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Journal of Virology, July 1999, p. 5671-5680, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Appearance of Mink Cell Focus-Inducing Recombinants
during In Vivo Infection by Moloney Murine Leukemia Virus (M-MuLV)
or the Mo+PyF101 M-MuLV Enhancer Variant: Implications for Sites
of Generation and Roles in Leukemogenesis
Jeffrey K.
Lander,1
Bruce
Chesebro,2 and
Hung
Fan1,*
Department of Molecular Biology and
Biochemistry and Cancer Research Institute, University of California,
Irvine, California 92697,1 and
Laboratory of Persistent Viral Diseases, Rocky Mountain
Laboratories, National Institute of Allergy and Infectious
Diseases, Hamilton, Montana 598402
Received 20 January 1999/Accepted 9 April 1999
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ABSTRACT |
One hallmark of murine leukemia virus (MuLV) leukemogenesis in mice
is the appearance of env gene recombinants known as mink cell focus-inducing (MCF) viruses. The site(s) of MCF recombinant generation in the animal during Moloney MuLV (M-MuLV) infection is
unknown, and the exact roles of MCF viruses in disease induction remain
unclear. Previous comparative studies between M-MuLV and an enhancer
variant, Mo+PyF101 MuLV, suggested that MCF generation or early
propagation might take place in the bone marrow under conditions of
efficient leukemogenesis. Moreover, M-MuLV induces disease efficiently
following both intraperitoneal (i.p.) and subcutaneous (s.c.)
inoculation but leukemogenicity by Mo+PyF101 M-MuLV is efficient
following i.p. inoculation but attenuated upon s.c. inoculation. Time
course studies of MCF recombinant appearance in the bone marrow,
spleen, and thymus of wild-type and Mo+PyF101 M-MuLV i.p.- and
s.c.-inoculated mice were carried out by performing focal
immunofluorescence assays. Both the route of inoculation and the
presence of the PyF101 enhancer sequences affected the patterns of MCF
generation or early propagation. The bone marrow was a likely site of
MCF recombinant generation and/or early propagation following i.p.
inoculation of M-MuLV. On the other hand, when the same virus was
inoculated s.c., the primary site of MCF generation appeared to be the
thymus. Also, when Mo+PyF101 M-MuLV was inoculated i.p., MCF generation
appeared to occur primarily in the thymus. The time course studies
indicated that MCF recombinants are not involved in preleukemic changes such as splenic hyperplasia. On the other hand, MCFs were detected in
tumors from Mo+PyF101 M-MuLV s.c.-inoculated mice even though they were
largely undetectable at preleukemic times. These results support a role
for MCF recombinants late in disease induction.
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INTRODUCTION |
Moloney murine leukemia virus
(M-MuLV) is a simple nonacute retrovirus that induces T-lymphoblastic
lymphoma in NIH Swiss mice. A frequent event during the development of
MuLV-induced leukemias is the formation of mink cell focus-inducing
(MCF) recombinant viruses (18). MCF viruses arise by
recombination in vivo between the env gene of an infecting
MuLV and endogenous polytropic or modified polytropic MuLV proviruses
present in virtually all mice (33). MCF envelope protein
binds to cells via a different receptor from that used by the original
ecotropic MuLVs (25). Several lines of evidence suggest that
MCF recombinants play important roles in MuLV-induced leukemogenesis in
mice, but the exact role(s) remains unclear (15). For
example, MCF recombinants may carry out late functions, including long
terminal repeat (LTR) activation of proto-oncogenes (19),
activation of cytokine receptors (21), and additional rounds
of superinfection of cells already infected with ecotropic MuLV
(20).
MCF recombinants may also be important early during leukemogenesis by
M-MuLV, as suggested by comparative studies of M-MuLV and an
enhancer variant of M-MuLV, Mo+PyF101 M-MuLV (15).
Mo+PyF101 M-MuLV contains enhancer sequences from the F101 strain of
polyomavirus inserted downstream from the M-MuLV enhancers
(23). Leukemogenicity of Mo+PyF101 M-MuLV is dependent on
the route of inoculation: Mo+PyF101 M-MuLV inoculated subcutaneously
(s.c.) is poorly leukemogenic, while Mo+PyF101 M-MuLV inoculated
intraperitoneally (i.p.) results in disease kinetics comparable to
those induced by wild-type M-MuLV (1, 6, 10). In contrast,
M-MuLV induces leukemia efficiently with the same kinetics regardless
of the route of inoculation (1). Experiments with Mo+PyF101
M-MuLV indicated that early high-level infection of the thymus (the
ultimate site of tumor formation) apparently was not required for
efficient disease induction by M-MuLV. Instead, efficient disease
induction was correlated with efficient early infection of the bone
marrow and the appearance of MCF recombinants (1, 2). Since
early bone marrow infection was well correlated with the development of
MCF recombinants, this suggested that infection of bone marrow might be
required for recombinant MCF virus formation. The association also
suggested that MCF recombinant formation and/or initial propagation may take place in the bone marrow. In addition, studies with Mo+PyF101 M-MuLV suggested that MCF recombinants might be involved in the establishment of preleukemic changes such as generalized hematopoietic hyperplasia in the spleen, defects in bone marrow hematopoiesis, and
accelerated thymic atrophy (11, 12, 22).
