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Peripheral Blood CD14 1 Cells From Healthy Subjects Carry a Circular
Conformation of Latent Cytomegalovirus Genome
By Cynthia A. Bolovan-Fritts, Edward S. Mocarski, and Jean A. Wiedeman
The majority of the human population harbors latent cytomegalovirus. Although CD141 peripheral blood mononuclear cells have been implicated as sites of latency, the
conformation of the latent viral genome in these cells is
unknown. In this study, the conformation of viral genomic
DNA was assessed in CD141 cells from healthy virus seropositive carriers using an electrophoretic separation on native
agarose gels in combination with polymerase chain reaction
detection. Here we show that the viral genome migrates as a
circular plasmid with a mobility equivalent to a circular
230-kb Shigella flexneri megaplasmid marker. Neither linear
nor complex or integrated forms of the viral genome were
detected. This report provides further evidence that the
CD141 cell population is an important site of viral latency in
the naturally infected human host. Detection of the viral
genome as a circular plasmid during latency suggests that
this virus maintains its genome in a manner analogous to
other herpesviruses where latent viral genome conformation has been studied.
r 1999 by The American Society of Hematology.
H
cally associated with a circular plasmid form of the viral
genome.10-14 In contrast, productive herpesviral infection is
associated predominantly with linear viral genome forms.12,15 It
has long been known that the latent Epstein-Barr virus (EBV) is
a circular extrachromasomal plasmid in cultured B-lymphoblastoid cells.10,11 The latent herpes simplex virus-1 genome exists
in an endless state based on DNA blot analysis16 and an endless
state has also been predicted for the latent varicella-zoster virus
genome.17 While these latter findings are consistent with a
circular genome conformation analogous to EBV, tandemly
integrated genomes or concatemers remain a possibility. To
analyze genome conformation specifically, a native agarose gel
system has been used to characterize large bacterial plasmids18
and circular or linear forms of herpesvirus saimiri, herpesvirus
ateles, EBV, and human herpesvirus 8 genomes.12-14 Rare cells
in the PB CD141 population, which can only be detected after
polymerase chain reaction (PCR) amplification, are known to
carry CMV without evidence of productive infection8,9,19 and
can reactivate CMV after allogeneic stimulation.7 The focus of
our study was to apply native agarose gel electrophoresis,
combined with PCR amplification,13 to determine the CMV
genome conformation in PB mononuclear CD141 cells from
healthy seropositive carriers. The results of this study show that
the CMV genome migrates as a circular plasmid form, suggesting that this virus maintains its genome in the PB CD141 cell
population in a manner analogous to other herpesviruses where
latent viral genome conformation has been studied.
UMAN CYTOMEGALOVIRUS (CMV), a large 230- to
240-kb dsDNA member of the betaherpesvirus subfamily, is harbored latently in 50% to 100% of healthy adults.1,2 The
virus can infect the human host directly through mucous
membrane contact, or by infected cells present in blood
transfusions or tissue transplants. Infections caused by CMV are
a leading infectious disease cause of morbidity in allograft
transplant recipients as well as other immunocompromised
hosts (eg, human immunodeficiency virus [HIV] infected).3
They are also the predominant cause of congenital infection in
the Western world.4 CMV infection is a significant risk after
blood transfusion, because circulating mononuclear cells of the
peripheral blood (PB) provide a reservoir for the latent virus.5-7
Reactivation of latent virus occurs in the face of diminished
immune surveillance and contributes more to the incidence of
serious disease in immunocompromised hosts than primary
infection. Despite the important role latency and reactivation
play in the pathogenesis of CMV disease, knowledge of the
underlying mechanisms controlling these processes remains
limited.
Although viral DNA has been detected in circulating PB
mononuclear cells (PBMCs) from healthy seropositive individuals,8,9 the conformation of this DNA has not been investigated.
