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Journal of Virology, April 2000, p. 3177-3187, Vol. 74, No. 7
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
DNA Methylation of Helper Virus Increases Genetic
Instability of Retroviral Vector Producer Cells
Won-Bin
Young,1,2,
Gary L.
Lindberg,3 and
Charles J.
Link Jr.1,2,*
Human Gene Therapy Research Institute, John
Stoddard Cancer Center, Des Moines, Iowa 50309,1
and Molecular, Cellular and Developmental Biology
Program2 and Department of Animal
Science,3 Iowa State University, Ames, Iowa
50011
Received 21 October 1999/Accepted 5 January 2000
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ABSTRACT |
Retroviral vector producer cells (VPC) have been considered
genetically stable. A clonal cell population exhibiting a uniform vector integration pattern is used for sustained vector production. Here, we observed that the vector copy number is increased and varied
in a population of established LTKOSN.2 VPC. Among five subclones of
LTKOSN.2 VPC, the vector copy number ranged from 1 to approximately 29 copies per cell. A vector superinfection experiment and Northern blot
analysis demonstrated that suppression of helper virus gene expression
decreased Env-receptor interference and allowed increased
superinfection. The titer production was tightly associated with helper
virus gene expression and varied between 0 and 2.2 × 105 CFU/ml in these subclones. In one analyzed subclone,
the number of integrated vectors increased from one copy per cell to
nine copies per cell during a 31-day period. Vector titer was reduced from 1.5 × 105 CFU to an undetectable level. To
understand the mechanism involved, helper virus and vectors were
examined for DNA methylation status by methylation-sensitive
restriction enzyme digestion. We demonstrated that DNA methylation of
helper virus 5' long terminal repeat occurred in approximately 2% of
the VPC population per day and correlated closely with inactivation of
helper virus gene expression. In contrast, retroviral vectors did not
exhibit significant methylation and maintained consistent transcription
activity. Treatment with 5-azacytidine, a methylation inhibitor,
partially reversed the helper virus DNA methylation and restored a
portion of vector production. The preference for methylation of helper
virus sequences over vector sequences may have important implications
for host-virus interaction. Designing a helper virus to overcome
cellular DNA methylation may therefore improve vector production. The
maintenance of increased viral envelope-receptor interference might
also prevent replication-competent retrovirus formation.
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INTRODUCTION |
Retroviral vectors are the most
commonly used gene delivery vehicles in human gene transfer trials
(50). Permanent modifications of a targeted cell's genotype
by the integration of a retroviral vector and the consistent production
of retroviral vectors from permanent packaging cells (5, 32)
offer significant advantages over other gene delivery vectors. After
infection of target cells, retroviral single-stranded RNA genome is
converted into double-stranded DNA (proviral DNA) by retroviral reverse
transcriptase (RT). The proviral DNA is permanently integrated into the
cellular chromosomes as a provirus and then replicates as part of the
host genome. The proviral DNA is then transmitted from one generation
to the next if the virus is integrated into a germ line cell. The
deletion of a provirus from its integration site is observed in only
approximately 1 of 106 cells (53, 59).
A major biosafety concern with retroviral vectors is
replication-competent retrovirus (RCR) outbreak (1). RCR has
been correlated with the occurrence of T-cell lymphomas in monkeys that
had received RCR-contaminated bone marrow cell transplants (7,
58). Vector and helper virus rearrangement caused by abnormal
template switches during the reverse transcription process has been
considered the major cause of RCR formation (46, 58). Without Env-receptor interference, cocultivation of vector producer cells (VPC) with different host ranges, such as ecotropic and amphotropic VPC, can cause RCR formation in 3 to 10 days depending on
the number of recombination events necessary. The driving force for
these recombination events is RT enzyme activity imported by the vector
infection from other VPC (38).
During the vector assembly process, viral Env protein produced by the
helper virus is transported to the cell membrane, where it binds the
cellular receptor used for virus entry (31, 54, 60). This
Env-receptor interference has been considered an efficient barrier to
the superinfection of vectors. The transduction efficiency of
amphotropic vector on amphotropic PA317 packaging cells is reduced by 6 orders of magnitude, because of the amphotropic Env-receptor interference mechanism, from the transduction efficiency of the same
vector on NIH 3T3 cells (33). However, Env-receptor
interference is not a complete block to superinfection. Most retroviral
packaging cells currently used in gene transfer studies are based on
either murine cells (i.e., NIH 3T3 cells in GP+E86
[29] and PA317 [32]) or human cells
(i.e., 293T cells in BOSC [47]) and express virus receptors. Thus, these packaging cells can still be infected by the
vector they produce, albeit at low frequency (33, 36). These
vector reentry events may lead to RCR formation.
Suicide gene therapy against cancer cells by the combination of
HSVtk and ganciclovir is a well-developed approach in human gene therapy trials (37, 43). We previously established
murine LTKOSN.2 VPC to transfer the HSVtk gene by
transducing an LTKOSN vector from GP+E86 to PA317 cells and
demonstrated effective suicide gene therapy against breast cancer cells
in vitro (27), colon carcinoma in vitro and in vivo
(41), and human ovarian adenocarcinoma xenografts in vivo
(28). We have previously found an HSVtk deletion mutant vector in addition to the original LTKOSN vector in the same VPC
(Fig. 1) (62). In the current study of LTKOSN.2 VPC and
derivative subclones, we observed significant genetic instability of
several types, including increased copies of both vectors, a deleted
provirus integration site, and a complete loss of vector production. To
understand the mechanisms involved in these observations, we analyzed
the DNA methylation status and the gene expression of vectors and
helper virus. We demonstrated that transcriptional inactivation
occurred in helper virus but not vectors. Significant DNA methylation
of the helper virus 5' long terminal repeat (LTR) region was observed
in VPC subclones that lacked helper virus gene expression. These same
VPC subclones also had a loss of vector production and high vector copy
number. A superinfection experiment demonstrated that these subclones
are very susceptible to the reentry of vector, compared to the other
subclones. These findings suggest that DNA methylation may lead to VPC
genome instability by reducing Env-receptor interference and permitting
high levels of vector reentry.
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MATERIALS AND METHODS |
Generation of LTKOSN.2 VPC and derived subclones.
The
construction of pLTKOSN and LTKOSN.2 VPC has been previously described
(28). Briefly, 1,201 bp of PCR-amplified HSVtk gene was cloned into the EcoRI site of pLXSN (35)
(kindly provided by A. Dusty Miller, Fred Hutchinson Cancer Research
Center, Seattle, Wash.) to produce pLTKOSN retroviral vector
(Fig. 1A). Ecotropic packaging cell line GP+E86 (29), a
generous gift from Arthur Bank, Columbia University, New York, N.Y.,
was transiently transfected with pLTKOSN. Supernatants from these
GP+E86 cells were used to transduce the amphotropic retroviral
packaging line PA317 (32), kindly provided by A. Dusty
Miller. Forty different VPC clones were isolated, and the LTKOSN.2 VPC
produced the highest titer, 1.6 × 106 CFU/ml
(28). The subclones of LTKOSN.2 in this study were obtained by limiting dilution of LTKOSN.2 VPC onto two 96-well plates and examined by light microscopy to ensure that only single-cell subclones were further analyzed. Six single-cell clones (1 to 5 and 10) were
identified and expanded for further study.
Cell culture and drug treatment.
Cell cultures were
maintained in Dulbecco's modified Eagle medium (DMEM; GIBCO BRL Life
Technologies, Gaithersburg, Md.)-10% fetal calf serum with 5%
CO2, at 37°C. To reverse the DNA methylation, cells were
grown in DMEM containing 5 µM 5-azacytidine (5-aza-C) (Sigma Chemical
Co., St. Louis, Mo.) for 72 h (26) and then subjected
to titer assay, vector RNA slot blotting, and DNA methylation analysis.
Retroviral infection.
Vector titers were determined by the
transduction of NIH 3T3 tk(
) cells (American Type Culture
Collection [ATCC] CRL1658), A375 cells (ATCC CRL1619; human
melanoma), and IGROV cells (human ovarian carcinoma
[57]) with 10-fold serial dilutions of vector stocks.
