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Journal of Virology, July 2001, p. 6007-6015, Vol. 75, No. 13
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.13.6007-6015.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mink Cell Focus-Forming Murine Leukemia Virus
Killing of Mink Cells Involves Apoptosis and Superinfection
Fayth K.
Yoshimura,1,2,*
Tao
Wang,1 and
Suparna
Nanua1
Department of Immunology and
Microbiology1 and the Karmanos Cancer
Institute,2 Wayne State University, Detroit,
Michigan 48201
Received 8 February 2001/Accepted 12 April 2001
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ABSTRACT |
Induction of apoptosis by different types of pathogenic
retroviruses is an important step in disease development. We have observed that infection of thymic lymphocytes by the mink cell focus-forming murine leukemia virus (MCF MLV) during the preleukemic period resulted in an enhancement of apoptosis of these cells. To
further study the ability of MCF MLVs to induce apoptosis and the role
of this process in viral pathogenesis, we have developed an in vitro
system of virus-induced apoptosis. MCF13 MLV infection of mink
epithelial cells resulted in the production of cytopathic foci. In
contrast, infection of mink cells with the 4070A amphotropic MLV did
not produce any cytopathic effects. Staining of MCF13 MLV-infected
cells with propidium iodide and annexin V-fluorescein isothiocyanate
indicated that virus-induced cell death was due to apoptosis. At 6 days
postinfection, the percentage of apoptotic MCF13 MLV-infected cells was
27% compared with 2 to 3% for mock- or amphotropic MLV-infected
cells, representing a 9- to 14-fold difference. Assays for caspase-3
activation confirmed the detection by flow cytometry of apoptosis of
MCF13 MLV-infected cells. Large amounts of unintegrated linear viral
DNA were detectable by Southern blot analysis during the acute phase of
infection, which indicated that MCF13 MLV is able to superinfect mink
cells. Unintegrated viral DNA of only the linear form was detectable in
thymic lymphocytes isolated from MCF13 MLV-inoculated mice during the
preleukemic period. These results indicated that the ability of MCF13
MLV to induce apoptosis is correlated with its ability to superinfect cells and that this occurs as an early step in thymic lymphoma development.
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INTRODUCTION |
The induction or inhibition of
apoptosis by viruses of different families is a critical determinant of
their pathogenicity (36, 42). Among retroviruses, the
induction of apoptosis has been shown to be important for the
generation of immunodeficient diseases as well as various forms of
neoplasia. Retroviruses for which apoptosis is thought to play an
important role in the generation of immunodeficiencies include the
human, simian, and feline immunodeficiency viruses (HIV, SIV, and
FeLV-FAIDS, respectively) (1, 15, 28, 34) and a mutant of
the Moloney murine leukemia virus (ts-1 Mo MLV)
(35). Examples of oncogenic retroviruses which induce cell
killing and apoptosis include the avian leukosis virus (ALV) of
subgroups B and D (10, 46, 47), avian
reticuloendotheliosis viruses (REVs) (22), Abelson MLV
(33, 43), and Mo MLV (9). It has recently
been shown that thymic lymphocytes isolated from preleukemic mice
inoculated with the mink cell focus-forming (MCF) MLV underwent
enhanced apoptosis (52). We observed that apoptosis of
thymic lymphocytes in these mice resulted in diffuse lymphocyte depletion mainly in the cortical region of the thymus. This region of
the thymus consists mainly of the subpopulation of cells that we and
others have shown are predominantly infected by MCF MLVs (12, 31,
51). Similar abnormalities of the preleukemic thymus have been
detectable by others during the development of spontaneous T-cell
lymphomas in the AKR mouse (30).
The relevance of MCF MLV-induced apoptosis of thymic lymphocytes to AKR
lymphomagenesis was observed in early studies by D. Metcalf, who noted
that cell death in the thymus was a requirement for leukemogenesis in
the AKR mouse (27). Moreover, work by Bonzon and Fan
(9) on the induction of thymic lymphoma by Mo MLV showed a
strong correlation between enhanced apoptosis of thymic lymphocytes
from mice inoculated with the wild-type virus and the reduced apoptosis
induced by a mutant which is attenuated in pathogenicity. Because of
the paradigm that tumorigenesis results from abnormal cellular
proliferation, it is not obvious why cell killing should be part of the
mechanism used by oncogenic retroviruses to induce tumors. It is
possible, however, that cells undergoing apoptosis may be rescued from
this initial crisis by genetic changes which may be provided by
proviral insertion adjacent to cellular genes involved in the
regulation of apoptosis.