Identification of the CMV genome form in blood cells can
provide insight into understanding mechanisms of viral persistence and dissemination in the human host. For herpesviruses
where this has been studied, latent infections are characteristi-
MATERIALS AND METHODS
From the Department of Pediatrics, Section of Infectious Diseases,
University of California Davis; and the Department of Microbiology
and Immunology, Stanford University School of Medicine, Stanford,
CA.
Submitted July 13, 1998; accepted September 4, 1998.
Supported by National Institutes of Health grants awarded to J.A.W.
(K08 AI01193) and E.S.M. (RO1 AI33852). J.A.W. is a recipient of UC
Davis Hibbard E. Williams Research Funds and UC Davis Medical
Center Children’s Miracle Network Telethon Research Funds.
Address reprint requests to Jean A. Wiedeman, Pediatric Infectious
Diseases, Neurosciences Bldg, 1515 Newton Ct, Room 600, Davis, CA,
95616; e-mail: [email protected].
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.
r 1999 by The American Society of Hematology.
0006-4971/99/9301-0043$3.00/0
394
Isolation of CD141 cells. Individuals were identified as CMV
seropositive using a CMV IgG ELISA (Sigma, St Louis, MO). Healthy
adult CMV-seropositive volunteers donated according to institutional
review board guidelines. Fifty to 100 mL of blood was obtained by
percutaneous venepuncture and anticoagulated with EDTA (final concentrations, 5 mmol/L). A volume of 250 to 300 mL of pooled blood was
prepared for each sample set. PBMCs were isolated on Lymphoprep
gradients (Nycomed, Oslo, Norway). CD141 cells were selected using
magnetic cell sorting with microbeads (Miltenyi Biotec, Auburn, CA)
according to manufacturer’s instructions, using two successive column
purifications for each preparation. Purity analysis was performed using
fluorescein isothiocyanate (FITC) anti-CD14 clone MfP9 (Becton
Dickinson, San Jose, CA) followed by flow cytometry.
Cells and viruses used in control samples. Primary human fibroblasts were infected at a multiplicity of infection (MOI) of 5 plaque
forming units (PFU) using CMV strain Toledo. Cells were obtained 7
days after infection, for preparation of positive controls. The bacteria
Shigella flexneri (gift from Stanley Falkow, Stanford University)
Blood, Vol 93, No 1 (January 1), 1999: pp 394-398
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CIRCULAR CONFORMATION OF LATENT CMV GENOME
contains a circular megaplasmid of approximately 230 kb, and served as
a circular plasmid size marker. Bacteria were cultured in Luria broth,
using appropriate biohazard precautions.
Preparation of agarose blocks for gel analysis. Cell samples were
prepared in agarose blocks using previously described methods,20 and
the CD141 cells were prepared at 2.5 3 106 cells per 100 µL volume in
each block. Two agarose blocks (stacked front to back) per lane were
cast directly into gels.
Electrophoretic gel analysis. Horizontal agarose gels were prepared according to methods previously described.12 Low melting point
(LMP) agarose (0.75%) in 0.53 Tris-Borate-EDTA buffer was used to
cast 25 3 20-cm gels. After the gel had solidified, a section of the gel at
the origin (3 3 15 cm) was excised, and the comb placed in the cutout
section. The section was filled with 0.8% LMP agarose containing 2%
sodium dodecyl sulfate (SDS) and 1 mg/mL of Proteinase K added after
gel temperature had cooled to 50°C. Bacteria samples were loaded live
onto gels using 108 cells per lane with previously described methods.12
Gels were run at 4°C for 3 hours at 0.8 V/cm, followed by 24 hours at
4.5 V/cm.
After electrophoresis, portions of the gel containing the controls were
stained in ethidium bromide solution (1 µg/mL) and photographed. For
PCR amplification, sample lanes were scored into sequential cores
starting from the origin, with the location of each recorded on a gel
template. Each agarose core was melted at 65°C for 15 minutes, then
digested with beta-agarase (FMC Bioproducts, Rockland, ME) according to the manufacturer’s instructions. DNA was precipitated with
glycogen (10 µg per sample), ammonium acetate (Sigma) added to a
final concentration of 2.5 mol/L, and 2.5 vol of 100% ethanol added.