Transduction was performed in 1 ml of DMEM by incubating 105 cells/well in six-well plates with vector in the
presence of 10 µg of protamine sulfate (Fujisawa USA Inc., Deerfield,
Ill.) per ml. After transduction for 24 h, the cells were selected
in medium containing G418 (1 mg/ml) for 10 to 14 days. Titers were obtained by multiplying the number of resistant colonies by the dilution factor.
To perform the superinfection assays on each subclone of LTKOSN.2 VPC,
a LEIN retroviral vector carrying an enhanced green fluorescent protein
(EGFP) (6, 10) reporter gene was used to transduce LTKOSN.2
VPC subclones. The LEIN vector is an LXSN vector with an internal
ribosomal entry site (IRES) (19) replacing the simian virus
40 (SV40) promoter. The EGFP gene was inserted in front of the IRES to
demonstrate transduction efficiency in target cells. Cells from NIH
3T3, PA317, parental LTKOSN.2 VPC, and LTKOSN.2 VPC subclones were
seeded at 5 × 105 cells per well on 12-well plates
and exposed to 0.5 ml of supernatant of LEIN vector (3.4 × 105 CFU/ml) in the presence of 10 µg of protamine sulfate
per ml 24 h after seeding. Two days after a single exposure to
LEIN vector, the transduction efficiency of these cell lines and VPC
subclones was determined by a fluorescence-activated cell sorter (FACS) analysis of EGFP expression (39) on an EPICS Profile II
Analyzer (Coulter Co., Miami, Fla.).
Southern blot analysis of genomic DNA.
Total cellular DNA
was extracted from cell cultures using phenol-chloroform extraction and
then dissolved in Tris-EDTA buffer (pH 8.0) overnight at 55°C
(51). Genomic DNA was digested by KpnI to release
nearly full-length LTKOSN (4.0-kb) and 
LTKOSN (2.5-kb) fragments
(Fig. 1) and then Southern blotted for Neor probe detection
of the copy number of integrated vectors. A 0.68-kb BstXI
fragment of pPAM3 was used as a probe to detect a 2.5-kb DNA fragment
of endogenous retroviral elements to demonstrate similar loading of DNA
samples. To analyze for multiple integration sites in each subclone,
BamHI was used to generate DNA fragments containing both
proviral vector sequences and flanking chromosomal sequences. Southern
blot analysis was performed, and membrane duplicates were hybridized
with Neor and HSVtk probes (Fig. 2).
RNA analysis of LTKOSN VPC and vector supernatants.
Total
cellular RNA was isolated from individual subclones using the RNAeasy
kit (Qiagen, Inc., Valencia, Calif.). Viral supernatants were subjected
to 20% sucrose gradient ultracentrifugation (125,000 × g) for 2 h at 4°C, and virion pellets were then extracted
for RNA with RNAzol (Biotecx, Houston, Tex.). Cellular and virion RNAs
from each subclone were subjected to Northern blot analysis on a 1%
agarose-0.4 M formaldehyde gel. Vector transcripts were detected by a
Neor probe, and helper viral transcripts were detected by a
1.4-kb env probe, which was digested from pPAM3 by
XhoI. RNA slot blotting for titer determination
(40) was performed by applying 200 µl of supernatant,
directly collected from VPC medium and centrifuged at 2,800 × g to eliminate the cellular debris, onto a slot blot apparatus
(SlotBlot; Hoefer Scientific Instruments, San Francisco, Calif.).
Vector RNA was UV cross-linked onto this membrane and detected by a
Neor probe.
Methylation analysis.
The methylation status of provirus and
vectors at the SmaI site in the 5' LTR was determined by
digestion of genomic DNA with DraI and EcoRV to
reduce the DNA fragment size. The DNA was then precipitated with
ethanol, redissolved in sterile water, and divided into two equal
portions, one of which was subjected to methylation-sensitive SmaI restriction endonuclease digestion for Southern
blotting analysis. The Southern blot membrane was hybridized with a
428-bp fragment of gag sequence
(PvuII/DraI) from pPAM3 to detect helper virus, a
273-bp EcoRI/EcoRV fragment of HSVtk
sequence for LTKOSN vector, and a Neor probe to detect both
LTKOSN and LTKOSN. This Southern blot was also probed with an
env probe to determine the methylation status of the
SmaI site in the helper virus RT region. DNAs derived from NIH 3T3 and PG13 (ATCC CRL 10686), a packaging cell line for gibbon ape
leukemia virus (GaLV) pseudotyped vector (34), were used as
negative controls. A 0.68-kb BstXI fragment of pPAM3 was
used as a probe to detect a 1.2-kb DNA fragment of endogenous
retroviral sequences to demonstrate similar loading of DNA samples.
Densitometric analyses were performed with a Hoefer Densitometer GS300
(Hoefer Scientific Instruments) to measure the densities of
SmaI-sensitive bands relative to those of
DraI/EcoRV bands. Due to the interference from
endogenous retroviral elements, the fraction of SmaI
methylation in 5' LTR was calculated as 1 minus the intensity ratio of
the SmaI-sensitive band (1.5 kb) divided by that of the
DraI/EcoRV band (1.8 kb).
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RESULTS |
Dynamics of proviral DNA integration.
We have previously
detected an HSVtk deletion mutant vector (LTKOSN)
coexisting with full-length LTKOSN vector (Fig.
1A) in our LTKOSN.2 VPC population
(62). To investigate whether these LTKOSN and LTKOSN
vectors coexisted within the same individual cellular genome or existed
separately in portions of the LTKOSN.2 VPC population, limiting
dilution cloning on LTKOSN.2 VPC was performed to isolate six
single-cell subclones (1 to 5 and 10). After expansion of the
subclones, each clone was then analyzed for integrated vectors by
KpnI digestion on both LTR regions to release the integrated
proviral vectors of LTKOSN (2.5 kb [Fig. 1A]) and LTKOSN
(4.0 kb [Fig. 1A]). Southern blot analysis demonstrated that
LTKOSN and LTKOSN coexisted in the same individual cellular genome,
since both vectors were detected in all six subclones (Fig. 1B).
Varying copy numbers of LTKOSN and LTKOSN were observed in subclones
2, 3, and 5 by comparing the intensities of detected signals of both
vectors. In contrast, only one single copy each of LTKOSN and LTKOSN
vector was detected in parental LTKOSN.2 VPC and subclones 1, 4, and
10. To estimate the copy numbers of integrated vectors, genomic DNA
from each subclone was subjected to BamHI digestion and
Southern blot analysis for the integration sites of both LTKOSN and
LTKOSN (Fig. 2). The BamHI
site is unique in the LTKOSN sequence, and restriction digestion yields
two fragments of the LTKOSN vector with adjacent chromosomal sequences.
Since the BamHI site was deleted with the HSVtk
gene in LTKOSN vector, only one genomic DNA fragment of integrated
LTKOSN vector with flanking chromosomal sequences is observed after
BamHI digestion (Fig. 2B). The copy number of LTKOSN was
approximately six copies per cell in subclone 3 and eight copies per
cell in subclone 2, while only one copy of LTKOSN exists per cell in
these two subclones. An increased copy number of LTKOSN was
observed only in subclone 5, which showed approximately 9 copies of
LTKOSN and approximately 20 copies of LTKOSN per cell.
Therefore, significant changes in the vector copy numbers have occurred
between the parental VPC and its subclones.
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FIG. 1.
Increased copy number of LTKOSN and LTKOSN in
LTKOSN.2 subclones. (A) Schematic diagram of LTKOSN and LTKOSN
vectors. LTKOSN contains an HSVtk gene, which was cloned
into the EcoRI site of LXSN.  , extended packaging signal
region; Neo, neomycin phosphate transferase gene; SV, promoter sequence
of SV40 early gene. Bp, BpmI; E, EcoRI; H,
HindIII; K, KpnI. (B) Genomic DNA was
extracted from GP+E86 transfected with LTKOSN (lane 1), parental
LTKOSN.2 (lane 3), and its derived subclones 1 to 5 and 10 (lanes 4 to
9, respectively). PA317-derived DNA (lane 2) was used as a control.