It has been shown that retroviral glycoproteins are involved in the
cytopathic effects of several different oncogenic retroviruses. A
direct involvement in apoptosis has been observed for the interaction of the ALV subgroup B glycoprotein with its cellular receptor CAR1, a
member of the tumor necrosis factor (TNF) receptor family (10). Activation of TNF receptor proteins induces
apoptosis (3), and a similar effect is observed for the
subgroup B ALV glycoprotein. For FeLV-C, genetic and mutagenesis
studies have mapped viral cytopathicity to the surface portion of the
envelope glycoprotein (34). However, the receptor for
FeLV-C, which has recently been identified, does not resemble any known
cellular protein that participates in apoptosis (32, 41).
Therefore, the mechanism used by FeLV-C to induce apoptosis may be
different from that used by ALV.
A close correlation has been observed between the ability of a
retrovirus to induce cytopathic effects and its ability to synthesize
high levels of unintegrated linear viral DNA in acutely infected cells
(22, 29, 46, 47). It has been demonstrated that the large
amounts of unintegrated viral DNA are the consequence of virus
superinfection for ALV of subgroups B and D (46, 47), REVs
(22), and FeLV-FAIDS (29). The relevance of
this observation to AKR lymphomagenesis is underscored by the detection
of high levels of unintegrated linear MLV DNA in thymuses of AKR mice during the preleukemic period of spontaneous lymphoma development (21). To better understand the role of MCF13 MLV-induced
apoptosis in the generation of thymic lymphoma, we have examined
whether this retrovirus resembles other cytopathic retroviruses in its ability to superinfect cells and synthesize high levels of unintegrated linear viral DNA. We summarize our studies employing cultured mink
epithelial cells in this report.
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MATERIALS AND METHODS |
Virus infection of cells.
Ten thousand CCL64 mink epithelial
cells (American Type Culture Collection) were plated in each well of a
24-well plate 1 day before infection with 1 ml of cell-free supernatant
containing MCF13 or 4070A amphotropic MLV and 2 µg of polybrene.
Mock-infected cells were treated with 1 ml of Dulbecco's minimal
essential medium containing 2 µg of polybrene. Cells were incubated
at 37°C for 6 h, after which virus was removed, cells were
rinsed once, and fresh Dulbecco's minimal essential medium containing
10% fetal bovine serum was added. Viable cells were enumerated by
trypan blue dye exclusion.
Detection of cytopathic foci and virus-infected cells.
Ten
thousand CCL64 mink cells were plated on a 6-cm culture dish and
infected with either MCF13 or 4070A amphotropic MLV or mock infected as
described above. Cytopathic foci were detected by phase microscopy
using a Leica DMIRB microscope connected to a digital camera from
DIAGNOSTIC Instrument, Inc. Detection of virus-infected cells was made
by an indirect immunofluorescence assay, which involved the initial
treatment of cells with 0.5 ml of hybridoma supernatant of monoclonal
antibody (MAb) 83A25, which recognizes the glycoprotein of MCF and
amphotropic MLVs (17), for 30 min at 37°C. This was
followed by the addition of 1 ml of a secondary fluorescein
isothiocyanate (FITC)-labeled anti-rat antibody (Sigma) for another 30 min. The MAb 83A25 hybridoma was a generous gift of L. H. Evans, Rocky
Mountain Laboratories, Hamilton, Mont.
Flow cytometric analysis of apoptotic cells.
One hundred
thousand mink cells were trypsinized and washed twice with
phosphate-buffered saline (PBS) containing 1% bovine serum albumin and
0.02% sodium azide. Cells were stained with 0.5 µg of propidium
iodide (PI) (Sigma) per ml and 0.6 µg of annexin V-FITC (Pharmingen)
per ml in 10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM
CaCl2 for 20 min at room temperature in the dark. Samples were analyzed within 1 h after staining on a Becton Dickinson FACScan. Data on 2 × 104 cells were analyzed using
the CELLQuest software (Becton Dickinson). Regions with populations of
cells that were negative for PI (PI
) and for annexin V
(annexin V) (live), PI and positive for
annexin V (annexin V+) (apoptotic), or positive for PI
(PI+) and annexin V+ (dead) were selected.
Analysis gates were set on single-color controls.
Caspase-3 assay.
