimulation with phorbol ester.22-25 Accordingly, we attempted to isolate HHV-6 from the cell lines that were infected latently with HHV-6B. The cell lines were cultured in RPMI 1640 medium
containing 10% FCS with or without the phorbol ester
12-0-tetradecanoylphorbol-13-acetate ([TPA] Sigma Chemical Co, St
Louis, MO) at a concentration of 1.6 × 10 7 mol/L for 7 days.
Isolation of HHV-6.
Isolation of HHV-6 from cell lines was performed as described
previously.9 Briefly, the virus-infected cell lines were frozen and thawed and then sonicated. The cell lysates were then added
to cord blood lymphocytes prestimulated with PHA for 3 days. The cells
were cultured in RPMI 1640 medium supplemented with 10% FCS, and HHV-6
replication was monitored as already described.
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RESULTS |
Morphology and cytochemical studies.
The morphology of SAS413 and SAS527 is shown in Fig 1. SAS413 grew as a
single-cell suspension with slight aggregation, and most SAS527 cells
showed adherent growth on the surface of a plastic flask.
May-Grünwald-Giemsa staining of SAS413 showed a myeloblastic appearance, with basophilic cytoplasm and a round nucleus without any
cytoplasmic granules. SAS527 was monoblastic in appearance, with
irregular-shaped cytoplasm and a convoluted nucleus. Results for
cytochemical studies of SAS413 were negative for myeloperoxidase, negative for naphthol AS-D chloroacetate esterase, weakly positive for
-naphthyl butyrate esterase, and negative for TdT, and results for
SAS527 were negative for myeloperoxidase, negative for naphthol AS-D
chloroacetate esterase, weakly positive for -naphthyl butyrate esterase, and weakly positive for TdT.
Cell-surface molecule expression.
Surface molecule expression of the original leukemic blasts and
HHV-6-infected and -uninfected SAS413 and SAS527 is shown in Table 1.
The original leukemic blasts were positive for CD13, CD14, and CD33,
suggesting that the lineage of the CML blast crisis was
myelomonocytoid. SAS413 showed expression of a megakaryocyte-associated antigen, CD41a, and an erythroblast-associated antigen, glycophorin A,
as well as the myelomonocytoid-associated antigens CD13, CD14, and
CD33, suggesting that this cell line may have the characteristics of a
multipotent hematopoietic precursor cell. In contrast, SAS527 was
positive for CD9, CD13, CD14, CD33, CD34, and CD56. No apparent changes
were observed in either SAS413 or SAS527 following infection with
HHV-6A or HHV-6B, except for increased expression of CD21 and
glycophorin A on SAS413 induced by HHV-6A and HHV-6B infection during
short-term culture and downregulation of CD34 on HHV-6A- and
HHV-6B-infected SAS527 during long-term
culture.
Genetic analyses.
The representative karyotypes of SAS413 and SAS527 were 60, XY, +1, +6,
+6, +8, +8, der(9)t(9;22)(q34;q11) × 2, +10, der(11)t(3;11)(p21;p13), +12, 18, +19, +19, +20, +20, +21, +21, 22, +2mar and 56, XY, +1,
+6, i(8)(q10), +i(8), t(9;22)(q34;q11), +der(9)t(9;22), +add(10)(q22), +13, +add(17)(p11), add(18)(p11), +19, +19, add(19)(q13), +21, respectively. Identical rearranged bands were detected by Southern blot
analysis of the bcr gene in all of the original leukemic cells
and SAS413 and SAS527, indicating that both SAS413 and SAS527 originated from leukemic cells (Fig 2).
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| Fig 2.
Southern blot analysis of DNA from original leukemic
cells (A), SAS413 (B), and SAS527 (C). DNA was digested with
BamHI (a) or BglII (b) and hybridized to a 3'
bcr probe. Arrows indicate rearranged bands.
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Replication of HHV-6A and HHV-6B in the cell lines.