KpnI releases a full-length proviral LTKOSN without the 5'
LTR (4.0 kb) and a LTKOSN without its 5' LTR (2.5 kb). Integrated
LTKOSN and LTKOSN were detected from parental LTKOSN.2 and all
subclones, but not PA317 cells. Only LTKOSN vector was detected in
LTKOSN-transfected GP+E86 cells. Note the relative variance in
LTKOSN copy number of different subclones compared to LTKOSN. (C)
The same Southern blot was hybridized with a BstXI fragment
probe (0.68 kb, located at gag coding region) that detects a
2.5-kb fragment of KpnI-digested endogenous retroviral
sequences showed similar loading of DNA samples.
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FIG. 2.
Genetic instability of integrated LTKOSN in VPC genome.
(A) Genomic DNA extracted from PA317 (lane 1), LTKOSN.2 VPC (lane 2),
and its subclones (lanes 3 to 8) was subjected to BamHI
digestion and detected by Neor and/or HSVtk
probes in Southern blot analysis. Only two integration sites were
detected on parental LTKOSN.2 and subclones 1, 4, and 10, but subclones
2, 3, and 5 showed more integration sites. The arrows indicate the
positions of integrated LTKOSN vector. Note the absence of these bands
from subclone 5 on both Neor and HSVtk probe
hybridizations. (B) Schema of LTKOSN and LTKOSN integration sites
shows the locations of BamHI sites on the flanking
chromosomal sequences. The BamHI site on LTKOSN is
deleted; therefore, digestion with BamHI excises the entire
LTKOSN from flanking chromosomal DNA sequences (4.5 kb) at the
integration site. BamHI digestion excises the LTKOSN
backbone 5' to the SV40 promoter and results in two fragments of 3.7 and 2.2 kb including 5' and 3' junctional chromosomal DNA sequences,
respectively.
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Deletion of proviral integration site.
In subclone 5, a
possible deletion of an integration site of LTKOSN vector was observed
(Fig. 2A). A 2.2-kb fragment (arrow) containing the 3' portion of
proviral LTKOSN fragment with adjacent chromosomal sequences was
detected by the Neor DNA probe on parental LTKOSN.2 VPC and
all of the subclones except subclone 5 (Fig. 2A, lane 7). A 4.5-kb
fragment containing the entire LTKOSN vector with adjacent cellular
sequences on both ends was detected by the same Neor DNA
probe on all of the subclones and parental LTKOSN.2 VPC. In addition to
the 4.5- and 2.2-kb bands detected in subclones 2 and 3, other bands
that are larger than 2.2 kb should be LTKOSN vector, since only one
integration site of LTKOSN (3.7 kb) was detected by the
HSVtk probe on the same membrane in subclones 2 and 3. This
3.7-kb fragment (arrow) containing the 5' portion of LTKOSN with
adjacent chromosomal sequences was detected in the LTKOSN.2 VPC and all
subclones except subclone 5 (Fig. 2A, lane 7). Taken together, the
original LTKOSN vector integration site, present in the parental
LTKOSN.2 VPC and the other subclones, appears absent in subclone 5 (Fig. 2A, lane 7). Since subclone 5 still contains the same integration
site (4.5-kb band) of LTKOSN as seen in parental LTKOSN.2 VPC and
the other subclones, subclone 5 is a true subclone derived from
parental LTKOSN.2 VPC. We also suspect that subclone 5 may be derived
from subclones 2 and 3, since subclone 5 has an integration pattern of
LTKOSN very similar to those of subclones 2 and 3.
Gene expression and packaging of LTKOSN and LTKOSN.
After Northern blot analysis of RNA transcripts from subclones 1, 3, 4, 5, and 10 (subclone 2 was lost to contamination), the results
revealed that the RNA transcript ratio of LTKOSN to LTKOSN in all of
the subclones was approximately 2:1 or greater (Fig.
3A). This ratio was observed in subclones
1, 4, and 10, which contain only one copy each of LTKOSN and
LTKOSN vector. This could result from differences in the integration
sites or the smaller size of LTKOSN. Previous reports
demonstrated that the internal SV40 promoter, present in LTKOSN but
deleted from LTKOSN, can interfere with transcription from the
5' LTR (48). Subclone 5, which contains the highest copy
number of both LTKOSN and LTKOSN vectors, showed more abundant
vector transcripts than the other subclones. Unexpectedly, these
abundant vector transcripts in subclones 1, 3, and 5 were not packaged
into virions (Fig. 3B).
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FIG. 3.
Differential gene expression of vectors and helper virus
in LTKOSN.2 VPC subclones. (A) Total RNA was extracted from LTKOSN.2
VPC subclones (lanes 3 to 7) and detected by Neor,
env, and human glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA probes. RNA transcripts of LTKOSN and LTKOSN are 4.0 and 2.5 kb in size, respectively. RNA extracted from NIH 3T3 (lane 8)
was used as a negative control. Hybridization with env probe
detected helper virus gene expression. Compared to the spliced
env transcript observed in all subclones, full-length MoMLV
transcript (gag-pol-env) was detected in only LTKOSN.2 VPC
(lane 2) and subclones 4 and 10 (lanes 5 and 7, respectively) but was
barely visible in other subclones and PA317 cells. (B) Viral RNA
extracted from virions was detected by Neor probe and
showed that LTKOSN and LTKOSN transcripts could be detected only in
the supernatants collected from parental LTKOSN.2 VPC and subclones 4 and 10. Thus, despite abundant LTKOSN and LTKOSN transcripts in the
VPC, little or no vector was packaged and released from subclones 1, 3, and 5.
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Transcriptional inactivation of helper virus.
To determine the
mechanism for the observed reduction of vector production in subclones
1, 3, and 5, Northern blots of subclones were probed with amphotropic
env DNA to evaluate the level of helper virus gene
expression (Fig. 3). Spliced env transcripts were detected
in all subclones, but significant levels of full-length helper virus
transcripts were observed only in subclones 4 and 10 (Fig. 3A), which
were previously shown to produce virions containing LTKOSN and
LTKOSN vectors (Fig. 3B). It has been reported that spliced RNA (e.g.,
env transcript) is more stable than precursor RNA (e.g.,
full-length helper virus) as a result of increased polyadenylation
efficiency on RNA 3' processing (13). Therefore, the overall
low-level gene expression of helper virus and faster degradation of
full-length helper virus transcripts may explain why only
env transcripts were observed in subclones 1, 3, and 5, not
full-length helper virus (Fig. 3A). These results demonstrated that the
reduction of packaged LTKOSN and LTKOSN vectors in subclones 1, 3, and 5 correlated closely with low levels of helper virus gene expression.
Superinfection of LTKOSN.2 VPC subclones.
To test our
hypothesis that the suppression of helper virus gene expression reduces
the Env-receptor interference and then results in vector reentry
(superinfection), LTKOSN.2 VPC subclones were transduced with an EGFP
expression vector, LEIN (see Materials and Methods). The transduction
efficiency of LEIN vector on these subclones was determined by FACS
analysis of EGFP expression compared to that in NIH 3T3, PA317, and
parental LTKOSN.2 VPC (Fig. 4). NIH 3T3
cells that do not express Env protein on the cell surface were
21.9% EGFP positive 2 days after single exposure to LEIN vector.
LTKOSN.2 VPC subclones with significant DNA methylation in the 5'
LTR region were transduced at rates comparable to those for NIH 3T3
cells (15.7 to 19.3% in subclones 1, 3, and 5). Subclones 4 and 10, which exhibited more detectable helper virus transcripts, were
transduced at lower rates of only 0.6 and 1.4%, which were comparable
to those of PA317 (0.2%) and parental LTKOSN.2 VPC (0.4%). These
results demonstrate that gene expression of helper virus in these
subclones correlated closely with the superinfection of LEIN vector.
These results also suggested that increased copy numbers of LTKOSN
and LTKOSN in subclones 3 and 5 were due to superinfection. Subclone
1 showed significant inactivation of helper virus gene expression and
superinfection of LEIN vector as well as subclones 3 and 5 but did not
exhibit increased copy number of vectors. We suspect that the
inactivation of helper virus gene expression by DNA methylation
occurred during or after the subcloning procedure, since we did observe
that the titer of subclone 4 was reduced to completely nondetectable
levels by DNA methylation during a 31-day period of cell culture (see
below).