The goals of this study were to characterize the initial sites of MCF
recombinant appearance in mice following M-MuLV and Mo+PyF101 M-MuLV
infection. Such a survey might suggest the sites of MCF recombinant
formation and propagation; it might also address the relevance of MCF
recombinant generation and propagation to leukemogenesis.
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MATERIALS AND METHODS |
Mice and viruses.
NIH Swiss mice (Hsd:NIHSThe mice) were
purchased from Harlan Sprague Dawley, Inc. (Indianapolis, Ind.) and
maintained as an outbred colony. Viral stocks were clarified cell
culture supernatants derived from NIH 3T3 fibroblasts productively
infected with either wild-type M-MuLV (22) or the Mo+PyF101
M-MuLV enhancer variant (23); portions of viral stocks were
individually frozen at 
70°C and were thawed and used only once
(17).
Virus and inoculation of NIH Swiss mice.
Virus infectivity
titer determinations were performed by both the UV-XC syncytial assay
(26) and the focal immunofluorescence assay (FIA)
(31) on NIH 3T3 cells. Neonatal NIH Swiss mice were inoculated either intraperitoneally (i.p.) or subcutaneously (s.c.) with 0.20 ml of virus stock (1.5 × 105 XC PFU, 3 × 105 FIU) within 48 h of birth.
MAbs.
The monoclonal antibodies (MAbs) used in this study
were MAb 514 and MAb 538. MAb 514 is specifically reactive with the SU proteins of polytropic (and modified polytropic) MuLVs, including the
two antigenic subclasses of M-MCF (7, 13). MAb 538 is specifically reactive with the SU protein of M-MuLV. MAb 514 and MAb
538 hybridoma cells were cultured in RPMI (28) supplemented with 10% fetal bovine serum. Cultures were grown to 106
cells/ml, and Ab-containing supernatants were collected 5 to 7 days
thereafter and used for infectious-center assays (see below).
MAb 538 was derived from the spleen of a (B10.A × A)F1 mouse immunized by intravenous inoculation with tissue
culture supernatant containing the M-MuLV-spleen focus-forming virus
(SFFV) complex. At 75 days after infection, this mouse was given an
intravenous booster immunization with 3 × 107 spleen
cells from the enlarged leukemic spleen of a (BALB/c × A)F1 mouse previously infected with the M-MuLV-SFFV
complex. At 16 days later, the spleen cells of the immunized mouse were
fused to NS1 cells to generate hybridomas as described previously
(7). Positive hybridoma wells were identified by indirect
membrane immunofluorescence on live trypsinized NIH 3T3 cells
chronically infected with M-MuLV. Ethanol-fixed cells gave negative
results. Hybridoma cells were cloned by limiting dilution, and
supernatant fluid was characterized for Ab specificity and
immunoglobulin (Ig) class. MAb 538 was an IgM antibody with reactivity
for only M-MuLV, and molecular clones 8.2 and 1387 were both positive. Over 30 other MuLVs were negative with this MAb, including ecotropic Friend, Rauscher and AKV MuLVs and numerous MuLVs in the amphotropic, xenotropic, and polytropic host range groups. Although MAb 538 was
unable to immunoprecipitate viral proteins efficiently or react in
Western blots, it was specific for viral envelope protein based on its
reactivity with the FMF chimeric MuLV, which has only the
SphI-ClaI env gene fragment derived
from M-MuLV (29).
Assays for infectious virus.
The presence of infectious
virus in hematopoietic organs was determined by infectious-center (IC)
assays on NIH 3T3 cells by the focal-immunofluorescence assay (FIA)
(31). Infected mice and uninfected control mice were
sacrificed at the times indicated, and single-cell suspensions were
prepared from their bone marrow, spleens, and thymuses. Bone marrow
cells were obtained by using a 1-ml syringe fitted with a 25G5/8 or
23G1 needle to flush each femur and tibia with sterile
phosphate-buffered saline (PBS). Spleen and thymus tissue was removed
and rinsed gently with sterile PBS. Cells were recovered by passing
tissue through a 94-mm 150-mesh screen (Bellco). The cells were washed
with PBS, and their viability and concentration were determined by
trypan blue exclusion. Tenfold serial dilutions of cell suspensions
were cocultivated for 24 h with NIH 3T3 cells (7 × 104 to 8 × 104 cells per 60-mm dish
seeded 1 day previously) in Dulbecco's modified Eagle's medium
containing 10% calf serum (CS) and 2 µg of Polybrene per ml.
Following cocultivation, the nonadherent hematopoietic cells were
aspirated, the NIH 3T3 monolayers were washed, the growth medium was
replaced with Dulbecco's modified Eagle's medium-10% CS, and the
cells were allowed to grow to confluency (4 to 5 days). The cultures
were then incubated for 30 min at room temperature with 0.25 ml of
undiluted MAb 538- or MAb 514-containing hybridoma supernatant, washed
twice with PBS-2% CS, and then treated for 30 min and room
temperature with fluorescein isothiocyanate (FITC)-conjugated goat
anti-mouse immunoglobulin G (IgG, IgA, and IgM [ICN Pharmaceuticals]) diluted 1:200 in PBS-2% CS. The cultures were then washed twice, and
infectious centers were visualized by fluorescence microscopy and counted.