SAS413 appeared to show a typical CPE of HHV-6 after 4 days of
inoculation with HHV-6A (Fig 3). At day 6 of virus inoculation, greater than 30% of the cells showed positive
immunofluorescence for HHV-6 antigen expression. Inoculation of SAS413
with HHV-6A produced large ballooning cells and resulted in cell lysis
15 days after virus inoculation. On the other hand, a CPE was scarcely detected in HHV-6A-inoculated SAS527 during culture for more than 10 months. Immunofluorescence assays performed on days 6 and 240 after
HHV-6A inoculation showed that only one in 500 to 1,000 SAS527 cells
were positive for HHV-6 antigen expression. In contrast to HHV-6A,
inoculation with HHV-6B did not induce any CPE in either SAS413 or
SAS527, and no HHV-6 antigen expression was detected in
HHV-6B-inoculated SAS413 or SAS527 during long-term culture.
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| Fig 3.
Replication of HHV-6 in SAS413 and SAS527.
HHV-6-inoculated SAS413 and SAS527 were observed with an inverted
microscope to detect CPE (A) and analyzed by indirect
immunofluorescence with HHV-6-seropositive human serum (B). Analyses
were performed at day 6 of virus inoculation for
HHV-6A-inoculated SAS413 and at day 240 for HHV-6B-inoculated SAS413,
HHV-6A-inoculated SAS527, and HHV-6B-inoculated SAS527.
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Effects of HHV-6 inoculation on cell growth.
The growth of both SAS413 and SAS527 did not change following
inoculation with HHV-6B (Fig 4). On the
other hand, SAS527 growth decreased following inoculation with HHV-6A,
although the cells were able to proliferate continuously. As detected
using an inverted microscope, the growth of SAS413 was severely
impaired by inoculation with HHV-6A. Two weeks after inoculation with
HHV-6A, greater than half of the SAS413 cells became moribund, showing
an apparent CPE.
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| Fig 4.
Growth curve of SAS413 and SAS527. The growth of
mock-infected ( ), HHV-6A-inoculated ( ), and HHV-6B-inoculated
( ) SAS413 and SAS527 was monitored by counting viable cells using
the trypan blue exclusion test.
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Detection of HHV-6 by transmission electron microscopy.
Transmission electron micrographs of HHV-6-inoculated SAS413 and
SAS527 are shown in Fig 5. Many HHV-6
particles were observed in the cytoplasm and nuclei of SAS413 at day 6 of HHV-6A inoculation when a marked CPE was detected. HHV-6 particles
were also detected in HHV-6A-inoculated SAS527 by electron microscopy
on day 240 after virus inoculation, although the frequency of cells
containing virus particles was markedly lower versus HHV-6A-inoculated
SAS413. On the other hand, no virus structures were detected in either SAS413 or SAS527 after inoculation with HHV-6B. These results were
identical to those obtained by the immunofluorescence assays.
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| Fig 5.
Transmission electron microscopy of HHV-6-inoculated
SAS413 and SAS527. Note that many HHV-6 particles are present in
HHV-6A-inoculated SAS413 (A and B) and SAS527 (C and D), whereas no
virus is detectable in HHV-6B-inoculated SAS413 (E) and SAS527 (F).
The highly magnified boxed areas in (A) and (C) are shown in (B) and
(D), respectively. HHV-6A-inoculated SAS413 cells were examined at day
6 of virus inoculation, and HHV-6B-inoculated SAS413,
HHV-6A-inoculated SAS527, and HHV-6B-inoculated SAS527 cells were
examined at day 240.
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Detection of HHV-6 genome in the cell lines.
We examined whether HHV-6B infected SAS413 and SAS527 or persistently
infected these cell lines using a PCR for the HHV-6 genome (Fig
6). PCR was performed repeatedly during
long-term culture, and the same results were obtained. HHV-6A and
HHV-6B genomes were not detected in the original SAS413 or SAS527.