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FIG. 4.
Superinfection susceptibility of LTKOSN.2 VPC subclones.
Cells from NIH 3T3, PA317, parental LTKOSN.2, and LTKOSN.2 VPC
subclones were seeded at 5 × 105 cells per well on
12-well plates and exposed to 0.5 ml of supernatant of EGFP-expressing
vector, LEIN (3.5 × 105 CFU/ml; see Materials and
Methods). FACS analysis of EGFP expression was performed 2 days after
the single exposure of LEIN vector. The average rate of three
transductions of each target cell line was as follows: NIH 3T3, 21.9%;
PA317, 0.2%; LTKOSN.2, 0.4%; subclone 1, 18.4%; subclone 3, 15.7%;
subclone 4, 0.6%; subclone 5, 19.3%; and subclone 10, 1.4%. Error
bars were calculated from triplicate data with each target cell line.
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Methylation status of helper virus and retroviral vectors.
DNA
methylation has been demonstrated to be associated with the repression
of retroviral vector gene expression in different cells and tissues in
vitro (12, 22, 24) and in vivo (3). The
methylation of CpG sequence(s) within an SmaI site by
mammalian methyltransferase will prevent SmaI digestion
(49). The methylation status of the SmaI site in
the 5' LTR has been employed to demonstrate an inverse correlation with
the gene expression levels of retroviral vector (3).
Therefore, the same analysis was performed to detect helper virus DNA
methylation (Fig. 5) compared to helper
virus gene expression in VPC subclones (Fig. 3A).
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FIG. 5.
DNA methylation status of helper virus and vectors.
Genomic DNA digested with DraI and EcoRV was
divided into two equal portions for SmaI digestion (lanes 1 to 9) or without SmaI digestion (lanes 10 to 18). (A)
Hybridization of gag DNA probe to detect helper virus 5'
LTR. SmaI digestion reduced the 1.8-kb band (lanes 11 to 18)
to 1.5 kb (lanes 2 to 9). DNA from NIH 3T3 cells was used to
demonstrate the presence of endogenous retroviral elements (lanes 1 and
10). Since a 1.8-kb band was generated from endogenous retroviral
element after SmaI digestion (lane 1), the values for
SmaI resistance were measured by densitometry and calculated
as 1 minus the relative intensities of the 1.5-kb bands
(SmaI digestion; lanes 1 to 9) and the 1.8-kb bands (without
SmaI digestion; lanes 10 to 18). Note the absence of helper
virus methylation in parental LTKOSN.2 (lane 3) and completely
methylated helper virus 5' LTR in subclone 5 (lane 7). (B)
Rehybridization of env DNA probe for the SmaI
methylation status in helper virus RT region. SmaI
resistance was measured as the relative intensities of the 4.6-kb and
3.3- plus 4.6-kb bands generated after SmaI digestion. Note
that multiple copies of helper virus env sequences were
detected in PA317 and derived cells. (C) HSVtk DNA probe was
used to detect the 5' LTR of LTKOSN vector. SmaI digestion
reduced the 1.5-kb fragment to 1.2 kb. The varied signal intensities
are secondary to the copy number of LTKOSN vector in each subclone. (D)
Neor probe was used to determine the SmaI
methylation status in both the HSVtk gene of LTKOSN and the
5' LTR region of LTKOSN. SmaI reduced the 2.4-kb fragment
of LTKOSN to 2.1 kb and the 2.1-kb band of LTKOSN to 1.5 kb. (E) A
0.68-kb DNA fragment from the gag gene digested by
BstXI was used as a probe to detect a 1.2-kb band of
endogenous retroviral sequence to demonstrate equivalent DNA loading in
paired samples of SmaI digestion. (F) Schema of helper virus
and vectors showing the locations of restriction enzyme sites and the
probes used for the methylation analysis. D, DraI; E,
EcoRV; S, SmaI; AAA, SV40 poly(A) signal.
Drawings are not to scale.
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Genomic DNA was first digested with
EcoRV and
DraI to generate a DNA fragment convenient for Southern blot
analysis. An equal
portion of this
EcoRV/
DraI-digested DNA was subjected to
SmaI
digestion. Since the
gag region is not
present in the retroviral
vector, a 428-bp
gag probe
hybridizes only to the helper virus
DNA (Fig.
5A and E). All of the
subclones and parental VPC showed
a 1.8-kb
EcoRV/
DraI band (lanes 11 to 18), which was
reduced to
a 1.5-kb band upon
SmaI digestion if no
methylation occurred at
the
SmaI site. This 1.5-kb band was
not detected in either NIH
3T3 cells or subclone 5 (Fig.
5A, lanes 1 and 7) but was observed
in all other samples. NIH 3T3 cells were used
to show the presence
of endogenous retroviral elements that hybridized
with the
gag probe (Fig.
5A, lanes 1 and 10). After
SmaI digestion of NIH 3T3-derived
DNA, a band of
approximately 1.8 kb was generated from endogenous
retroviral elements,
complicating our analysis (Fig.
5A, lane
1). Therefore, the methylation
status of the
SmaI site was evaluated
by the relative
intensities of the observed 1.5-kb bands from
SmaI/
EcoRV/
DraI digestions (lanes 2 to
9) compared to 1.8-kb bands
from
EcoRV/
DraI
digestions (lanes 11 to 18), which do not overlap
in size with
endogenous sequences (Fig.
5A, lane 10). Although
40% of helper virus
5' LTR in PA317 is methylated (Fig.
5A, lane
2), complete digestion by
SmaI shows the apparent absence of methylation
of the helper
virus 5' LTR in parental LTKOSN.2 VPC (Fig.
5A,
lane 3). This suggests
that the prior selection of LTKOSN.2 VPC,
a clone that was chosen for
its high titer compared to those of
the other VPC clones, is likely the
result of both high-level
expression of vector and low methylation of
helper virus. A high
degree (60%) of helper virus methylation is also
found in the
5' LTR of PG13, a GaLV pseudotyped VPC (Fig.
5A, lanes 9 and 18).
This indicates that the methylation of helper virus 5' LTR in
PA317 is not a single packaging cell line phenomenon. In contrast
to
the low level of helper virus methylation in parental LTKOSN.2
VPC,
higher degrees of helper virus DNA methylation were observed
in the
subclones (Fig.
5A). This high level of methylation was
inversely
proportional to the helper virus gene expression (Fig.
3A) and
subsequently to the packaged vector titer (Fig.
3A and
Table
1). Subclone 5 has an absence of the
1.5-kb band, which
indicates that the methylation of the
SmaI site in the helper
virus 5' LTR (Fig.
5A, lane 7)
occurred in the entire subclone
5 population.
The methylation status of the
SmaI site in the RT region of
the helper virus was evaluated by the rehybridization of this
Southern
blot with a 1.4-kb
env probe (Fig.
5B). In subclones
1, 3, and 5, which had highly methylated helper viral 5' LTR,
the methylation
level of
SmaI in the RT region ranged from 46
to 100%. In
contrast, only 3 to 10% methylation was observed in
parental LTKOSN.2
VPC and subclones 4 and 10. Thus, a strong correlation
exists between
the observed methylation of the
SmaI site in 5'
LTR and
within the RT gene (Fig.
5A and B). In addition to the
4.6-kb band in
the DNA samples without
SmaI digestion (lanes 10
to 18),
three additional bands were observed in parental LTKOSN.2
VPC and all
VPC subclones (lanes 2 to 8 and 10 to 17) but not
in NIH 3T3 and PG13
(lanes 1 and 10 and lanes 9 and 18). These
results demonstrate that the
env probe is specific for the
env sequence of
helper virus pPAM3 and does not detect GaLV
env or
Moloney
murine leukemia virus (MoMLV)
gag-pol from PG13 cells.
Furthermore, at least four copies of
env sequence are
present
in the PA317 genome. These multiple copies of helper virus
env sequence were confirmed by Southern blot analysis of
PA317 genomic
DNA with different methylation-insensitive restriction
endonuclease
digestions (data not shown). In contrast, hybridization
with probes
for either the HSV
tk (Fig.