One million mink cells which were either
infected with virus or mock infected were trypsinized, washed twice
with PBS, and lysed in 200 µl of 50 mM Tris-HCl (pH 7.5) containing
0.03% Nonidet P-40 and 1 mM dithiothreitol. Nuclei were removed by
centrifugation at 1,200 × g for 5 min at 4°C.
Caspase-3 activity was measured by using an EnzChek caspase-3 assay kit
(Molecular Probes) in a 96-well plate format. Fluoromethylcoumarin
fluorescence, released from the Z-DEVD-AMC peptide substrate by
caspase-3 activity, was detected at 450 nm for 30 min with a Spectra
MAX Gemini fluorometer (Molecular Devices). A 7-amino-4-methyl coumarin
standard curve was prepared in the range of 0.137 to 17.5 µg. Protein
amounts were measured using the bicinchoninic acid protein assay
(Pierce). Units of caspase-3 activity are expressed as picomoles of
substrate hydrolyzed per microgram of protein per half hour.
Hirt extraction of unintegrated viral DNA.
Hirt extractions
of mink epithelial cells growing on 10-cm tissue culture plates were
performed as described previously (53). Briefly, cells
were rinsed twice with PBS and lysed by the addition of 3 ml of lysis
buffer (10 mM Tris-HCl [pH 7.5], 10 mM EDTA, 100 mM NaCl, 1% sodium
dodecyl sulfate) at 37°C for 20 min. Cell lysates were then made to 1 N NaCl final concentration and incubated overnight at 4°C. The salt
precipitate was pelleted at 27,000 × g for 1 h at
4°C. DNA was precipitated from the supernatant, which had been
extracted twice with chloroform, with the addition of 2.5 volumes of
ethyl alcohol at 
20°C overnight. DNA was subsequently pelleted by
centrifugation at 1,100 × g at 4°C and dissolved in 200 µl of Tris-EDTA buffer (10 mM Tris-HCl [pH7.5], 1 mM EDTA), after which 20 µl of 3 M sodium acetate, 60 µg of glycogen, and 3 volumes of ethyl alcohol were added with an overnight precipitation at
20°C. DNA was pelleted by centrifugation at 16,000 × g for 30 min at 4°C and dissolved in 15 µl of Tris-EDTA buffer.
Hirt extractions were also performed on 2 × 108
thymic lymphocytes that were isolated as described previously
(51) from AKR/J mice that were inoculated neonatally with
106 infectious units of MCF13 MLV. Control mice were
inoculated with tissue culture medium.
Southern blot detection of unintegrated viral DNA.
Hirt-extracted DNA was electrophoresed through a 1% agarose gel in
Tris-borate EDTA buffer (89 mM Tris-borate [pH 8.0], 2 mM EDTA) at
150 V for 3 to 5 h at 4°C. One-kilobase ladder DNA was
electrophoresed in one lane of each agarose gel to provide size
markers. Transfer of DNA to a GeneScreen Plus nylon membrane (New
England Nuclear Life Sciences) was performed as described before
(49). The DNA probe for hybridization was either a 8.2-kb linear plasmid DNA corresponding to the complete MCF13 MLV genome or a
1.6-kb fragment consisting of MCF13 MLV gag sequences. Both DNA probes were 32P-labeled with the DNA Nick Translation
Kit (Boehringer Mannheim). Five million counts per minute of labeled
probe was used for hybridization of the membrane overnight at 68°C in
a roller bottle hybridization oven (Boekel Scientific). After
hybridization, nylon filters were rinsed as previously described
(49). DNA bands were detected and analyzed with a
Molecular Dynamics PhosphorImager and Storm 840 scanner.
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RESULTS |
In a previous study we observed that thymic lymphocytes isolated
during the preleukemic period from AKR/J mice inoculated with MCF13 MLV
underwent enhanced apoptosis compared with thymic lymphocytes from
uninoculated animals (52). To facilitate studies of the
mechanism by which MCF13 MLV induces apoptosis, we have developed an in
vitro system employing the CCL64 mink epithelial cell line. The
rationale for performing these studies in vitro is that experiments
with mice are costly and require long incubation periods to detect
apoptotic effects (approximately 4 to 8 weeks postinoculation) and that
mink cells are easily grown in culture, unlike primary lymphocytes,
which rapidly die when cultured. Moreover, we wished to determine
whether MCF13 MLV could induce apoptosis of other cell types besides
thymic T cells and to facilitate further studies to elucidate the
mechanism of viral cytopathicity.