Similarly, the genome for HHV-7, a virus closely related to HHV-6, was
not detected in either of the cell lines (data not shown). As expected, HHV-6 DNA was detected in HHV-6A-infected SAS413. Similarly, the HHV-6
genome was detected in SAS527 cultured for more than 10 months after
HHV-6A inoculation. Although apparent CPE and HHV-6 antigen expression
were not detected in HHV-6B-inoculated SAS413 and SAS527, the HHV-6
genome was detected in both cell lines that were cultured for more than
10 months after HHV-6B inoculation. These data suggest that HHV-6A
productively infected and caused cell death in SAS413 and persistently
infected SAS527 during long-term culture. On the other hand, HHV-6B
latently infected both SAS413 and SAS527 without viral expression.
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| Fig 6.
Detection of HHV-6 genome in SAS413 and SAS527 by PCR. A
PCR product of the expected size was detected in HHV-6A-inoculated
SAS413 (lane 1), HHV-6B-inoculated SAS413 (lane 2), HHV-6A-inoculated
SAS527 (lane 4), and HHV-6B-inoculated SAS527 (lane 5), but not in
uninfected SAS413 (lane 3) or SAS527 (lane 6). DNA was extracted from
HHV-6A-inoculated SAS413 at day 6 after virus inoculation, and from
HHV-6B-inoculated SAS413, HHV-6A-inoculated SAS527, and
HHV-6B-inoculated SAS527 at day 240. Lane M shows marker DNAs.
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Expression of HHV-6 mRNA.
We examined whether HHV-6 infected SAS413 and SAS527 latently without
HHV-6 gene expression or whether transcription of the HHV-6 gene
occurred in virus-inoculated cell lines using RT-PCR for the
immediate-early gene. As expected, mRNA for the immediate-early gene
was detected in HHV-6A-inoculated SAS413 and SAS527, whereas it was
not detectable in HHV-6B-inoculated SAS413 or SAS527 (Fig 7). These results suggest that HHV-6A
infected productively and HHV-6B infected latently in these cell lines.
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| Fig 7.
Expression of HHV-6 mRNA in SAS413 and SAS527. (A)
Expression of the HHV-6 immediate-early gene in the cell lines was
examined by RT-PCR. cDNA synthesized from cord blood mononuclear cells
(lane 1), SAS413 (lane 2), and SAS527 (lane 3) inoculated with HHV-6A
or HHV-6B was amplified using primers corresponding to the
immediate-early gene. (B) RT-PCR for -actin gene. Lane M shows
marker DNAs.
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Reactivation of HHV-6B by treatment with phorbol ester.
We sought to determine whether HHV-6B could be reactivated in SAS413
and SAS527 latently infected with HHV-6B. As it has been reported that
phorbol ester treatment of cells latently infected with virus results
in reactivation of the virus, we attempted to reactivate HHV-6B in
SAS413 and SAS527 by treatment with the phorbol ester TPA. Experiments
were performed 120 and 200 days after HHV-6B inoculation, and identical
results were obtained. After 7 days of TPA treatment, a CPE was
detected in SAS527 inoculated with HHV-6B (Fig
8A). Immunofluorescence assays showed that
about one in 300 cells were positive for HHV-6 antigen expression (Fig 8B). Transmission electron microscopy also revealed HHV-6 particles in
the cytoplasm of SAS527 (data not shown). In addition, HHV-6B was
isolated from TPA-treated, but not from untreated, SAS527 by
cocultivation with PHA-stimulated cord blood lymphocytes as demonstrated by the appearance of a marked CPE and positive reactivity with anti-HHV-6 antibody using indirect immunofluorescence (Fig 8C and
D). As expected, although morphologic change was induced, no HHV-6
antigen was detected in mock-infected SAS527 after treatment with TPA.