5C) or
Neo
r (Fig.
5D) gene reveals no significant methylation
within either
LTKOSN or
LTKOSN vectors. Extremely low methylation of
the 5'
LTR of LTKOSN and
LTKOSN vectors was observed only in
subclone
5 (Fig.
5D, lane 7), which showed 100% methylation of the
SmaI
sites in both the helper virus 5' LTR and RT region
(Fig.
5A and
B, lane
7).
Parental LTKOSN.2 VPC do not show significant methylation of either
helper virus or vectors. However, the subclones derived
from LTKOSN.2
VPC show various methylation levels of the helper
virus but not of
their retroviral vectors. These results suggest
a sequential time line
of methylation that developed within the
helper virus DNA in these five
subclones. These data also support
our previously theorized cell
lineage relationship, based upon
the similarity of vector integration
patterns observed in Fig.
2A.
Reversal of DNA methylation.
To reactivate silenced helper
viruses, subclones were treated with 5-aza-C, a cytidine analog that
can inhibit DNA methylase and reverse methylation to restore gene
expression (8, 21, 23, 26). After coculture with 5-aza-C for
72 h, the rescue of vector RNA from these VPC subclones was
demonstrated by RNA slot blot analysis of supernatants (Fig.
6). Partial recovery of vector production
was also demonstrated by titer assay (Table 1) along with the partial
reversal of DNA methylation by 5-aza-C treatment (Fig.
7). The titers of subclones 1, 3, and 5 were increased by 2 orders of magnitude from 
10 CFU/ml to up to
6 × 102 CFU/ml. No significant change was observed in
subclones 4 and 10, which exhibited high vector production and less
helper virus methylation before 5-aza-C treatment. The most significant
reduction of SmaI resistance was observed in subclone 5, in
which the methylation of the 5' LTR and RT regions was reduced from 100 to about 45% on a Southern blot (Fig. 7). In this model system, the
methylation of helper virus is responsible for the reduction of vector
production.
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FIG. 6.
Treatment with 5-aza-C partially restores the vector
production ability in subclones. Cell cultures were coincubated with 5 µM 5-aza-C for 72 h. Supernatant (200 µl) collected from each
of the cell cultures with or without 5-aza-C treatment was loaded onto
the slot blot apparatus and UV cross-linked onto a nylon membrane.
Neor probe was used to detect the LTKOSN and LTKOSN
vector transcripts to quantify the vector production ability of each
subclone. Supernatant collected from NIH 3T3 was used as a negative
control.
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|
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FIG. 7.
Treatment with 5-aza-C reverses the DNA methylation of
helper virus. (A and B) Genomic DNA digestion and hybridization with
the gag (A) and env (B) DNA probes were performed
the same way as described in the legend to Fig. 5 to evaluate the
SmaI methylation status in the helper virus 5' LTR and RT
regions. Note the new appearance of a 1.5-kb band (A) and a 3.3-kb band
(B) in subclone 5 (lane 4) that were not seen in Fig. 5, lane 7, before
5-aza-C treatment. (C) A 0.68-kb DNA fragment from the gag
gene digested by BstXI was used as a probe to detect a
1.2-kb band of endogenous retroviral sequence to demonstrate equivalent
DNA loading in paired samples of SmaI digestion.
|
|
DNA methylation rate of helper virus 5' LTR.
To study the DNA
methylation rate of helper virus 5' LTR, we examined the DNA
methylation of subclone 4, which showed the least DNA methylation with
the most highly activated gene expression among these subclones. During
31 days of continuous cell culture, DNA methylation of helper virus 5'
LTR in subclone 4 increased rapidly from 36 to 100% (Fig.
8A). The methylation rate of the SmaI site in helper virus 5' LTR was calculated to be as
high as 2% of the cell population per day. The copy numbers of vector were increased from one copy (parental LTKOSN.2 VPC and subclone 4 on
day 0) to five copies (day 7) and subsequently to 9 copies (days 23 and
31) per cell (Fig. 8B and C). The titer of subclone 4 was reduced from
1.5 × 105 CFU/ml (day 0) to 7 × 104
CFU/ml (day 7) and then to undetectable levels by days 23 and 31. The
reduction in vector titer correlated with increasing inactivation of
helper virus gene expression (Fig. 9A).
However, vector gene expression still remained at high levels (Fig.
9B). Only minimal DNA methylation (10%) of 5' LTR was observed in
LTKOSN vector (Fig. 8B), and no detectable DNA methylation was noted in
the LTKOSN vector (Fig. 8C).
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FIG. 8.
Increased DNA methylation in helper virus of subclone 4 over time. Genomic DNA was first digested with DraI and
EcoRV and divided into two equal portions for
SmaI digestion as described in the legend to Fig. 5. (A)
Hybridization of gag DNA probe to detect helper virus 5'
LTR. SmaI digestion reduced the 1.8-kb band (even-numbered
lanes 4 to 14) to 1.5 kb (odd-numbered lanes 3 to 13). SmaI
resistance was calculated as described for Fig. 5. (B) HSVtk
DNA probe was used to detect the 5' LTR of LTKOSN vector.
SmaI digestion reduced the 1.5-kb fragment to 1.2 kb. The
values for SmaI resistance were calculated as described for
Fig. 5. The varied signal intensities are secondary to increasing copy
numbers of LTKOSN vector over time. Copy number was estimated by
comparing the relative intensity of HSVtk signals and
intensities detected by BstXI fragment probe from endogenous
retroviral elements in panel D to standardize loading. Increased copy
numbers were observed on days 7 (five copies, lane 10) and 23 and 31 (nine copies, lanes 12 and 14), while only one copy in parental
LTKOSN.2 and subclone 4 was detected on day 0 (lanes 6 and 8). (C)
Neor probe was used to determine the SmaI
methylation status in both the HSVtk gene of LTKOSN and the
5' LTR region of LTKOSN. (D) A 0.68-kb DNA fragment from the
gag gene digested by BstXI was used as probe to
detect a 1.2-kb band of endogenous retroviral sequence to demonstrate
equivalent DNA loading in paired samples of SmaI
digestion.
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FIG. 9.
Transcription activity of helper virus and retroviral
vectors in LTKOSN.2 VPC subclone 4. (A) Northern blot analysis of
cellular RNA extracted from LTKOSN.2 VPC subclone 4 at different time
points and hybridized with env probe. Unspliced MoMLV
transcripts (gag-pol-env) were detected at different sizes
as a result of the presence of at least four different integrated pPAM3
plasmids detected in PA317 with different adjacent sequences. (B)
Gene expression of LTKOSN and LTKOSN vectors. Rehybridization
of this Northern blot membrane with Neor probe to detect
LTKOSN vector (4.0 kb), LTKOSN vector (2.5 kb), and a
Neor RNA transcript (1.2 kb) derived from internal SV40
promoter activity was carried out. (C) Hybridization with
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe
demonstrated similar levels of RNA loading.
|
|
| |
DISCUSSION |
The genetic stability of retroviral vectors and a VPC genome was
examined in LTKOSN.2 VPC and derived subclones. We also compared the
methylation status of helper virus and vectors in these individual subclones. Our results demonstrate that DNA methylation occurred in
helper viral sequences rather than vector in subclones derived from
LTKOSN.2 VPC (Fig. 5). The increase in methylation of the helper virus
(Fig. 5 and 8) correlated directly with the inactivation of helper
virus gene expression (Fig. 3 and 9), which resulted in a loss of
vector production. In contrast, vector gene expression was constant,
and no significant methylation of vector sequences was detected. We
conclude that the key limitation for vector production in LTKOSN.2 VPC
is the inactivation of helper virus gene expression by DNA methylation.
This conclusion was supported by the partial restoration of vector
production (Table 1) and the reversal of DNA methylation (Fig. 7) from
VPC subclones treated with 5-aza-C. In previous studies, de novo DNA
methylation has been suggested to cause transcriptional inactivation of
retroviral vectors in infected embryonic cells (3, 12, 22).