The resulting pellets were resuspended in 10 µL of TE buffer (10
mmol/L TRIS-HCl pH 8, 1 mmol/L EDTA), and the entire volume was
added to each PCR reaction.
PCR detection assays. To reduce the risk of losing PCR amplification due to viral sequence heterogeneity among different isolates,
primer pairs directed to the major early beta 2.7 gene were selected.
This primer set recognizes a conserved region of the CMV genome, and
has been applied successfully to detection of CMV isolates from eight
different individuals and one laboratory strain (AD 169) as previously
reported.21 Forward primer, reverse primer, and probe oligonucleotides
are as previously reported.21 PCR amplifications (50-µL vol) used
reaction mix as reported with the following exceptions: Tris-HCl pH
8.3, 3 mmol/L MgCl2, 1.25 U of Amplitaq Gold (Perkin Elmer,
Norwalk, CT), and 1.25 U of Taq Extender (Stratagene, La Jolla, CA).
The primary PCR amplification product was 315 bp. PCR reactions
were performed in a Perkin Elmer 2400 thermocycler under the
following conditions: 10 minutes at 94°C was used to activate the
Amplitaq polymerase, followed by 50 cycles of 20 seconds at 94°C, 30
seconds at 55°C, and 30 seconds at 70°C. After the cycles were
completed, a final extension at 70°C for 7 minutes was performed. For
nested PCR conditions, the forward primer 58 CCG GTC GGC TTC
TGT TTT AT 38 and reverse primer 58 TCT CTT GTT GGG AAT CGT
CG 38 were used. PCR amplifications (30 cycles, 20-µL vol) used
reaction mix as reported above with the following exceptions: Tris-HCl
pH 8.7, 0.1% Triton X-100, and 0.1 U of native Taq polymerase
(Promega, Madison, WI). One microliter of the primary PCR reaction
was added as template. PCR conditions were 5 minutes at 94°C,
followed by 30 cycles of 20 seconds at 94°C, 30 seconds at 60°C, and
30 seconds at 72°C. A final extension at 72°C for 7 minutes was then
performed. Nested PCR yielded a 202-bp product. The internal
oligonucleotide probe used was the same as above. CD141 cell samples
were confirmed to contain CMV DNA before gel analysis, using total
cell DNA prepared and PCR amplified as described above. When using
limiting dilutions of positive control viral DNA as template, PCR
reactions were sensitive enough to detect two template copies, without
requiring a nested amplification.
The detection limits of the combined gel resolution and PCR
395
approach were determined, using limiting dilutions of CMV infected
cells in a background of negative cells. Viral DNA was detected down to
a dilution of <3 infected cells in a background of 106 uninfected
PBMCs collected from a seronegative donor (see Fig 2E). This amount
of viral DNA is expected to be in the range of 3 3 103 to 3 3 104 copies,
well below the detection limits for direct blot hybridization.22 To further
establish the limit of detection by PCR after recovery of DNA after gel
electrophoresis and beta agarase digestion, known copy numbers of
CMV cosmid clones were used as template. It was determined that the
level of sensitivity was 50 copies (data not shown). Detection was lost
below this level.
No inhibition of the PCR reactions was observed when low copy
numbers of CMV DNA were tested in the presence of melted agarose
gels collected from linear regions of uninfected cell DNA. Cores
collected adjacent to low copy positive sample lanes were also routinely
negative. No inhibition of the PCR reactions was observed, when tested
using low copy positive controls in the presence of beta agarase
digested agarose cores (data not shown).