Treatment of HHV-6A-infected SAS527 with TPA resulted in the
augmentation of HHV-6A replication as determined by the appearance of a
CPE and positive reactivity to indirect immunofluorescence (data not
shown). In contrast to SAS527, typical CPE or HHV-6 antigen expression
was hardly detected in HHV-6B-inoculated SAS413 even after treatment
with TPA (data not shown).
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| Fig 8.
Reactivation of HHV-6B in SAS527. HHV-6B-inoculated
SAS527 cells were cultured in the presence of TPA at a concentration of
1.6 × 107 mol/L for 7 days. The cells were then
observed with an inverted microscope for detection of CPE (A) and
analyzed by indirect immunofluorescence with HHV-6-seropositive human
serum (B). The cells were also sonicated and added to PHA-stimulated
cord blood lymphocytes. After 6 days, the cells were observed with an
inverted microscope for detection of CPE (C) and analyzed by indirect
immunofluorescence with HHV-6-seropositive human serum (D).
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DISCUSSION |
HHV-6 was originally defined as a B-lymphotropic virus,1
but subsequent studies showed that it preferentially infects
CD4+ T lymphocytes.26 The presence of HHV-6 DNA
sequences has also been shown in several cases of lymphoid
neoplasm.21,27-30 Although it has been reported that
nonlymphoid cell lines also support the replication of
HHV-6,31-35 HHV-6 infectivity against myeloid cell lines
has not been extensively examined. The present study shows that both
HHV-6A and HHV-6B can infect myeloid cells with different replication
patterns. Recent studies have shown that the major sites for latent
infection of HHV-6 in vivo are monocytes24 and salivary
glands.19 The present data demonstrating that HHV-6 can
infect myeloid cell lines suggest that myeloid precursor cells in bone
marrow also support HHV-6 latency. The finding that HHV-6 is frequently
isolated from bone marrow cells of posttransplant patients36 supports this hypothesis.
Although the HHV-6A and HHV-6B genomes were detected in both SAS413 and
SAS527, their replication patterns in these cell lines were distinctly
different. That is, HHV-6A markedly replicated in SAS413 with a
remarkable CPE and persistently infected SAS527 with little CPE,
whereas HHV-6B latently infected both cell lines without apparent viral
replication. Although the infectivity of HHV-6A against various cell
lines has been shown, it has been reported that lytic infection of
HHV-6A occurs only in T lymphocytes. SAS413 is the first myeloid cell
line reported to permit lytic infection of HHV-6A. Viral replication
requires various factors derived from host cells. Thus, analysis of
SAS413 may provide important information to clarify the intracellular
factors essential for HHV-6 replication.
Activation of HHV-6 is frequently observed in patients who have
received bone marrow transplantation, and this inhibits marrow engraftment.11,12,15,37,38 Although the precise mechanisms of bone marrow suppression mediated by HHV-6 are still obscure, frequent isolation of HHV-6 from bone marrow and the suppressive effect
of HHV-6 on in vitro colony formation by bone marrow
cells13,14 suggest that direct infection of hematopoietic
precursor cells with HHV-6 may cause suppression of bone marrow
progenitor cell differentiation. Since it has been reported that the
vast majority of HHV-6 isolates from posttransplant patients are
subgroup B,36,39 the SAS413 and SAS527 cell lines latently
infected with HHV-6B seem to be a useful model to investigate the
mechanisms of bone marrow suppression mediated by HHV-6B. It has been
reported that phorbol ester treatment of cells latently infected with
virus occasionally results in viral reactivation.22-25 In
view of these previous findings, we attempted to induce HHV-6B
reactivation in these cell lines by stimulation with the phorbol ester
TPA. HHV-6B was indeed reactivated, and the infectious virus was
isolated from TPA-treated SAS527 but not from SAS413. The unsuccessful isolation of HHV-6B from SAS413 may be due to the fact that SAS527, but
not SAS413, has monocyte-lineage characteristics, since HHV-6 is known
to show tropism to monocytes.24 This is the first
experimental system using cell lines for HHV-6 reactivation, and it
should provide important information for clarifying the mechanisms of HHV-6 reactivation and bone marrow suppression induced by HHV-6 reactivation.