Cytopathic effects of MCF13 MLV infection of CCL64 mink epithelial
cells.
When CCL64 cells were infected with MCF13 MLV at different
multiplicities of infection (MOI), we detected a depletion of cells over time. Figure 1A shows that a
reduction in cell number was detectable starting at 3 days
postinfection (p.i.) and continued through 5 days p.i. at MOI of 1 and
7. Cell depletion level correlated with the amount of virus added. In
comparison, we did not detect a similar reduction in the number of mink
cells that were infected with the 4070A amphotropic MLV, another MLV
that infects CCL64 cells (11), at an MOI of 7 (Fig. 1B).
At this MOI, however, a slight decrease in cell number was detectable
for the amphotropic MLV.
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FIG. 1.
Effect of MCF13 MLV infection on cell growth. (A) Ten
thousand CCL64 mink epithelial cells were infected with MCF13 MLV at an
MOI of 1 ( ) or 7 ( ) or mock infected with medium ( ). (B) Mink
cells were infected with MCF13 MLV () or 4070A amphotropic MLV ( )
at an MOI of 7 or mock infected (). Cells were trypsinized at
different days after infection, and viable cells were enumerated by
trypan blue dye exclusion. Values represent the means and standard
deviations calculated from counting duplicate samples from four
independent experiments.
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Starting at approximately 5 days after MCF13 MLV infection, cytopathic
foci were detectable by phase microscopy throughout
the mink cell
culture (Fig.
2A).
These foci consisted of cells
with altered morphology including
pycnotic cells and resembled
the foci which were described for the
initial isolation of MCF
MLVs (
19). Detection of MCF
glycoprotein expression on the cell
surface by indirect
immunofluorescence staining demonstrated that
cells constituting the
cytopathic foci were infected with MCF13
virus (Fig.
2B). Cytopathic
foci were not detectable with cells
that were either infected with the
4070A amphotropic MLV (Fig.
2C and D) or mock infected (Fig.
2E and F).
Cells infected with
the amphotropic MLV showed positive staining by the
indirect immunofluorescence
assay, thereby demonstrating that the
absence of cell death and
cytopathic foci was not due to the inability
of this virus to
infect mink cells (Fig.
2D).
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FIG. 2.
MCF13 MLV infection of mink epithelial cells produces
cytopathic foci. Ten thousand CCL64 mink epithelial cells were infected
with MCF13 MLV (A and B) or 4070A amphotropic MLV (C and D) at an MOI
of 1. Cells were also mock infected with medium (E and F). Six days
after virus infection, cells were examined by phase microscopy (A, C,
and E) and fluorescence microscopy (B, D, and F). Fluorescence assays
were performed by first staining cells with MAb 83A25, a monoclonal
antibody that recognizes nearly all MLV glycoproteins, and subsequently
with an FITC-labeled secondary anti-rat antibody.
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Mink cell killing by MCF13 MLV occurs by apoptosis.
Because we
had observed that MCF13 MLV infection of thymic lymphocytes resulted in
an enhancement of apoptosis, we investigated whether apoptosis was also
the cause of mink cell killing. It has been shown that flow cytometric
analysis of cells stained with PI and FITC-labeled annexin V (annexin
V-FITC) is an effective method to distinguish between live
(PI, annexin V), apoptotic
(PI, annexin V+), and dead (PI+,
annexin V+) cells (23). Using this method, we
observed an increase over time in the percentage of apoptotic mink
cells infected with MCF13 MLV (Fig. 3A).
The greatest percentage of apoptotic MCF13 MLV-infected cells was
detectable at 6 days p.i. when 27% of cells were apoptotic. This
represented a 14-fold increase over the percentage of apoptotic cells
that were detectable for mock-infected cells or cells infected with the
4070A amphotropic MLV. As a correlate, we also examined the percentage
of live cells by flow cytometric analysis and observed that the
percentage of live cells that were infected with MCF13 MLV decreased
steadily over time compared with that of mock-infected cells (Fig. 3B).
In contrast, no difference in the percentage of live cells infected
with the amphotropic MLV was detectable.
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FIG. 3.