DNA blot analysis of PCR products. Ten percent of each PCR
reaction volume was analyzed on 6% polyacrylamide gels, with a 1-kb
ladder marker (GIBCO-BRL, Gaithersburg, MD). DNA was transferred
to Zetaprobe membrane (Boehringer Mannheim, Indianapolis, IN), by
capillary transfer in 0.4 N sodium hydroxide. The oligonucleotide probe
(above) was prepared using previously described methods.9
RESULTS
Resolution of viral genome forms using native agarose gels.
The Southern blot in Fig 1 shows that circular forms of DNA are
clearly resolved from those of unit-length linear structures. The
results obtained from a megaplasmid of approximately 230 kb
Fig 1. Southern blot showing resolution of DNA forms. Lanes
loaded as the following: lanes 1 and 2, duplicate samples of 108 cells S
flexneri bacteria carrying E230-kb circular megaplasmid, loaded live
in each lane; lanes 3 and 4, duplicate samples of EBV cell line B95-8
with 106 cells loaded per lane; lane 5, 106 CMV lytically infected cells.
Southern blot was sectioned, and each section hybridized with probe
specific for those particular samples. The blots were then realigned
for figure. The (p
p) symbol marks the lower edge of the proteinase
K/SDS/agarose trough.
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396
(similar in size to the CMV genome) carried in S flexneri
bacteria are shown in duplicate lanes 1 and 2. Probe-positive
linear and circular forms of the megaplasmid are observed. The
linearized form seen is caused by the harsh lysozyme treatment
and shearing of some of the megaplasmid DNA.18 Duplicate
lanes 3 and 4 show the distinct separation of probe-positive
linear and circular forms for EBV, a herpesvirus that is closely
related in size to CMV (<170 kb). For EBV analyses, the B95-8
cell line was used. In these cells, both circular and linear forms
of the EBV viral genome are present, because 5% of the cells
are known to be producing virus with the remaining number
carrying latent circular viral genome.13,23 Finally, in lane 5,
CMV productively infected cells (collected 7 days postinfection) show only a distinct probe-positive unit-length linear
230-kb band and no circular form, as would be predicted in a
productive infection. In addition, some viral DNA is retained at
the well origin for all lanes. The linearized megaplasmid form
(lanes 1 and 2) migrates with a similar mobility as the unit
length linear form of the CMV genome and closely with the
linear EBV form. Overall, the clear resolution of circular versus
linearized genome forms is shown using the native agarose gel
system.
Purity of isolated CD141 cell populations and detection of
CMV DNA before native agarose gel analysis. CD141 cells
were isolated from PBMCs using positive selection as described
in Materials and Methods. The purity of the CD141 cells was
routinely 95% or higher, based on flow cytometry analysis (data
not shown). Typical yields ranged from 10% to 20% of the
original PBMC numbers. The cells displayed a monocyte type
morphology, and were able to adhere to glass or plastic.
BOLOVAN-FRITTS, MOCARSKI, AND WIEDEMAN
Demonstration of free episomal form of CMV DNA from
CD141 cell populations in healthy carriers. To characterize
the conformation of the CMV genome in PB mononuclear
CD141 cells, we analyzed three independent sets of pooled cells
from healthy seropositive individuals. Figure 2 shows the
results obtained after selection of CD141 cells, electrophoretic
separation, and PCR amplification of DNA recovered from
individual gel cores followed by blot hybridization. A clear
CMV probe-positive signal was observed in each of three
analyses, sets 1 through 3 (panels B through D), from respective
single cores with similar mobility to that of the circular 230-kb
megaplasmid marker control (panel A). In all three sets, a CMV
DNA positive signal was detected in only one discrete agarose
core.
A clear CMV probe-positive signal was observed in sample
set 1 (panel B; core 8), at a region comigrating with the circular
230-kb megaplasmid marker (panel A). In addition, DNA blot
analyses typically showed both the primary PCR amplification
product (315 bp) as well as the nested amplification product
(202 bp). Neither the region where linear DNA would migrate
nor the well origin showed positive signal. For comparison, the
relative mobility of the linear CMV genome on this type of gel
system is represented in Fig 2E, starting with <3 CMV-infected
fibroblasts in a background of 106 CMV-negative PB cells.