It has been shown that the expression of various cell surface molecules
is altered following infection with HHV-6. For example, de novo
expression of CD4 is induced in CD4 lymphocytes by
HHV-6A infection, rendering them susceptible to infection with
HIV-1.40-42 Induction of CD4 mRNA synthesis and surface CD4
expression by HHV-6 infection has also been reported recently in the
lymphomyeloid progenitor cell line KG-1.43 Although examination after long-term culture was not performed, KG-1 cells inoculated with HHV-6 showed HHV-6 antigen expression without a CPE or
loss of cell viability, resembling HHV-6-inoculated SAS527. In light
of these previous reports, we examined the expression levels of various
surface molecules on SAS413 and SAS527, before and after HHV-6
infection. Although a slight increase of CD21 and glycophorin A
expression and downregulation of CD34 were observed on SAS413 and
SAS527, respectively, following infection with HHV-6A and HHV-6B, no
apparent alteration of other surface molecules, including CD4
expression, was detected. Although the reason for the different effects
of HHV-6 infection on CD4 expression in KG-1 and our present cell lines
is unknown, it may be due to the dependency of de novo CD4 expression
induced by HHV-6 infection on certain intracellular factor(s) produced
by specific cells.
In summary, we have established the first myeloid cell line susceptible
to lytic infection with HHV-6A and two myeloid cell lines that have
contained the HHV-6B genome for more than 10 months without apparent
CPE or HHV-6 antigen expression. We have also succeeded in reactivating
HHV-6B from one of the cell lines by treatment with phorbol ester.
These cell lines should be useful for two avenues of research. One is
the mechanism of myeloid lineage differentiation, since these cell
lines possess distinct hematologic characteristics with an identical
genetic background. The other is the mechanism of HHV-6 latent
infection and reactivation in myeloid precursor cells. Further
investigations using these cell lines should provide important
information to clarify these important issues in hematology and virology.
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ACKNOWLEDGMENT |
We gratefully acknowledge Drs J. Nicholas, J.B. Black, and C. Lopez for
providing the viruses. We also thank M. Shudo for technical support.
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FOOTNOTES |
Submitted January 27, 1998; accepted September 25, 1998.
Supported by a Grant-in-Aid for Scientific Research from the Ministry
of Education, Science, and Culture of Japan, and by the Mochida
Foundation for Medical and Pharmaceutical Research, the Inamori
Foundation, and the Suzuken Memorial Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Masaki Yasukawa, MD, PhD, First Department
of Internal Medicine, Ehime University School of Medicine, Shigenobu,
Ehime 791-0295, Japan; e-mail: yasukawa{at}m.ehime-u.ac.jp.
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REFERENCES |
1.
Salahuddin SZ, Ablashi DV, Markham PD, Josephs SF, Sturzenegger S, Kaplan M, Halligan G, Biberfeld P, Wong-Staal F, Kramarsky B, Gallo RC:
Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders.
Science
234:596, 1986[Abstract/Free Full Text]
2.
Ablashi DV, Balachandran N, Josephs SF, Hung CL, Krueger GRF, Kramarsky B, Salahuddin SZ, Gallo RC:
Genomic polymorphism, growth properties, and immunologic variations in human herpesvirus-6 isolates.
Virology
184:545, 1991[Medline]
[Order article via Infotrieve]
3.
Schirmer EC, Wyatt LS, Yamanishi K, Rodriguez WJ, Frenkel N:
Differentiation between two distinct classes of viruses now classified as human herpesvirus 6.
Proc Natl Acad Sci USA
88:5922, 1991[Abstract/Free Full Text]
4.