Flow cytometric analysis of mink epithelial cells
stained with PI and annexin V-FITC after virus infection. One hundred
thousand mink epithelial cells were infected with either MCF13 MLV
() or 4070A amphotropic MLV () or mock infected (). Cells were
trypsinized at different days after virus infection at an MOI of 4 and
stained with PI and annexin V-FITC. Twenty thousand cells were analyzed
on a FACScan flow cytometer. Values are the means and standard
deviations calculated from cell numbers from four independent
experiments. (A) Percentage of apoptotic cells (PI,
annexin V+). (B) Percentage of live cells
(PI, annexin V).
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As an independent assay for apoptosis, we monitored caspase-3 activity
in mink cells at various times after infection with
MCF13 MLV.
Activation of caspase-3 occurs in apoptotic cells by
specific cleavage
of procaspase-3 by other caspases, such as caspase-8
and caspase-9,
which themselves are activated by more upstream
events in apoptotic
signaling (
44,
45). For this assay, we
monitored the
production of fluoromethylcoumarin fluorescence,
which is released from
the Z-DEVD-AMC peptide substrate by caspase-3
cleavage. As shown in
Fig.
4, we detected greater caspase-3
activity
in MCF13 MLV-infected cells than in mock-infected and
amphotropic
MLV-infected cells. The peak of caspase-3 activity occurred
at
4 days p.i., which is slightly before the peak of apoptotic cell
number that was detectable by flow cytometry analysis (Fig.
3A).
This
result is consistent with the observation that caspase-3
activation is
a relatively early event in apoptosis and occurs
in some cell types
before the detection of phosphatidylserine
on the outer plasma membrane
of the cell (
6,
16).
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FIG. 4.
Caspase-3 activity is increased in cells infected with
MCF13 MLV. Cytosolic extracts were prepared from MCF13 MLV-infected
(), 4070A amphotropic MLV-infected (), or mock-infected ()
cells on different days p.i. Caspase-3 activity was measured by using
the EnzChek kit (Molecular Probes), which uses the Z-DEVD-AMC peptide
as a substrate. Fluoromethylcoumarin fluorescence was detected with a
SpectraMAX Gemini fluorometer (Molecular Devices). Units of caspase-3
activity are expressed as picomoles of substrate hydrolyzed per
microgram of protein per half hour.
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Detection of unintegrated linear viral DNA in MCF13 MLV-infected
cells.
It has been shown that there is a strong correlation
between cell killing by a retrovirus and its ability to superinfect
cells (22, 29, 46, 47). A consequence of superinfection by
these retroviruses is the accumulation predominantly of the linear form of unintegrated viral DNA during the acute phase of infection when
cytopathic effects are most prominent (22, 29, 46, 47). We
therefore wished to determine whether MCF13 MLV infection also produced
high levels of unintegrated linear viral DNA.
After mink epithelial cells were infected with MCF13 for 6 days, we
performed Hirt extractions to isolate unintegrated viral
DNA. By
Southern blot analysis of Hirt-extracted DNA from cells
infected with
MCF13 MLV, we detected an intense band of unintegrated
viral DNA (Fig.
5A, lane 3) that comigrated with a linear
DNA
plasmid fragment corresponding to the cloned genomic length of
MCF13 MLV (lane 1) (
50). The smear also detectable with
this
probe and migrating above the unintegrated linear viral DNA
corresponds
to genomic DNA, which often is present in Hirt-extracted
DNA preparations
and was detectable by ethidium bromide staining of the
agarose
gel. Hybridization to this high-molecular-weight DNA could be
due to the presence of either integrated proviral DNA or unintegrated
viral DNA, which can copurify with genomic DNA during Hirt extraction
(reference
47 and our personal observation). It does not
appear
to be due to nonspecific hybridization, since both the Hirt DNA
extractions from mock-infected and amphotropic MLV-infected cells
contained comparable amounts of genomic DNA as shown by ethidium
bromide staining. Cells that were infected with the amphotropic
MLV
lacked detectable unintegrated viral DNA (lane 4) as did mock-infected
cells (lane 2). The hybridization probe used to detect amphotropic
MLV
DNA consisted of highly conserved
gag sequences of MCF13
MLV,
which should detect the two viral DNAs equally well. However,
to
ensure that no amphotropic MLV unintegrated DNA was present,
we also
hybridized the same filter with an amphotropic-specific
env
probe and obtained no signal in lane 4 (data not shown). Cleavage
of
unintegrated viral DNA with
EcoRI, which makes a single cut
in the MCF13 MLV genome, produced fragments with sizes that were
also
consistent with a linear DNA molecule (data not shown).
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FIG. 5.