Set 2 (panel C) exhibited a slightly slower migrating CMV
probe-positive core than that of the circular megaplasmid
marker or the migration of the CMV DNA probe positive core in
sample set 1. For set 2, core 5 (panel C) migrated approximately
1 cm slower than core 8 (panel B), which was the core migrating
with similar mobility to the circular megaplasmid. Once again,
Fig 2. CMV genome conformation in PB CD141
cells. For all panels the top (Origin) of the gel is
represented on the right and the bottom of the gel is
depicted on the left. Symbols 1 and 2 indicate
positive and negative controls for PCR. Numbers 8,
5, and 4 label the probe-positive cores for panels (B),
(C), and (D), respectively. Size markers (315 bp and
202 bp) for the primary and nested PCR products,
respectively, are represented. (A) Ethidium bromide–
stained gel showing a representative lane containing 108 cells of S flexneri bacteria carrying the
E230-kb circular megaplasmid marker. Ethidium bromide staining of bacterial chromosomal DNA is
associated with the region near the well origin
(Origin). The lower edge of the proteinase K/SDS/
agarose trough is seen as a brightly stained region
midway between the circular marker and the well
origin. The circular megaplasmid control migrated at
core 8 for all gels shown in panels (B), (C), (D), and
(E). The apparent linear megaplasmid band (Linear)
invariably comigrated with the CMV linear band as
exemplified in the positive cores after PCR amplification of low copy CMV-infected cells (E). (B) DNA blot
analysis of nested PCR products, from an inclusive
series of sequential agarose cores collected from the
gel lane prepared from set 1 of pooled CD141 cells
from five donors. (C) DNA blot analysis similar to (B),
from set 2 (3 donors). (D) DNA blot analysis of
primary PCR products, from set 3 (5 donors) and
using only 10% of the proteinase K normally used in
block preparation. (E) Ethidium bromide–stained gel
of PCR amplification of low copy CMV-infected cells.
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CIRCULAR CONFORMATION OF LATENT CMV GENOME
there was no evidence of a PCR-positive signal in the region of
the gel representing linear 230-kb CMV genome and none seen
at the well origin, even after nested PCR amplification and DNA
blot analysis. For both sets 1 and 2 (panels B and C) the primary
PCR amplification showed CMV DNA migrating at a mobility
similar to the circular megaplasmid marker DNA and nested
PCR was not necessary to see this signal (see panel D where
only primary PCR was undertaken). Nested PCR was used to
increase the sensitivity of the assay for viral DNA in other
regions of the gel. Our results suggest that viral DNA was not
present in the linear region of the gel or near the well origin.
One explanation for the mobility differences between set 1
and set 2 might have been varying efficiency of proteinase K
digestion due to the fact that the blocks were prepared from
such large numbers of cells.12 To address this possibility, an
additional pooled set of CD141 cells was prepared from
seropositive individuals similar in average age, gender, and
serostatus as those of the first pooled set. The cells were
prepared in agarose blocks at the same density (2.5 3 106 cells
each) as in the first two sets, but only 10% of the proteinase K
was used in the digestion step. As in previous gels, two agarose
cell blocks (total of 5 3 106 cells) were loaded per lane. The
results (panel D) show the probe-positive signal was detected at
the slowest observed mobility, core 4 as compared with that of
the circular megaplasmid (core 8) and both previous CMV
sample sets, but still in the region of the gel where a circular
form would be expected to migrate. Only primary amplification
was undertaken in this analysis (panel D), because it had
previously been established that a single round of PCR was
sufficient to amplify viral DNA in these samples. These results
suggest that incomplete proteolysis may contribute to reduced
mobility of circular CMV DNA, although these differences in
migration may also have other explanations, such as variation in
the electrophoretic conditions. Taken together, all three pooled
sets of CD141 cells showed probe-positive signal from cores in
the region where a circular form would migrate. Given the fact
that these gels separate primarily on the basis of structure,12,18
the slight mobility differences do not detract from the overall
conclusion.