Yasukawa M, Yakushijin Y, Furukawa M, Fujita S:
Specificity analysis of human CD4+ T-cell clones directed against human herpesvirus 6 (HHV-6), HHV-7, and human cytomegalovirus.
J Virol
67:6259, 1993[Abstract/Free Full Text]
5.
Lusso P, Malnati M, De Maria A, Balotta C, DeRocco SE, Markham PD, Gallo RC:
Productive infection of CD4+ and CD8+ mature human T cell populations and clones by human herpesvirus 6: Transcriptional down-regulation of CD3.
J Immunol
147:685, 1991[Abstract]
6.
Furukawa M, Yasukawa M, Yakushijin Y, Fujita S:
Distinct effects of human herpesvirus 6 and human herpesvirus 7 on surface molecule expression and function of CD4+ T cells.
J Immunol
152:5768, 1994[Abstract]
7.
Ensoli B, Lusso P, Schachter F, Josephs SF, Rappaport J, Negro F, Gallo RC, Wong-Staal F:
Human herpes virus-6 increases HIV-1 expression in co-infected T cells via nuclear factors binding to the HIV-1 enhancer.
EMBO J
8:3019, 1989[Medline]
[Order article via Infotrieve]
8.
Lusso P, Ensoli B, Markham PD, Ablashi DV, Salahuddin SZ, Tschachler E, Wong-Staal F, Gallo RC:
Productive dual infection of human CD4+ T lymphocytes by HIV-1 and HHV-6.
Nature
337:370, 1989[Medline]
[Order article via Infotrieve]
9.
Yamanishi K, Okuno T, Shiraki K, Takahashi M, Kondo T, Asano Y, Kurata T:
Identification of human herpesvirus-6 as a causative agent for exanthem subitum.
Lancet
1:1065, 1988[Medline]
[Order article via Infotrieve]
10.
Asano Y, Yoshikawa T, Suga S, Nakashima T, Yazaki T:
Reactivation of herpesvirus type 6 in children receiving bone marrow transplants for leukemia.
N Engl J Med
324:634, 1991[Medline]
[Order article via Infotrieve]
11.
Drobyski WR, Dunne WM, Burd EM, Knox KK, Ash RC, Horowitz MM, Flomenberg N, Carrigan DR:
Human herpesvirus-6 (HHV-6) infection in allogeneic bone marrow transplant recipients: Evidence of a marrow-suppressive role for HHV-6 in vivo.
J Infect Dis
167:735, 1993[Medline]
[Order article via Infotrieve]
12.
Carrigan DR, Knox KK:
Human herpesvirus 6 (HHV-6) isolation from bone marrow: HHV-6-associated bone marrow suppression in bone marrow transplant patients.
Blood
84:3307, 1994[Abstract/Free Full Text]
13.
Knox KK, Carrigan DR:
In vitro suppression of bone marrow progenitor cell differentiation by human herpesvirus 6 infection.
J Infect Dis
165:925, 1992[Medline]
[Order article via Infotrieve]
14.
Isomura H, Yamada M, Yoshida M, Tanaka H, Kitamura T, Oda M, Nii S, Seino Y:
Suppressive effects of human herpesvirus 6 on in vitro colony formation of hematopoietic progenitor cells.
J Med Virol
52:406, 1997[Medline]
[Order article via Infotrieve]
15.
Yoshikawa T, Suga S, Asano Y, Nakashima T, Yazaki T, Sobue R, Hirano M, Fukuda M, Kojima S, Matsuyama T:
Human herpesvirus-6 infection in bone marrow transplantation.
Blood
78:1381, 1991[Abstract/Free Full Text]
16.
Seabright M:
A rapid banding technique for human chromosomes.
Lancet
2:971, 1971[Medline]
[Order article via Infotrieve]
17.
Yakushijin Y, Yasukawa M, Kobayashi Y:
T-cell immune response to human herpesvirus-6 in healthy adults.