MCF13 MLV infection produces high levels of unintegrated
linear viral DNA. Southern blot analysis was performed on
Hirt-extracted DNA from mink epithelial cells (A and B) and thymic
lymphocytes from virus-inoculated mice (C). (A) Hirt-extracted DNA from
mink cells that were either mock infected (lane 2) or infected with
MCF13 MLV (lane 3) or with 4070A amphotropic MLV (lane 4) after 6 days
p.i. Plasmid DNA (2.5 ng) corresponding to the genomic-length MCF13 MLV
was electrophoresed in lane 1. DNA was hybridized with a
32P-labeled 1.6-kb DNA fragment corresponding to MCF13 MLV
gag sequences. (B) Mink cells were infected with MCF13 MLV
at an MOI of 1 for 2 days (lane 2), 4 days (lane 3), 11 days (lane 4),
and 24 days (lane 5). Lane 1 contains 10 ng of linear plasmid MCF13 MLV
genomic-length DNA. A 32P-labeled probe consisting of 8.2 kb of MCF13 MLV genomic DNA was used for hybridization. (C) Hirt DNA
was prepared from thymic lymphocytes which were isolated from thymuses
of either uninoculated age-matched control AKR mice (lanes 2 and 4) or
MCF13 MLV-inoculated mice (lanes 3 and 5) at 4 and 8 weeks
postinoculation of virus. Lane 1 contains 2.5 ng of linear plasmid
MCF13 MLV DNA and Hirt DNA from a control mouse. DNA was hybridized
with a 32P-labeled probe consisting of 8.2-kb MCF13 MLV
DNA. Arrows, 8.8-kb linear MCF13 MLV DNA.
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A time course analysis of the appearance of unintegrated viral DNA
indicated that synthesis peaked at 2 to 4 days p.i. (Fig.
5B). After
that time, we observed a decrease in the amount of
unintegrated viral DNA at 11 and 24 days p.i. At 2 days p.i.,
there
were 55 copies of unintegrated viral DNA per cell, and this
number
decreased to 10 copies per cell at 24 days p.i. Copy numbers
of
unintegrated viral DNA were derived from a standard curve generated
by
plotting different amounts of a purified plasmid DNA fragment
comprising the complete MCF13 genome against their band intensities
measured from the Southern blots using the ImageQuant software
(Molecular Dynamics). These values were divided by the number
of cells
used to obtain the Hirt-extracted DNA to obtain the copy
number per
cell. These results indicated that large amounts of
unintegrated viral
DNA are synthesized primarily during the acute
phase of virus
infection. Thus, similar to what has been observed
for other cytopathic
retroviruses, MCF13 MLV produces large amounts
of unintegrated viral
DNA, which is predominantly linear in form,
during the acute phase of
virus infection of mink cells. We did
not detect any covalently closed
circular DNA molecules even upon
longer exposure of the Southern
blots.
To determine whether MCF13 MLV infection of thymic lymphocytes in
virus-inoculated mice also produced unintegrated linear
viral DNA, we
performed Hirt extractions of thymic lymphocytes
at 4 and 8 weeks
postinoculation. These two time points during
the preleukemic period
were those at which a previous study had
detected enhanced apoptosis of
thymic cells isolated from MCF13
MLV-inoculated mice (
52).
Southern blotting analysis of Hirt
DNA extracted from thymic
lymphocytes isolated from either MCF13
MLV-inoculated or uninoculated
control mice produced the results
shown in Fig.
5C. A band
corresponding to MCF13 MLV unintegrated
DNA (shown by the arrow) was
detectable only for virus-inoculated
mice. The more slowly migrating
thick bands detectable in all
lanes corresponded to the positions of
high-molecular-weight cellular
DNA. Hybridization of cellular DNA with
our probe was due to the
presence of multiple copies of endogenous MCF
MLV-related proviruses
in the genome of the AKR mouse (
2,
14,
39). Similar to
what was seen in mink cells, we observed that
the unintegrated
viral DNA corresponded to the linear form since it
comigrated
with the genome-length linear plasmid MCF13 MLV DNA present
in
lane 1. Hirt-extracted DNA from lymphocytes of control mice was
added to the lane containing the plasmid DNA because of the effect
that
different amounts of DNA have on its electrophoretic mobility.
Unintegrated linear viral DNA was detectable as early as 4 weeks
postinoculation (lane 3) and increased slightly in amount at 8
weeks
after virus inoculation (lane
5).
Inhibition of superinfection resulted in a decrease in cell killing
and lower levels of unintegrated viral DNA.