DNA isolation and Southern blot hybridization.
DNA was
extracted from single-cell suspensions or cell lines by a modification
of standard methods (8) and suspended in 10 mM Tris-0.1 mM
EDTA (pH 8). Restriction endonuclease digestion of
high-molecular-weight DNA, gel electrophoresis, transfer, and hybridization were performed as previously described (2, 4, 6). DNA fragments used as radioactive probes for Southern blot analyses included the 199-bp PyF101 enhancer-containing
PvuII-4 fragment inserted at the XbaI site of
Mo+PyF101 M-MuLV as previously described (23); the 620-bp
BamHI-EcoRI fragment from the 5' env
region of the M-MCF recombinant that is reactive with the M-MCF
recombinant and endogenous polytropic and modified polytropic viruses
(6), and the 1.1-kb BamHI-ClaI
fragment from the 3' env region of M-MuLV that is reactive
with both ecotropic M-MuLV and polytropic M-MCF recombinants
(4). Random primer [32P]dATP- (or
[32P]dCTP)-labeled probes were prepared by using the
DECAprime II DNA labeling kit (Ambion, Austin, Tex.) as specified by
the manufacturer. When indicated, the probes were stripped from the
membranes by incubation at 95 to 100°C in 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1% sodium dodecyl sulfate for 40 to 60 min with gentle agitation and the membranes were rehybridized as
described above.
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RESULTS |
MCF recombinant appearance and propagation in M-MuLV
i.p.-inoculated mice.
The site(s) of MCF recombinant
generation during M-MuLV infection is unknown. Our previous studies
suggested that the bone marrow might be involved in MCF virus formation
or propagation (2, 6), but other candidate hematopoietic
compartments included the spleen and thymus. High levels of ecotropic
virus infection are established in all three tissue compartments at
early times postinfection (<14 days) (1), and MCF
recombinants have been detected in the spleen and thymus of
M-MuLV-infected mice as early as 3-4 weeks, although the bone marrow
was not examined (14, 20). Our initial strategy to identify
the site(s) of MCF recombinant generation was to search different
tissues for the earliest appearance of MCF recombinants.
Immediately after MCF recombinant formation and initial propagation, it
is likely that only a small fraction of cells will
express MCF virus.
However, at later times, MCF recombinants will
have amplified in vivo
by several orders of magnitude. We evaluated
the sensitivity, range of
detection, and reproducibility of several
different methods of MCF
recombinant detection including Southern
blot hybridization
(
32), PCR amplification (
24,
27,
28),
and IC
assays (FIAs) (
31). FIA proved to be the most sensitive
and
reliable technique, allowing measurements of MCF virus ICs
over a range
of 6 to 7 orders of magnitude (0.00002 to 100% of
cells infected).
Another advantage of the FIA was that it measured
infectious MCF
recombinants.
We infected neonatal mice i.p. with M-MuLV and quantified the
appearance of MCF virus-infected cells in the bone marrow, spleens,
and
thymuses of infected mice during the preleukemic period by
measuring
MAb 514-reactive ICs by FIA of cells from these hematopoietic
compartments. MAb 514 is specifically reactive with the SU proteins
of
polytropic (and modified polytropic) MuLVs, including the two
antigenic
subclasses of recombinant MCFs that appear during M-MuLV
infection
(
7,
13). We also measured the appearance of ecotropic
M-MuLV-infected cells in the same tissues by FIA with MAb 538.
MAb 538 is specifically reactive with the ecotropic SU protein
of M-MuLV. As
shown in Fig.
1A, mice inoculated i.p.
with M-MuLV
showed a rapid appearance of ecotropic ICs in the bone
marrow,
spleen, and thymus. The appearance of M-MuLV ecotropic ICs in
the thymus was slightly delayed compared to that in bone marrow
and
spleen: maximum numbers of ecotropic ICs were detected in
the bone
marrow and spleen by 7 to 10 days, in contrast to 13
to 15 days for the
thymus. At later times, the numbers of ecotropic
ICs in the thymus were
somewhat higher (approximately 5- to 10-fold)
than in the bone marrow
and spleen. These results were consistent
with previous reports on
M-MuLV-infected mice (
1,
14).
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FIG. 1.
Numbers of ecotropic and MCF recombinant ICs in
preleukemic mice inoculated i.p. with M-MuLV. Neonatal mice were
inoculated i.p. with 1.5 × 105 XC PFU (3 × 105 FIU) of M-MuLV; bone marrow cells, splenocytes, and
thymocytes were plated onto NIH 3T3 fibroblasts; and the ICs were
quantified by FIA as described in Materials and Methods. The results
are plotted as the number of MAb 538 (ecotropic) or MAb 514 (MCF
recombinant)-reactive ICs per 106 cells as a function of
age. MAb 538-reactive (ecotropic) IC numbers (A) and MAb 514-reactive
(MCF recombinant) IC numbers (B) from bone marrow cells, splenocytes,
and thymocytes are shown. All datum points represent the IC number from
an individual mouse. Datum points resting on the x axis
indicate animals with an undetectable IC number (<0.2 IC per
106 cells plated).