Microbiol Immunol
35:655, 1991[Medline]
[Order article via Infotrieve]
18.
Inoue Y, Yasukawa M, Fujita S:
Induction of T-cell apoptosis by human herpesvirus 6.
J Virol
71:3751, 1997[Abstract]
19.
Sada E, Yasukawa M, Itoh C, Takeda A, Shiosaka T, Tanioka H, Fujita S:
Detection of human herpesvirus 6 and human herpesvirus 7 in submandibular gland, parotid gland, and lip salivary gland by polymerase chain reaction.
J Clin Microbiol
34:2320, 1996[Abstract]
20.
Yamamoto T, Mukai T, Kondo K, Yamanishi K:
Variation of DNA sequence in immediate-early gene of human herpesvirus 6 and variant identification by PCR.
J Clin Microbiol
32:473, 1994[Abstract/Free Full Text]
21.
Bandobashi K, Daibata M, Kamioka M, Tanaka Y, Kubonishi I, Taguchi H, Ohtsuki Y, Miyoshi I:
Human herpesvirus 6 (HHV-6)-positive Burkitt's lymphoma: Establishment of a novel cell line infected with HHV-6.
Blood
90:1200, 1997[Abstract/Free Full Text]
22.
zur Hausen H, O'Neill FJ, Freese UK, Hecker E:
Persisting oncogenic herpesvirus induced by the tumor promoter TPA.
Nature
272:373, 1978[Medline]
[Order article via Infotrieve]
23.
Harada S, Kobayashi Y, Nakashima N, Yamamoto N:
Tumor promoter, TPA, enhances replication of HTLV-III/LAV.
Virology
154:249, 1986[Medline]
[Order article via Infotrieve]
24.
Kondo K, Kondo T, Okuno T, Takahashi M, Yamanishi K:
Latent human herpesvirus 6 infection of human monocyte/macrophages.
J Gen Virol
72:1401, 1991[Abstract/Free Full Text]
25.
Renne R, Zhong W, Herndier B, McGrath M, Abbey N, Kedes D, Ganem D:
Lytic growth of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in culture.
Nat Med
2:342, 1996[Medline]
[Order article via Infotrieve]
26.
Takahashi K, Sonoda S, Higashi K, Kondo K, Takahashi H, Takahashi M, Yamanishi K:
Predominant CD4 T-lymphocyte tropism of human herpesvirus 6-related virus.
J Virol
63:3161, 1989[Abstract/Free Full Text]
27.
Jarrett RF, Gledhill S, Qureshi F, Crae SH, Madhok R, Brown I, Evans I, Krajewski A, O'Brien CJ, Cartwright RA, Venables P, Onions DE:
Identification of human herpesvirus 6-specific DNA sequences in two patients with non-Hodgkin's lymphoma.
Leukemia
2:496, 1988[Medline]
[Order article via Infotrieve]
28.
Josephs SF, Buchbinder A, Streicher HZ, Ablashi DV, Salahuddin SZ, Guo H-G, Wong-Staal F, Cossman J, Raffeld M, Sundeen J, Levine P, Biggar R, Krueger GRF, Fox RI, Gallo RC:
Detection of human B-lymphotropic virus (human herpesvirus 6) sequences in B cell lymphoma tissues of three patients.
Leukemia
2:132, 1988[Medline]
[Order article via Infotrieve]
29.
Torelli G, Marasca R, Luppi M, Selleri L, Ferrari S, Narni F, Mariano MT, Federico M, Ceccherini-Nelli L, Bendinelli M, Montagnani G, Montorsi M, Artusi T:
Human herpesvirus-6 in human lymphomas: Identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction.
Blood
77:2251, 1991[Abstract/Free Full Text]
30.
Luka J, Pirruccello SJ, Kersey JH:
HHV-6 genome in T-cell acute lymphoblastic leukaemia.
Lancet
338:1277, 1991[Medline]
[Order article via Infotrieve]
31.