It has been proposed
that cell killing and the presence of high levels of unintegrated
linear viral DNA are due to the ability of some retroviruses to
reinfect the same cell (22, 29, 46, 47). We wished to
determine whether a similar mechanism was responsible for the
cytopathic effects of MCF13 MLV. To inhibit superinfection of mink
cells, we added neutralizing antibody (MAb 83A25) (reference
17 and unpublished data) at 6 h after the addition of
virus to cells, which is the time when medium is normally changed after
virus infection. The numbers of virus-infected mink cells grown either
in the absence or in the presence of different amounts of neutralizing
antibody were monitored over time (Fig. 6). We observed that inhibition of
superinfection by MCF13 MLV resulted in a decrease in cell killing and
that the level of cell killing decreased with increasing amounts of MAb
83A25 in the supernatant.
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FIG. 6.
Neutralizing antibody inhibits MCF13 MLV-induced cell
killing. Mink cells were infected with MCF13 MLV at an MOI of 7 for
6 h, after which fresh medium containing no (), 1% ( ), 5%
(), or 10% () of hybridoma supernatant containing neutralizing
MAb 83A25 was added to cells. Mock-infected cells () were also
included. Cells were trypsinized at different days after infection, and
viable cells were enumerated by trypan blue dye exclusion. Values
represent the means and standard deviations calculated from two
independent experiments.
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To examine the effect of superinfection inhibition on the synthesis of
unintegrated viral DNA, we performed Southern blotting
analysis on
Hirt-extracted DNA from cells that were similarly
treated with
neutralizing antibody. Our results showed that unintegrated
linear
viral DNA was undetectable in virus-infected mink cells
grown in the
presence of MAb 83A25 at 2 and 4 days p.i. but was
present in untreated
cells (Fig.
7). At 7 days p.i. we did
detect
a small amount of unintegrated viral DNA for antibody-treated
cells (lane 5), but it was much less than the amount detectable
in
untreated cells (lane 6). Again, some hybridization was detectable
for
high-molecular-weight DNA from cells that also had detectable
unintegrated viral DNA. Taken together, these results indicated
that
mink cell killing by MCF13 MLV and the presence of high levels
of
unintegrated viral DNA were the results of viral superinfection.
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FIG. 7.
Neutralizing antibody inhibits the production of MCF13
MLV unintegrated viral DNA. Mink cells were infected with MCF13 MLV at
an MOI of 1 and treated with medium with 1% neutralizing antibody or
without antibody. Hirt-extracted DNA was prepared from antibody-treated
(+) and untreated () cells at days 2, 4, and 7 after virus
infection.
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DISCUSSION |
We observed previously that during the preleukemic period of
thymic lymphoma development by MCF13 MLV inoculation in AKR mice, thymus cellularity was reduced mainly by lymphocyte depletion of the
cortical region (52). The role of MCF13 MLV infection in
lymphocyte depletion was supported by our observations that this virus
predominantly infected the subpopulation of cells that reside in the
thymic cortex and that thymic lymphocyte depletion in animals
correlated with enhanced apoptosis of virus-infected lymphocytes in
culture. Depletion of cortical cells in the thymus has also been
detected in AKR mice undergoing spontaneous thymic lymphoma
development, arguing for the relevance of this process to tumorigenesis
(30). A strong correlation of thymic cell killing and the
development of spontaneous tumors in AKR mice has been noted
(27). Although cell killing appears to be a characteristic of other oncogenic retroviruses, such as the ALV of subgroups B and D,
the REVs, and Mo MLV (9, 10, 22, 46, 47), it is not
understood what role this phenomenon plays in tumor development.
Therefore, we have established an in vitro system to better understand
the role of apoptosis in thymic lymphoma development.
Mink epithelial cells were first used to isolate MCF MLVs, which
produced cytopathic foci of infected cells (19). We
therefore used a mink epithelial cell line (CCL64) to further study the apoptotic effects of MCF13 MLV. By different independent assays we
demonstrated that the cytopathic effects of this retrovirus were due to
cells undergoing apoptosis and were a direct consequence of virus
infection. We also showed that similar to other cytopathic retroviruses, MCF13 MLV produces high levels of unintegrated linear DNA
during the acute phase of infection and that this appears to be the
result of virus superinfection. It is curious that only the linear form
of the unintegrated viral DNA is detectable in cells infected by
cytopathic retroviruses. Acute infection by retroviruses normally
produces unintegrated closed circular DNA molecules as well as linear
molecules (13), as has been observed for Mo MLV infection
of mouse fibroblasts (53). Whether the explanation for
this observation resides with the infected cell type, which may be
missing cellular enzymes thought to be responsible for the generation
of the closed circles from the linear DNA, or with a peculiarity of the
viral DNA remains to be determined.