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Figure
1B displays the appearance of MCF virus ICs in M-MuLV
i.p.-infected mice. MCF virus ICs were detected beginning 13
to 22 days
postinoculation. At very early times, the fractions
of cells scoring
positive for polytropic ICs in each tissue were
generally very low (0.2 to 10 per million). By 24 days postinfection,
MCF virus ICs were
detected in all three hematopoietic compartments
in all animals. Thus,
they had been generated and disseminated
by that time. If MCF virus
generation took place in only one tissue,
propagation to the other two
compartments occurred
rapidly.
In mice infected i.p. with M-MuLV, the numbers of MCF virus ICs reached
plateau levels by 4 to 5 weeks, most probably reflecting
a rapid
amplification of MCF virus infection between 3 and 5 weeks.
It was
noteworthy that the maximal numbers of polytropic ICs were
substantially larger (10- to 100-fold) in the thymus than in the
bone
marrow and spleen. Thus, MCF recombinant amplification was
most
efficient in the
thymus.
Sites of the earliest appearance of MCF recombinants following i.p.
inoculation.
The patterns of ecotropic and polytropic ICs shown in
Fig. 1 did not readily suggest a site for MCF recombinant generation, since high-level ecotropic infection was established in all tissues before MCF recombinant ICs were detected in any of them. Furthermore, MCF recombinants were amplified in the bone marrow, spleen, and thymus
with similar rapid kinetics. In most animals tested at 1 to 4 weeks,
MCF recombinants were either absent from all three hematopoietic
compartments or present in all of them. However, in a subset of
animals, MCF recombinants were detected in one or two but not all three
compartments. Thus, MCF recombinants apparently had not spread
completely in these animals. The MCF recombinant IC profiles in these
animals might suggest where MCF recombinant were generated.
Table
1 summarizes the polytropic IC data
from the animals that were "discordant" for detectable MCF
recombinants, i.e.,
those that showed MCF recombinant ICs in at least
one but not
all tissues. MCF recombinant ICs were detected in the bone
marrow
in 14 of 15 discordant animals. In three animals, only the bone
marrow sample showed detectable ICs. In contrast, only 4 of 15
discordant animals showed ICs in the spleen, although in 1 animal
they
were detected in only the spleen. An intermediate fraction
of
discordant animals (8 of 15) showed detectable ICs in the thymus;
however, none of these 8 animals showed ICs only in the thymus.
These
findings were consistent with the possibility that the bone
marrow was
the primary site of MCF recombinant generation.
MCF recombinant appearance and propagation in M-MuLV
s.c.-inoculated mice.
Previous studies of wild-type and
Mo+PyF101 M-MuLV indicated that the route of inoculation (i.p.
versus s.c.) affected the efficiency of Mo+PyF101 M-MuLV-induced
leukemogenesis but not that of M-MuLV-induced leukemogenesis
(1). Since leukemogenesis by M-MuLV is not dependent on the
route of inoculation, studying MCF appearance in mice infected either
i.p. or s.c. with M-MuLV might reveal common features important for
efficient leukemogenesis.
We infected neonatal mice with M-MuLV s.c. and measured the appearance
of polytropic and ecotropic reactive ICs. Relative
to i.p. inoculation,
s.c. inoculation resulted in a slight delay
in establishment of
ecotropic-virus infection in the bone marrow
and spleen but had little
effect on establishment of infection
in the thymus (Fig.
2A). We first detected MCF ICs at 19 to
28
days postinoculation, 1 week later than for i.p.-inoculated mice
(Fig.
2B). Polytropic ICs were first detected in bone marrow cells
after 24 days, versus 19 days in splenocytes and thymocytes. As
in
M-MuLV i.p.-inoculated animals, the numbers of polytropic ICs
in each
tissue at very early times were generally very small (0.2
to 10 per
million cells plated). By 30 days postinfection, MCF
recombinant ICs
were detected in all three tissues in all animals,
indicating general
dissemination by that time. MCF recombinant
ICs were detected 1 to 2 weeks later than when ecotropic M-MuLV
IC numbers plateaued, and they
amplified rapidly regardless of
the inoculation route.
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FIG. 2.
s.c. inoculation of M-MuLV. Neonatal mice were
inoculated s.c. with M-MuLV, and the numbers of ecotropic and
polytropic ICs were measured as described in the legend to Fig. 1. The
data for s.c.-infected mice are shown in the large shaded circles. For
comparison, data from Fig. 1 (i.p.-infected animals) are shown in the
small solid circles.
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The apparent 3- to 5-day delay in the appearance of MCF recombinants in
the bone marrow with respect to the splenocytes and
thymocytes of
s.c.-inoculated mice suggested that they might form
in some tissue
other than bone marrow. Results for s.c.-inoculated
animals discordant
for MCF virus appearance are shown in Table
2. MCF recombinant ICs were detected in
the bone marrow in only
two of eight discordant animals; furthermore,
none of the discordant
animals tested showed MCF recombinants only in
bone marrow cells.
Four of eight discordant animals showed detectable
MCF recombinant
ICs in the spleen, and none showed infection only in
the spleen.