DNA methylation had not been a focus of investigation with regard to
the genetic instability of retroviral VPC that are designed for
continuous vector production. A cascade of complex events resulting in
the genetic instability of VPC caused by the methylation of helper
virus 5' LTR is summarized in Fig. 10.
We propose that the cascade occurs in the following sequence: (i)
repression of helper virus 5' LTR transcriptional activity by
methylation reduces the production of vector, (ii) decreased Env
protein synthesis reduces Env-receptor interference and allows vector
superinfection to cause increased vector copy number in VPC, and (iii)
random integration of vectors caused by superinfection results in a
more fluid VPC cellular genome.
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FIG. 10.
Cascade reactions initiated by increased DNA
methylation of helper virus 5' LTR. DNA methylation inactivates helper
virus gene expression, and then vector production is reduced. Without
sufficient Env-receptor interference, susceptibility to superinfection
of vector from its own or other VPC in the same population is increased
and is seen in the increased vector copy number.
|
|
The preference for methylation of the helper virus rather than the
retroviral vectors is not clear and requires further study. However,
chromosomal location has been suggested as a key factor for
facilitating methylation (12, 20). In PA317, four copies of
env sequences of pPAM3 helper virus were observed (Fig. 5B). Restriction analysis of these helper virus sequences demonstrated that
the sequences are located at different chromosomal sites (data not
shown). The probability that all four copies of helper virus sequences
are located within a hypermethylation region of PA317 seems remote. No
significant methylation of vectors was observed in subclones 3 and 5, even though these subclones contain multiple copies of vectors in
different integration sites. About 100 copies of integrated MoMLV
provirus have been observed in infected murine embryonic carcinoma
cells, and these proviruses were hypermethylated with no significant
gene expression (55). Presumably, these 100 copies of
provirus were not all located in hypermethylation regions. Therefore,
the preference of methylation for helper virus is probably related to
both helper virus sequence and chromosomal location. The idea that
sequence has an important role is also suggested by the findings that
the presence of retrotransposon and provirus sequences enhances the de
novo DNA methylation of adjacent sequences (11, 16). Most
recently, evidence that DNA methylation acts as a defense system
against retrotransposons invading the mammalian genome has been shown
elsewhere (44). Infection of retrovirus, such as human
immunodeficiency virus, increased the overall DNA methyltransferase
gene expression and activity, which resulted in suppression of cytokine
gene expression for evading immune surveillance (30). The
spreading of de novo methylation from a provirus into adjacent cellular
sequences was observed in either the 5' or 3' region of the integration
site in Mov-derived mice carrying a MoMLV provirus in a distinct
chromosomal location (16). In the pPAM3 LTR, the methylation
observed could be the result of spreading of methylation from adjacent
gag sequences, since the LTR in either LTKOSN or LTKOSN
vector did not demonstrate any significant methylation. However, we
have not excluded a role of chromosomal position and preferred DNA
methylation (12). These subclones of VPC may provide a
useful model to study DNA methylation preferences that are key in
host-retrovirus interactions (11, 17, 18, 30, 44).
Alternatively, G418 selection of VPC could have eliminated VPC cells
with methylated vectors. After the establishment of LTKOSN.2 VPC, VPC
were not maintained on G418 selection. Other LTKOSN parental lines
require only a single copy of the Neor gene for G418
resistance. Subclones 3 and 5 contained multiple copies of LTKOSN and
LTKOSN. Even if partial methylation of the vector, as noted for the
helper virus, had occurred, adequate Neor gene expression
would nevertheless provide G418 resistance. However, almost no
methylation was observed in either LTKOSN or LTKOSN. An extremely
low degree of methylation was observed only in subclone 5, which
contained approximately 29 copies of integrated vectors. This could be
attributed to the random integration of some vectors into
hypermethylation regions of the cellular genome. In contrast to the
moderate degree of methylation (40%) of the helper virus 5' LTR in
PA317, the helper virus in parental LTKOSN.2 VPC did not show
significant methylation of SmaI sites in the 5' LTR. This
demonstrates that a diverse range of methylation statuses exists within
PA317 cells and that the selection of the highest-titer LTKOSN VPC
(LTKOSN.2) from among the other 39 clones (see Materials and Methods)
may have selected for a clone with hypomethylated helper viruses.
It has been well documented that Env-receptor interference is important
to reducing the risk of superinfection (31, 33, 54, 60).
Recent data demonstrated that at least four copies of retrovirus per
cell are required for sufficient Env protein synthesis to interfere
with the cellular receptor (42). Superinfection experiments
on these LTKOSN.2 subclones (Fig. 4) demonstrated that DNA methylation
(Fig. 5) and helper virus mRNA level (Fig. 3) are highly correlated
with the reduction of Env-receptor interference. The reduction of
env gene expression by DNA methylation would logically
result in the reentry of multiple vectors into subclones 3 and 5. Subclone 1 may represent a transitional state between the parental
LTKOSN.2 and subclone 3, since only one copy of each LTKOSN
and LTKOSN vector is present while helper virus gene
expression is low.
DNA methylation in subclones 4 and 10 (38 and 50%, respectively) and
PA317 (40%) is significantly higher than that in parental LTKOSN.2
cells (0%). However, the resistance to superinfection in subclones 4, 10, and PA317 is still comparable to that observed in parental LTKOSN.2
cells (Fig. 4). A significant increase in superinfection was observed
in subclones only when at least 60% methylation of the helper virus 5'
LTR occurred. This suggests a possible threshold effect. It has been
demonstrated that the amount of Env product per cell, the interference,
and virion release did not increase linearly but increased abruptly
once an infected cell reached the threshold of proviral copy number
which correlated with mRNA level (42). This is due to the
fact that oligomerization of Env protein is essential for functional
virus assembly and interference (2, 14, 42). Since the
concentration of Env protein determines the kinetics of
oligomerization, reduction of helper virus mRNA level by DNA
methylation resulted in low Env protein concentration and
oligomerization and, therefore, significantly decreased the
interference and virion release. Compared to the previously reported
high level of resistance to the superinfection of PA317, which was
transduced with the vector collected from another PA317 VPC
(33), the apparent reentry frequency of vectors in LTKOSN.2
VPC is relatively high (50% of population, three out of six subclones
[Fig. 1]). This superinfection was enhanced by both the DNA
methylation of helper virus and the presence of extensive vector
exposure in cell culture. It has also been observed that human
immunodeficiency virus provirus was accumulated in infected cells to
multiple copies by superinfection of 70% of cells in the culture over
time (45).
Amphotropic murine leukemia virus vectors are used widely for human
gene transfer. However, most amphotropic murine leukemia virus VPC are
derived from murine cells and are susceptible to vector reentry. The
consequence of vector reentry is the import of active RT enzyme which
may lead to RCR formation and the random integration of additional
vectors that can interrupt functional genes or activate proto-oncogenes
(9, 15, 25, 52). Our results imply that DNA methylation
decreases the Env-receptor interference, which results in vector
reintegration and subsequent further instability of the VPC genome.
Recently, progress in this direction has been made: PG13 VPC were
established using NIH 3T3 cells for packaging xenotropic
GaLV-pseudotyped vector. GaLV packaged vector will not reinfect PG13
cells that lack the receptor for GaLV envelope (34, 61).
PG13 VPC may diminish the vector reentry problem; however, helper virus
gene expression in PG13 is still not monitored and selected by a
selection marker. Our data with PG13 show significant DNA methylation
(60%) of helper virus 5' LTR (Fig. 5A, lanes 9 and 18). The
observations in this study strongly suggest that improvement of the
helper virus gene expression in overcoming DNA methylation in VPC is
important for sustained, stable vector production. Treatment with
5-aza-C of LTKOSN.2 subclones did not completely restore the titer
(Table 1). The effect of 5-aza-C treatment is transient (8),
and the treatment cannot be applied to long-term cell culture since
5-aza-C is toxic to the treated cells (21). Several
alternative strategies that ensure sustained vector gene expression in
target cells might improve the gene expression of helper virus in VPC.