The ability of MCF13 MLV to superinfect cells was suggested by viral
interference studies of the receptor used by both MCF and xenotropic
MLVs (26), variously referred to as X-receptor (40), XPR1 (5), and Rmc1 protein
(48). This proposal was based on results which showed that
coexpression of the MCF glycoprotein and the MCF and xenotropic
receptor isolated from mink cells did not cause substantial
interference of infection by either a xenotropic MLV or MCF MLV
(26). In the reciprocal experiment, expression of the
xenotropic glycoprotein produced efficient interference of infection by
both MLVs. Thus, as noted in this report, these observations suggest a
strong correlation between the ability of a retrovirus to superinfect
cells and its oncogenic potential since xenotropic MLVs are not
associated with neoplastic diseases (25).
We propose two different mechanisms by which MCF13 MLV can induce
apoptosis. A mechanism that involves glycoprotein-receptor interactions
is supported by work on the ALV subgroup B glycoprotein, which induces
apoptosis upon binding to its cellular receptor CAR1, a protein that
belongs to the TNF receptor family (10). The possibility
that the MCF and xenotropic receptor may also signal apoptosis is based
on its amino acid similarity with a yeast protein that induces cell
cycle arrest in a G protein-coupled manner in response to pheromone
signaling (5, 37, 40, 48). Thus, this receptor is a good
candidate for the signaling of cell cycle arrest, which often leads to
apoptosis, by MCF glycoprotein binding. This mechanism, however, does
not adequately explain why superinfection is required for cell death. A
possible explanation for this requirement is taken into account by a
second mechanism, which is based on the strong correlation between cell
killing and the presence of high levels of unintegrated linear viral
DNA. Other cytopathic retroviruses also produce large amounts of
unintegrated linear DNA (22, 29, 46, 47). We, in this
report, and other investigators previously have shown that these large
amounts of unintegrated linear viral DNA are due to the ability of
cytopathic retroviruses to superinfect susceptible cells. We propose
that linear molecules of unintegrated viral DNA may resemble damaged DNA with double-strand breaks and thus initiate apoptotic signaling through the intrinsic pathway involving mitochondrial damage
(18). Another potential consequence of the synthesis of
large amounts of unintegrated viral DNA is increased provirus
integration, which also may be lethal to a cell. It is also possible
that both of our proposed mechanisms may be relevant to MCF MLV-induced
apoptosis, similar to what has been observed for the subgroup B ALV
(10, 46, 47).
The role of unintegrated viral DNA in cell killing has been examined
for HIV-1, for which it was concluded that cell killing could occur in
the absence of large amounts of unintegrated viral DNA synthesis
(7, 24). In these studies, however, HIV-1 produced predominantly unintegrated closed circular molecules and hence, differs
from other cytopathic retroviruses described above. Therefore, the
mechanism of HIV-induced apoptosis may be more dependent upon viral
proteins, such as gp120, Vpr, and Tat (4, 8, 20, 38).
Further studies of MCF MLV-induced apoptosis are required to understand
the role of the unintegrated linear viral DNA and glycoprotein-receptor
interactions in this process and the role that apoptosis plays in tumorigenesis.
| |
ACKNOWLEDGMENTS |
We are grateful to R. Skoff for use of the Leica DMIRB microscope
and camera and to members of the WSU Flow Cytometry Core Facility for
processing our samples. We also thank H.-R. Kim for help in doing the
caspase-3 assays and the Center for Molecular Medicine and Genetics for
use of the phosphorimager. Comments by T. R. Reddy and Chris
Roberts on the manuscript were much appreciated.
This work was supported by Public Health Service grant CA44166 to
F.K.Y. from the National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Immunology and Microbiology, Wayne State University, 540 E. Canfield Ave., Detroit, MI 48201. Phone: (313) 577-1571. Fax: (313) 577-1155. E-mail: fyoshi{at}med.wayne.edu.
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Journal of Virology, July 2001, p. 6007-6015, Vol. 75, No. 13
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.13.6007-6015.2001
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Top
Abstract
Introduction