In contrast, all eight discordant animals showed MCF
recombinant
ICs in the thymus and two of these animals showed MCF
recombinant
ICs only in the thymus. In mouse 8 (Table
2), a substantial
number
of MCF recombinant ICs was measured in the thymus but none were
detected in the bone marrow or spleen. These results suggested
that a
site other than the bone marrow (perhaps the thymus) might
function as
an MCF recombinant generation site following s.c.
inoculation.
MCF virus appearance in Mo+PyF101 M-MuLV i.p.-inoculated mice.
Since wild-type and Mo+PyF101 M-MuLV induce disease with similar
efficiencies following i.p. inoculation (1), comparison of
MCF recombinant appearance in mice infected i.p. by these two viruses
was of interest. As shown in Fig. 3A,
i.p. inoculation of Mo+PyF101 M-MuLV resulted in a slight delay
relative to M-MuLV i.p. inoculation in the establishment of ecotropic
virus infection in each tissue, but the delay was more evident in the
thymus, consistent with results of previous studies (1). At
later times, the numbers of ecotropic ICs in the thymus were somewhat
larger than in the bone marrow and spleen, but the difference was not as great as in M-MuLV i.p.-inoculated mice.
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FIG. 3.
Inoculation of Mo+PyF101 M-MuLV. Neonatal mice were
inoculated i.p. with Mo+PyF101 M-MuLV, and the numbers of ecotropic and
polytropic ICs were measured as described in the legend to Fig. 1
(solid triangles). Data from mice inoculated s.c. by Mo+PyF101 M-MuLV
are shown in shaded triangles. Note that the timescale in this figure
is different than in Fig. 1 and 2. Any animals that developed leukemia
were excluded from the analysis.
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Figure
3B shows the appearance of MCF recombinant ICs in cells of
Mo+PyF101 i.p.-infected mice. Polytropic ICs were first
detected after
40 days on average (compared to 13 days for M-MuLV
i.p.-infected mice),
and the time of appearance was also more
variable than that for
M-MuLV-infected mice. In addition, not
all animals showed MCF
recombinant ICs: only two-thirds of the
animals tested between 39 and
96 days postinoculation showed detectable
MCF recombinant ICs, and only
one-fourth of the animals at these
times showed MCF recombinant ICs in
all three tissues. Finally,
MCF recombinant IC titers measured in the
bone marrow and spleen
were 100- to 1,000-fold lower than in M-MuLV
i.p.-infected mice.
Thus, either MCF virus generation occurred much
later or propagation
of MCF recombinants to detectable levels was less
efficient. As
in M-MuLV-infected animals, however, MCF recombinant
amplification
was most efficient in the thymus in Mo+PyF101
i.p.-infected mice
(generally 100-fold higher levels than in the bone
marrow and
spleen). The delay in appearance of MCF recombinants was
noteworthy,
since previous experiments had suggested an early role for
MCF
recombinants in efficient leukemogenesis by Mo+PyF101 M-MuLV
(
2,
6,
22).
We also studied animals discordant for MCF virus appearance following
i.p. inoculation with Mo+PyF101 M-MuLV (Table
3). Only
one discordant animal showed
detectable MCF recombinant ICs in
bone marrow. MCF recombinant ICs were
detected in the spleens
in six of nine and in the thymuses in eight of
nine discordant
animals. In three mice, appreciable MCF recombinant IC
numbers
were detected only in the thymus.
MCF recombinant proviruses in Mo+PyF101 M-MuLV i.p.-inoculated
mice.
The delay in appearance and the inefficient amplification of
MCF recombinants in Mo+PyF101 M-MuLV i.p.-inoculated mice was unexpected from our previous experiments (2, 6). Although unlikely, it was possible that Mo+PyF101 M-MuLV-infected mice developed
significant levels of polytropic viruses that did not score as MAb
514-reactive ICs by FIA. To rule out this possibility, we tested DNAs
from six Mo+PyF101 M-MuLV i.p.-infected preleukemic mice for
recombinant MCF proviruses by Southern blot analysis (2, 4).
As shown in Fig. 4, digestion of the DNA
with BamHI and ClaI and hybridization with a 3'
env probe yielded a 1.1-kb fragment diagnostic for ecotropic
provirus and a 1.9-kb fragment diagnostic for MCF provirus (Fig. 4A).
The 1.9-kb MCF recombinant-specific band could be detected in DNAs from
Mo+PyF101 M-MuLV- and M-MuLV-infected tissue that showed MCF
recombinant IC titers of >103 per million cells in most
cases (Fig. 4B). Three additional preleukemic Mo+PyF101 M-MuLV
i.p.-infected animals (28, 56, and 70 days postinoculation) were
tested, and similar results were observed: ecotropic but not polytropic
provirus-specific bands were detected, and the numbers of MCF
recombinant ICs were less than 1,000/106 cells.
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FIG. 4.