DISCUSSION
Using a native agarose gel electrophoresis system in conjunction with PCR amplification, we were able to show that CMV
genome in CD141 cells of healthy seropositive adult carriers is
detected only as a circular form without evidence of linear or
integrated CMV genome. The detection of circular CMV in the
CD141 cell population from PB of healthy hosts indicates that
this is the form in which the latent viral genome persists in this
population of cells. Because the predicted viral DNA copy
number present in healthy subjects lies below the detection
limits of direct blot hybridization analysis,8 PCR amplification
was used as a means to extend the sensitivity of the detection
limits. In our results, the circular form of CMV DNA was
consistently detected after only one round of initial PCR
amplification. Viral DNA was not detected in the linear region
of the gels or in the well origins in any of these analyses, despite
the fact that nested PCR conditions and DNA blot hybridization
analyses were used in a majority of analyses.
397
Viral DNA from productive or persistent infections is associated with both large complex structures, such as concatemers or
branched forms, as well as linear forms of the viral genome, the
products of concatemers cleaved into unit-length linear viral
DNA during the packaging and assembly process. In the type of
gel analysis used here, branched, highly structured or very large
DNA forms are retained at the well origin of the gel, while
linear forms of <50 to 700 kb migrate with a mobility similar to
linear CMV DNA.12,24,25 Both large complex and/or concatemeric viral DNA and unit-length linear forms were detected at
the well origin and in the linear region of the gel, respectively,
for high copy and low copy CMV productively infected cell
controls (Fig 1 and Fig 2, respectively). These linear, large
concatemeric or integrated viral DNA do not migrate in the
same region of the gel with circular forms. If other forms exist
in the viral DNA reservoir during latency, the copy number was
below our detection limits.
Studies in healthy carriers have shown the presence of CMV
DNA throughout the myeloid lineage.8,26,27 Viral DNA has been
detected in CD341 cell populations that include early bone
marrow hematopoietic progenitors27 and CMV DNA and RNA
from bone marrow myeloid-committed progenitors coexpressing CD33/15.26 In addition, CD141 cells from PB, but generally
not CD142 cell types in PB, are the predominant site of CMV
viral genome.9,21 CMV virus can also be reactivated from
CD141 PB cells from healthy subjects after allogeneic stimulation and prolonged in vitro culture.7 Our results have extended
these observations and indicate that latent viral DNA is
maintained in CD141 PB cells as an extrachromosomal circular
plasmid.
Finally, a circular plasmid conformation for the CMV
genome is consistent with that of other latent herpesviral
genomes, and supports studies suggesting the CMV genome is
harbored in a latent state in immature and more differentiated
myeloid cells.26-29 Future studies should be undertaken to
extend the analyses of the CMV genome conformation to
include these progressively earlier stages of the myeloid cell
lineage.
Taken together, these results are consistent with the proposed
model that the latent CMV genome resides in early CD341 and
CD331 progenitor cells in the bone marrow, and is partitioned
to the monocyte or dendritic lineage, where it can be detected in
CD141 cells of the PB.30 In support of this, we have reported the
first successful characterization of the CMV genome conformation in CD141 cells from normal CMV seropositive individuals
and demonstrated that the genome persists as a circular plasmid
comigrating with a 230-kb circular megaplasmid marker.
ACKNOWLEDGMENT
We thank Mike McVoy, Lenore Pereira, Lucy Rasmussen, and Steve
St. Jeor for their critical review of this manuscript.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1999 93: 394-398
Peripheral Blood CD14+ Cells From Healthy Subjects Carry a Circular
Conformation of Latent Cytomegalovirus Genome
Cynthia A. Bolovan-Fritts, Edward S. Mocarski and Jean A. Wiedeman
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