Insertion of a demethylation fragment of the murine Thy-1 gene in front of 5' LTR can inhibit methylation (4, 56) and may be useful for helper virus. Another strategy is to ligate an IRES (19) with a selection marker gene downstream of the helper virus so that
drug selection would ensure active enhancer-promoter function of helper
virus for high vector packaging and preventing superinfection by
enhancing Env-receptor interference (W.-B. Young and C. J. Link,
Jr., submitted for publication).
| |
ACKNOWLEDGMENTS |
We thank John Levy, A. Dusty Miller, and Tatiana Seregina for
helpful discussions. We also thank Ginger Dreifurst and Julie Seiwert
for technical assistance with flow cytometry.
This work was supported in part by a grant from the Iowa Health System,
Des Moines, and Research Project Grant RPG-98-091-01-MBC from the
American Cancer Society. W.-B. Young is a recipient of a competitive
research fellowship from the Molecular, Cellular and Developmental
Biology Program, Iowa State University, Ames.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Human Gene
Therapy Research Institute, John Stoddard Cancer Center, 1415 Woodland
Ave., Des Moines, IA 50309. Phone: (515) 241-8787. Fax: (515) 241-8788. E-mail: linkcj{at}ihs.org.
Present address: Division of Hematology-Oncology, Harvard Medical
School, Beth Israel Deaconess Medical Center, Boston, MA 02215.
| |
REFERENCES |
| 1.
|
Anderson, W. F.,
G. J. McGarrity, and R. C. Moen.
1993.
Report to the NIH Recombinant DNA Advisory Committee on murine replication-competent retrovirus (RCR) assays.
Hum. Gene Ther.
4:311-321[Medline].
|
| 2.
|
Bernstein, H. B.,
S. P. Tucker,
S. R. Kar,
S. A. McPherson,
D. T. McPherson,
J. W. Dubay,
J. Lebowitz,
R. W. Compans, and E. Hunter.
1995.
Oligomerization of the hydrophobic heptad repeat of gp41.
J. Virol.
69:2745-2750[Abstract].
|
| 3.
|
Challita, P. M., and D. B. Kohn.
1994.
Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo.
Proc. Natl. Acad. Sci. USA
91:2567-2571[Abstract/Free Full Text].
|
| 4.
|
Challita, P. M.,
D. Skelton,
A. el-Khoueiry,
X. J. Yu,
K. Weinberg, and D. B. Kohn.
1995.
Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells.
J. Virol.
69:748-755[Abstract].
|
| 5.
|
Cone, R. D., and R. C. Mulligan.
1984.
High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range.
Proc. Natl. Acad. Sci. USA
81:6349-6353[Abstract/Free Full Text].
|
| 6.
|
Cormack, B. P.,
R. H. Valdivia, and S. Falkow.
1996.
FACS-optimized mutants of the green fluorescent protein (GFP).
Gene
173:33-38[CrossRef][Medline].
|
| 7.
|
Donahue, R. E.,
S. W. Kessler,
D. Bodine,
K. McDonagh,
C. Dunber,
S. Goodman,
B. Agricola,
E. Byrne,
M. Raffeld,
R. Moen,
J. Bacher,
K. M. Zsebo, and A. W. Nienhuis.
1992.
Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer.
J. Exp. Med.
176:1125-1135[Abstract/Free Full Text].
|
| 8.
|
Gram, G. J.,
S. D. Nielsen, and J. E. Hansen.
1998.
Spontaneous silencing of humanized green fluorescent protein (hGFP) gene expression from a retroviral vector by DNA methylation.
J. Hematother.
7:333-341[Medline].
|
| 9.
|
Gray, D. A.
1991.
Insertional mutagenesis: neoplasia arising from retroviral integration.
Cancer Investig.
9:295-304[Medline].
|
| 10.
|
Haas, J.,
E. C. Park, and B. Seed.
1996.
Codon usage limitation in the expression of HIV-1 envelope glycoprotein.
Curr. Biol.
6:315-324[CrossRef][Medline].
|
| 11.
|
Hasse, A., and W. A. Schulz.
1994.
Enhancement of reporter gene de novo methylation by DNA fragments from the alpha-fetoprotein control region.
J. Biol. Chem.
269:1821-1826[Abstract/Free Full Text].
|
| 12.
|
Hoeben, R. C.,
A. A. Migchielsen,
R. C. van der Jagt,
H. van Ormondt, and A. J. van der Eb.
1991.
Inactivation of the Moloney murine leukemia virus long terminal repeat in murine fibroblast cell lines is associated with methylation and dependent on its chromosomal position.
J. Virol.
65:904-912[Abstract/Free Full Text].
|
| 13.
|
Huang, M. T., and C. M. Gorman.
1990.
Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.
Nucleic Acids Res.
18:937-947[Abstract/Free Full Text].
|
| 14.
|
Hunter, E.
1994.
Macromolecular interactions in the assembly of HIV and other retroviruses.
Semin. Virol.
5:71-83.
|
| 15.
|
Ihle, J. N.,
B. Smith-White,
B. Sisson,
D. Parker,
D. G. Blair,
A. Schultz,
C. Kozak,
R. D. Lunsford,
D. Askew,
Y. Weinstein, and R. J. Isfort.
1989.
Activation of the c-H-ras proto-oncogene by retrovirus insertion and chromosomal rearrangement in a Moloney leukemia virus-induced T-cell leukemia.
J. Virol.
63:2959-2966[Abstract/Free Full Text].
|
| 16.
|
Jahner, D., and R. Jaenisch.
1985.
Retrovirus-induced de novo methylation of flanking host sequences correlates with gene inactivity.
Nature
315:594-597[CrossRef][Medline].
|
| 17.
|
Jähner, D., and R. Jäenisch.
1985.
Chromosomal position and specific demethylation in enhancer sequences of germ line-transmitted retroviral genomes during mouse development.
Mol. Cell. Biol.
5:2212-2220[Abstract/Free Full Text].
|
| 18.
|
Jähner, D.,
H. Stuhlmann,
C. L. Stewart,
K. Harbers,
J. Lohler,
I. Simon, and R. Jaenisch.
1982.
De novo methylation and expression of retroviral genomes during mouse embryogenesis.
Nature
298:623-628[CrossRef][Medline].
|
| 19.
|
Jang, S. K.,
T. V. Pestova,
C. U. Hellen,
G. W. Witherell, and E. Wimmer.
1990.
Cap-independent translation of picornavirus RNAs: structure and function of the internal ribosomal entry site.
Enzyme
44:292-309[Medline].
|
| 20.
|
Jolly, D. J.,
R. C. Willis, and T. Friedmann.
1986.
Variable stability of a selectable provirus after retroviral vector gene transfer into human cells.
Mol. Cell. Biol.
6:1141-1147[Abstract/Free Full Text].
|
| 21.
|
Juttermann, R.,
E. Li, and R. Jaenisch.
1994.
Toxicity of 5-aza-2'-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation.
Proc. Natl. Acad. Sci. USA
91:11797-11801[Abstract/Free Full Text].
|
| 22.
|
Kempler, G.,
B. Freitag,
B. Berwin,
O. Nanassy, and E. Barklis.
1993.
Characterization of the Moloney murine leukemia virus stem cell-specific repressor binding site.
Virology
193:690-699[CrossRef][Medline].
|
| 23.
|
Kuriyama, S.,
T. Sakamoto,
M. Kikukawa,
T. Nakatani,
Y. Toyokawa,
H. Tsujinoue,
K. Ikenaka,
H. Fukui, and T. Tsujii.
1998.
Expression of a retrovirally transduced gene under control of an internal housekeeping gene promoter does not persist due to methylation and is restored partially by 5-azacytidine treatment.
Gene Ther.
5:1299-1305[CrossRef][Medline].
|
| 24.
|
Laker, C.,
J. Meyer,
A. Schopen,
J. Friel,
C. Heberlein,
W. Ostertag, and C. Stocking.
1998.
Host cis-mediated extinction of a retrovirus permissive for expression in embryonal stem cells during differentiation.
J. Virol.
72:339-348[Abstract/Free Full Text].
|
| 25.
|
Lazo, P. A., and P. N. Tsichlis.
1988.
Recombination between two integrated proviruses, one of which was inserted near c-myc in a retrovirus-induced rat thymoma: implications for tumor progression.