Southern blot hybridization for ecotropic and polytropic
proviruses. (A) Restriction endonuclease cleavage site maps of M-MuLV
and Mo+PyF101 M-MuLV (top) and M-MCF and Mo+PyF101 M-MCF recombinant
(bottom) proviruses. Only the BamHI and ClaI
sites pertinent to the analysis are shown. The heavy bar indicates the
probe. The box in the env gene shows the portion involved in
the recombination. Digestion of proviral DNA with
BamHI-ClaI yields a 1.1-kb fragment from the
ecotropic M-MuLV provirus and a 1.9-kb fragment from the polytropic MCF
recombinant provirus; both fragments hybridize with an ecotropic 3'
env probe. (B) High-molecular-weight DNA was prepared from
bone marrow cells (lanes B), splenocytes (lanes S), and thymocytes
(lanes T) from three mice inoculated with Mo+PyF101 M-MuLV i.p. and two
mice inoculated with M-MuLV. The ages of the animals are indicated. A
10-µg sample of each DNA was digested with
BamHI-ClaI, separated by agarose gel
electrophoresis, and subjected to Southern blot hybridization with the
3' env probe shown in panel A. Lanes: 1, M-MuLV producer
cell line DNA; 2, Mo+PyF101 M-MuLV producer cell line DNA; 3, NIH 3T3
cell line DNA; 4, uninoculated control thymus DNA; 5 to 13, DNAs from
Mo+PyF101 M-MuLV-infected tissue; 14 to 19, DNAs from M-MuLV-infected
tissue. Continuous lines above the lanes indicate tissues from the same
animal. The tissue samples were also tested by FIA, and the numbers of
ecotropic and MCF recombinant ICs per 106 cells plated are
indicated below lanes 4 to 19. bdl: below the detection limit of the
assay (<0.2 MCF recombinant ICs/106 cells).
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MCF recombinants in tumors of Mo+PyF101 M-MuLV s.c.-inoculated
mice.
In previous studies we did not detect MCF recombinants by
Southern blot analysis in Mo+PyF101 M-MuLV s.c.-infected mice at preleukemic times or in end-stage tumors if they developed
(6). We infected neonatal mice with Mo+PyF101 M-MuLV s.c.
and tested them for MCF recombinant and ecotropic ICs at late
preleukemic times and in animals that developed tumors (Fig. 3). Of 20 animals tested at times from 39 to 148 days, 4 had developed large
thymic tumors and other characteristic signs of Mo+PyF101
M-MuLV-induced disease. Of the remaining 16 animals, 12 showed
undetectable numbers of MCF recombinant ICs in all tissues. However,
three showed very small but detectable numbers of MCF recombinant ICs
in one or two tissue compartments (0.2 × 106 to
3/106 cells) and one showed substantial numbers of MCF
recombinant ICs in the thymus (2 × 103/106 cells). The MCF recombinant IC results
on the preleukemic animals were generally consistent with our previous
studies (2) in that the percentage of animals that showed
detectable MCF recombinant ICs was much lower following s.c. than i.p. inoculation.
Somewhat unexpectedly, we detected large numbers of MCF ICs in the
thymic tumors (5 × 10
2 to 2 × 10
5/10
6 thymocytes) and in some instances
significant numbers in the
bone marrow and spleens (7 × 10
4/10
6 cells) of the four leukemic Mo+PyF101
M-MuLV s.c.-inoculated
mice. If MCF recombinant IC numbers and the MCF
proviral content
in these animals obeyed the same correlation as
observed in Fig.
4, Southern blot analysis would be sensitive enough to
detect
MCF recombinant proviruses in these tumor DNAs. These four
tumors
and six additional Mo+PyF101 M-MuLV s.c.-induced tumors from
previous
experiments were tested for MCF proviruses by Southern blot
hybridization
(Fig.
5). The 1.9-kbMCF
recombinant-specific
BamHI-
ClaI fragment
was
readily detected in 7 of 10 tumor DNAs from mice inoculated
s.c. with
Mo+PyF101 M-MuLV. A faint 1.9-kb hybridizing band was
evident in one
additional DNA (Fig.
5, lane 15), and no MCF provirus-specific
band was
visualized in two tumor DNAs. However, one of these DNAs
(lane 13) was
partially degraded in this experiment and was judged
MCF recombinant
positive in two additional Southern blot assays.
In sum, 9 of the 10 tumor DNAs contained MCF proviruses at levels
detectable by Southern
blot hybridization. Thus, the experiments
on Mo+PyF101 s.c.-inoculated
animals indicated that MCF recombinant
formation and propagation at
preleukemic times was substantially
reduced or undetectable.
Nevertheless, the majority of the animals
that developed tumors showed
infection by MCF recombinants. For
these animals, MCF recombinant
formation was correlated with a
late step in leukemogenesis.
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|
FIG. 5.