Ablashi DV, Lusso P, Hung C-L, Salahuddin SZ, Josephs SF, Llana T, Kramarsky B, Biberfeld P, Markham PD, Gallo RC:
Utilization of human hematopoietic cell lines for the propagation and characterization of HBLV (human herpesvirus 6).
Int J Cancer
42:787, 1988[Medline]
[Order article via Infotrieve]
32.
Luka J, Okano M, Thiele G:
Isolation of human herpesvirus-6 from clinical specimens using human fibroblast cultures.
J Clin Lab Anal
4:483, 1990[Medline]
[Order article via Infotrieve]
33.
He J, McCarthy M, Zhou Y, Chandran B, Wood C:
Infection of primary human astrocytes by human herpesvirus 6.
J Virol
70:1296, 1996[Abstract]
34.
Inagi R, Guntapong R, Nakao M, Ishino Y, Kawanishi K, Isegawa Y, Yamanishi K:
Human herpesvirus 6 induces IL-8 gene expression in human hepatoma cell line, Hep G2.
J Med Virol
49:34, 1996[Medline]
[Order article via Infotrieve]
35.
Cermelli C, Concari M, Carubbi F, Fabio G, Sabbatini AMT, Recorari M, Pietrosemoli P, Meacci M, Guicciardi E, Carulli N, Portolani M:
Growth of human herpesvirus 6 in HEPG2 cells.
Virus Res
45:75, 1996[Medline]
[Order article via Infotrieve]
36.
Kadakia MP, Rybka WB, Stewart JA, Patton JL, Stamey FR, Elsawy M, Pellett PE, Armstrong JA:
Human herpesvirus 6: Infection and disease following autologous and allogeneic bone marrow transplantation.
Blood
87:5341, 1996[Abstract/Free Full Text]
37.
Rosenfeld CS, Rybka WB, Weinbaum D, Carrigan DR, Knox KK, Andrews DF, Shadduck RK:
Late graft failure due to dual bone marrow infection with variant A and B of human herpesvirus-6.
Exp Hematol
23:626, 1995[Medline]
[Order article via Infotrieve]
38.
Knox KK, Carrigan DR:
Chronic myelosuppression associated with persistent bone marrow infection due to human herpesvirus 6 in a bone marrow transplant recipient.
Clin Infect Dis
22:174, 1996[Medline]
[Order article via Infotrieve]
39.
Frenkel N, Katsafanas GC, Wyatt LS, Yoshikawa T, Asano Y:
Bone marrow transplant recipients harbor the B variant of human herpesvirus 6.
Bone Marrow Transplant
14:839, 1994[Medline]
[Order article via Infotrieve]
40.
Lusso P, De Maria A, Malnati M, Lori F, DeRocco SE, Baseler M, Gallo RC:
Induction of CD4 and susceptibility to HIV-1 infection in human CD8+ T lymphocytes by human herpesvirus 6.
Nature
349:533, 1991[Medline]
[Order article via Infotrieve]
41.
Lusso P, Malnati MS, Garzino-Demo A, Crowley RW, Long EO, Gallo RC:
Infection of natural killer cells by human herpesvirus 6.
Nature
362:458, 1993[Medline]
[Order article via Infotrieve]
42.
Lusso P, Garzino-Demo A, Crowley RW, Malnati MS:
Infection of  / T lymphocytes by human herpesvirus 6: Transcriptional induction of CD4 and susceptibility to HIV infection.
J Exp Med
181:1303, 1995[Abstract/Free Full Text]
43.
Furlini G, Vignoli M, Ramazzotti E, Re MC, Visani G, La Place M:
A concurrent human herpesvirus-6 infection renders two human hematopoietic progenitor (TF-1 and KG-1) cell lines susceptible to human immunodeficiency virus type-1.
Blood
87:4737, 1996[Abstract/Free Full Text]
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Abstract
Introduction