M-MCF proviruses in tumor DNAs from Mo+PyF101 M-MuLV
s.c.-inoculated mice. (A) Restriction endonuclease cleavage site maps
of the M-MuLV (top) and env gene MCF recombinant (bottom)
proviruses. Only the 1.9-kb MCF recombinant-specific fragment
hybridizes with the polytropic 5' env probe. (A large
hybridizing fragment from the ecotropic M-MuLV provirus was not in the
region of the gel shown.) (B) A 10-µg sample of high-molecular-weight
DNA obtained from end-stage tumors in Mo+PyF101 M-MuLV s.c.-inoculated
mice was digested with BamHI-ClaI and analyzed by
Southern blot hybridization with the polytropic 5' env probe
shown in panel A. Lanes: 1, NIH 3T3 cell line DNA; 2, M-MuLV producer
cell line DNA; 3, Mo+PyF101 M-MuLV producer cell line DNA; 4, uninoculated control thymus DNA; 5, tumor DNA from an M-MuLV
s.c.-inoculated animal; 6 to 15, tumor DNAs from Mo+PyF101 M-MuLV
s.c.-inoculated mice; 16, cell line infected with a Mo+PyF101 MCF
recombinant molecular clone. The ages of the animals are indicated
below the lanes. Thymocytes from four tumor samples (lanes 9 to 12)
were also tested by FIA (see Materials and Methods). The MAb 514 (polytropic)-reactive IC titers were calculated as ICs per
106 cells plated and are indicated below the lane numbers.
ND, not done.
|
|
| |
DISCUSSION |
In this study, we investigated MCF recombinant appearance during
infection by M-MuLV. MCF recombinant formation in vivo most probably
requires two infection events: (i) ecotropic M-MuLV infection of a cell
expressing an endogenous replication-defective polytropic MuLV,
resulting in the production of heterozygous virions; and (ii) infection
of a second cell, in which template switching during reverse
transcription leads to formation an integrated replication-competent MCF provirus (5, 9). The latter cell would produce MCF
recombinant virions (or ecotropic-pseudotyped MCF recombinants if it
was also infected by M-MuLV). Cells that score as MCF recombinant ICs
reflect the second cells involved in MCF recombinant generation or
cells that subsequently propagate MCF recombinants. While MCF
recombinant IC data cannot distinguish between "generator" and
"propagator" cells, in animals where MCF recombinant have not
disseminated to all tissues, MCF recombinant ICs will most probably
reflect those involved in generation or the earliest stages of propagation.
For mice infected i.p. by M-MuLV, the fact that ecotropic M-MuLV
infection established in the bone marrow and spleen more rapidly than
in the thymus suggested that MCF recombinant generation might be more
likely to occur in these tissues. MCF recombinant ICs first appeared at
approximately the same time in all three tissues, i.e., beginning at 13 to 15 days. However, as shown in Table 1, in discordant animals the
bone marrow was included as a site of first appearance of MCF
recombinants in virtually every case. This supported the hypothesis
that the bone marrow is a likely site of MCF recombinant generation in
M-MuLV-infected mice (2). At the same time, these
experiments did not rule out the possibility that MCFs were generated
in multiple tissues in the same or different animals. Another
conclusion from the M-MuLV i.p.-inoculated mice was that once formed,
MCF recombinants propagate to higher levels in the thymus, i.e., 1 to 2 log units higher than in bone marrow or spleen. This was consistent
with results of previous studies (20).
It was interesting and somewhat surprising that the patterns of initial
appearance of MCF recombinants were different for mice inoculated with
M-MuLV s.c. vs. i.p. While the plateau levels of MCF recombinant
infection were similar for s.c.- and i.p.-infected mice,
s.c.-inoculated animals discordant for MCF recombinant infection did
not show patterns consistent with generation in the bone marrow. Only
25% of discordant mice showed MCF recombinant ICs in the bone marrow,
while 100% showed MCF virus infection in the thymus (Table 2). Thus,
it seems more likely that MCF recombinant generation occurs
predominantly in the thymus after s.c. inoculation by M-MuLV.
Studies with Mo+PyF101 M-MuLV provided additional insights into MCF
recombinant generation and early propagation. When this virus was
injected by the i.p. route, MCF recombinants formed, although with a
substantial delay relative to that in wild-type-M-MuLV-infected mice
(Fig. 3). The delay might reflect the fact that in general the viral
loads in animals infected i.p. by Mo+PyF101 M-MuLV were somewhat lower
than in those infected by wild-type M-MuLV. Amplification of MCF
recombinants in the Mo+PyF101 M-MuLV i.p.-infected animals occurred in
the thymus at later times, the same as for wild-type-M-MuLV-infected mice. When animals discordant for MCF virus infection were analyzed, the patterns favored generation and initial propagation primarily in
the thymus, not in the bone marrow (Table 3). Thus, the nature of the
M-MuLV enhancer sequences appeared to affect the site(s) of MCF
recombinant generation, even when the same route of inoculation was
used. When mice were inoculated with Mo+PyF101 M-MuLV by the s.c. route
(where they show attenuated leukemogenicity), very few preleukemic
animals showed MCF recombinants even by the sensitive FIA, consistent
with results of our previous studies (6).
Another goal of these experiments was to test the hypothesis that MCF
recombinants are important for early (preleukemic) events in M-MuLV
leukemogenesis. We previously suggested that MCF recombinants are
involved in preleukemic M-MuLV-induced events such as
splenic hyperplasia, bone marrow hematopoietic defects, and accelerated thymic atrophy (15). The detailed analyses carried out here indicate that this hypothesis is unlikely to be correct. While MCF
recombinants first appeared in M-MuLV-infected mice well before the
preleukemic events (13 to 15 days), the levels of infection were low,
i.e., plateau levels of 103 to 104 ICs per
106 cells (0.1 to 1% MCF recombinant infection) in the
bone marrow and spleen. Thus, in these tissues that were undergoing
substantial preleukemic changes, most of the cells were not infected by
MCF recombinants.
The lack of a role for MCF recombinants in M-MuLV-induced preleukemic
changes was further substantiated by the studies on mice infected
by Mo+PyF101 M-MuLV. Mo+PyF101 M-MuLV i.p.-infected animals develop
leukemia with the same efficiency as wild-type-M-MuLV-infected mice
and show the same preleukemic changes (1). The substantial delay in the appearance of MCF recombinants in Mo+PyF101 M-MuLV i.p.-infected mice was not reflected in a delay of preleukemic events.
Moreover, several individual animals that showed clear evidence of
preleukemic splenomegaly had no detectable MCF recombinants. Similarly,
several mice infected by Mo+PyF101 M-MuLV that showed preleukemic
thymic atrophy showed no detectable MCF recombinants in the thymus
(3). A similar lack of correlation between MCF recombinants
and induction of splenomegaly (erythroid hyperplasia) induced by Friend
MuLV has been reported (30). However, in the experiments
described here, only the nonadherent cells from the spleen and thymus
were analyzed. If MCF recombinant infection of adherent (stromal)
elements led to preleukemic changes, this might have escaped detection.
On the other hand, for the bone marrow, all of the cells (including
stromal cells) were analyzed, but it is still possible that MCF virus
infection of a minority subset of cells in the bone marrow is important.
At the same time, these experiments provide strong support for a role
of MCF recombinants in later steps in leukemogenesis. In particular, in
mice inoculated with Mo+PyF101 M-MuLV by the s.c. route, the great
majority that did not have tumors had undetectable or extremely small
numbers of MCF recombinant ICs. Nevertheless, most of the tumors in the
individuals that developed leukemia were MCF virus infected. Thus, for
these animals, there was a strong selection for MCF recombinants in the
tumors. The appearance of MCFs may have been the final
rate-limiting event for disease development. The overall reduced
rate of leukemogenesis by Mo+PyF101 M-MuLV after s.c. inoculation may
have resulted from both the low levels of MCF recombinants formed and a
lower level of ecotropic virus load (compare Fig. 3 with Fig. 2).
As mentioned in Results, we previously did not detect MCF recombinants
in Mo+PyF101 M-MuLV s.c.-infected tumors by Southern blot analysis
(6), whereas in the present experiments we did. This was
probably due to the restriction endonucleases used. Previously, a
BamHI-XbaI digest was used to test for a 2.3-kb
MCF virus-diagnostic fragment that hybridized to an MCF virus
env-specific probe (6). The diagnostic fragment
was only slightly smaller than restriction fragments corresponding to
endogenous MuLV proviruses in NIH Swiss mice. In retrospect, two
classes of LTR changes could have resulted in failure to detect the MCF
virus-diagnostic BamHI-XbaI fragment. Multimerization of M-MuLV enhancer sequences in the Mo+PyF101 M-MuLV
LTR could result in comigration of the diagnostic MCF virus fragment
with an endogenous MuLV-related fragment. Alternatively, deletion of
polyomavirus enhancer sequences and the adjacent XbaI site
could have converted the MCF virus-diagnostic fragment to viral-cellular junction fragments of indeterminate size. In subsequent studies, we observed both classes of LTR alterations (2, 4). The BamHI-ClaI restriction digestion used in
these experiments is not influenced by alterations in the Mo+PyF101
M-MuLV LTR. Indeed, several of the previously isolated tumor samples
illustrated in Fig. 5 that showed the MCF virus-diagnostic 1.9-kb
BamHI-ClaI fragment had been previously
classified as MCF virus negative on the basis of the
BamHI-XbaI digest (6). Additional
Southern blot digestions confirmed that the Mo+PyF101 M-MuLV
s.c.-induced tumors had LTR rearrangements consisting of either or
deletions or expansions of the enhancer sequences (results not shown).
In summary, several conclusions resulted from these studies. First,
both the route of inoculation and the nature of the enhancers appeared
to affect the sites of MCF recombinant generation and/or early
propagation in M-MuLV-infected mice. Second, the initial site(s) of MCF
recombinant appearance does not appear to affect the efficiency of
leukemogenesis. Third, MCF recombinants do not appear to be involved in
establishing M-MuLV-induced preleukemic changes. Finally, MCF
recombinants are more likely to be involved in late steps in
leukemogenesis such as activation of proto-oncogenes, induction of
cytokine autocrine loops, and activation of tumor progression genes
(15, 16).
| |
ACKNOWLEDGMENTS |
We thank Christine Lee, Tina Pham, Jeremy Shaw, and Grace Lee for
technical assistance.
J.K.L. was supported by NIH training grants T32 GM 07134 and T32
CA09054. This work was supported by grant RO1 CA-32455 from the
National Cancer Institute. Support by the UCI Cancer Research Institute
and the UCI Cancer Center is acknowledged.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Biochemistry, University of California, Irvine, CA 92697. Phone: (949) 824-5554. Fax: (949) 824-4023. E-mail: HYFAN{at}UCI.EDU.
| |
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Journal of Virology, July 1999, p. 5671-5680, Vol. 73, No. 7
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Abstract
Introduction