J. Virol.
62:788-794[Abstract/Free Full Text].
|
| 26.
|
Lengauer, C.,
K. W. Kinzler, and B. Vogelstein.
1997.
DNA methylation and genetic instability in colorectal cancer cells.
Proc. Natl. Acad. Sci. USA
94:2545-2550[Abstract/Free Full Text].
|
| 27.
|
Link, C. J., Jr.,
E. M. Kolb, and R. R. Muldoon.
1995.
Preliminary in vitro efficacy and toxicity studies of the herpes simplex thymidine kinase gene system for the treatment of breast cancer.
Hybridoma
14:143-147[Medline].
|
| 28.
|
Link, C. J., Jr.,
D. Moorman,
T. Seregina,
J. P. Levy, and K. J. Schabold.
1996.
A phase I trial of in vivo gene therapy with the herpes simplex thymidine kinase/ganciclovir system for the treatment of refractory or recurrent ovarian cancer.
Hum. Gene Ther.
7:1161-1179[Medline].
|
| 29.
|
Markowitz, D.,
S. Goff, and A. Bank.
1988.
A safe packaging line for gene transfer: separating viral genes on two different plasmids.
J. Virol.
62:1120-1124[Abstract/Free Full Text].
|
| 30.
|
Mikovits, J. A.,
H. A. Young,
P. Vertino,
J. P. Issa,
P. M. Pitha,
S. Turcoski-Corrales,
D. D. Taub,
C. L. Petrow,
S. B. Baylin, and F. W. Ruscetti.
1998.
Infection with human immunodeficiency virus type 1 upregulates DNA methyltransferase, resulting in de novo methylation of the gamma interferon (IFN- ) promoter and subsequent downregulation of IFN- production.
Mol. Cell. Biol.
18:5166-5177[Abstract/Free Full Text].
|
| 31.
|
Miller, A. D.
1996.
Cell-surface receptors for retroviruses and implications for gene transfer.
Proc. Natl. Acad. Sci. USA
93:11407-11413[Abstract/Free Full Text].
|
| 32.
|
Miller, A. D., and C. Buttimore.
1986.
Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.
Mol. Cell. Biol.
6:2895-2902[Abstract/Free Full Text].
|
| 33.
|
Miller, A. D., and F. Chen.
1996.
Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry.
J. Virol.
70:5564-5571[Abstract/Free Full Text].
|
| 34.
|
Miller, A. D.,
J. V. Garcia,
N. Von Shur,
C. M. Lynch,
C. Wilson, and M. V. Eiden.
1991.
Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus.
J. Virol.
65:2220-2224[Abstract/Free Full Text].
|
| 35.
|
Miller, A. D., and G. J. Rosman.
1989.
Improved retroviral vectors for gene transfer and expression.
BioTechniques
7:980-982[Medline], 984-986, 989-990.
|
| 36.
|
Miller, A. D.,
D. R. Trauber, and C. Buttimore.
1986.
Factors involved in production of helper virus-free retrovirus vectors.
Somat. Cell Mol. Genet.
12:175-183[CrossRef][Medline].
|
| 37.
|
Moolten, F. L.
1986.
Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy.
Cancer Res.
46:5276-5281[Abstract/Free Full Text].
|
| 38.
|
Muenchau, D. D.,
S. M. Freeman,
K. Cornetta,
J. A. Zwiebel, and W. F. Anderson.
1990.
Analysis of retroviral packaging lines for generation of replication-competent virus.
Virology
176:262-265[CrossRef][Medline].
|
| 39.
|
Muldoon, R. R.,
J. P. Levy,
S. R. Kain,
P. A. Kitts, and C. J. Link, Jr.
1997.
Tracking and quantitation of retroviral-mediated transfer using a completely humanized, red-shifted green fluorescent protein gene.
BioTechniques
22:162-167[Medline].
|
| 40.
|
Nelson, D. M.,
J. J. Wahlfors,
L. Chen,
M. Onodera, and R. A. Morgan.
1998.
Characterization of diverse viral vector preparations, using a simple and rapid whole-virion dot-blot method.
Hum. Gene Ther.
9:2401-2405[Medline].
|
| 41.
|
Nielsen, C. S.,
D. W. Moorman,
J. P. Levy, and C. J. Link, Jr.
1997.
Herpes simplex thymidine kinase gene transfer is required for complete regression of murine colon adenocarcinoma.
Am. Surg.
63:617-620[Medline].
|
| 42.
|
Odawara, T.,
M. Oshima,
K. Doi,
A. Iwamoto, and H. Yoshikura.
1998.
Threshold number of provirus copies required per cell for efficient virus production and interference in Moloney murine leukemia virus-infected NIH 3T3 cells.
J. Virol.
72:5414-5424[Abstract/Free Full Text].
|
| 43.
|
Oldfield, E. H.,
Z. Ram,
K. W. Culver,
R. M. Blaese,
H. L. DeVroom, and W. F. Anderson.
1993.
Gene therapy for the treatment of brain tumors using intra-tumoral transduction with the thymidine kinase gene and intravenous ganciclovir.
Hum. Gene Ther.
4:39-69[Medline].
|
| 44.
|
O'Neill, R.,
M. O'Neill, and J. A. Graves.
1998.
Undermethylation associated with retroelement activation and chromosome remodelling in an interspecific mammalian hybrid.
Nature
393:68-72[CrossRef][Medline].
|
| 45.
|
Ott, D. E.,
S. M. Nigida, Jr.,
L. E. Henderson, and L. O. Arthur.
1995.
The majority of cells are superinfected in a cloned cell line that produces high levels of human immunodeficiency virus type 1 strain MN.
J. Virol.
69:2443-2450[Abstract].
|
| 46.
|
Otto, E.,
A. Jones-Trower,
E. F. Vanin,
K. Stambaugh,
S. N. Mueller,
W. F. Anderson, and G. J. McGarrity.
1994.
Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences.
Hum. Gene Ther.
5:567-575[Medline].
|
| 47.
|
Pear, W. S.,
G. P. Nolan,
M. L. Scott, and D. Baltimore.
1993.
Production of high-titer helper-free retroviruses by transient transfection.
Proc. Natl. Acad. Sci. USA
90:8392-8396[Abstract/Free Full Text].
|
| 48.
|
Robbins, P. B.,
X. J. Yu,
D. M. Skelton,
K. A. Pepper,
R. M. Wasserman,
L. Zhu, and D. B. Kohn.
1997.
Increased probability of expression from modified retroviral vectors in embryonal stem cells and embryonal carcinoma cells.
J. Virol.
71:9466-9474[Abstract].
|
| 49.
|
Roberts, R. J.
1990.
Restriction enzymes and their isoschizomers.
Nucleic Acids Res.
18(Suppl.):2331-2365.
|
| 50.
|
Roth, J. A., and R. J. Cristiano.
1997.
Gene therapy for cancer: what have we done and where are we going?
J. Natl. Cancer Inst.
89:21-39[Abstract/Free Full Text].
|
| 51.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed., p. 9.14-9.23.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 52.
|
Schwartz, J. R.,
S. Duesberg, and P. Duesberg.
1995.
DNA recombination is sufficient for retroviral transduction.
Proc. Natl. Acad. Sci. USA
92:2460-2464[Abstract/Free Full Text].
|
| 53.
|
Seperack, P. K.,
M. C. Strobel,
D. J. Corrow,
N. A. Jenkins, and N. G. Copeland.
1988.
Somatic and germ-line mutation rates of the retrovirus-induced dilute coat-color mutation of DBA mice.
Proc. Natl. Acad. Sci. USA
85:189-192[Abstract/Free Full Text].
|
| 54.
|
Sommerfelt, M. A., and R. A. Weiss.
1990.
Receptor interference groups of 20 retroviruses plating on human cells.
Virology
176:58-69[CrossRef][Medline].
|
| 55.
|
Stewart, C. L.,
H. Stuhlmann,
D. Jahner, and R. Jaenisch.
1982.
De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells.
Proc. Natl. Acad. Sci. USA
79:4098-4102[Abstract/Free Full Text].
|
| 56.
|
Szyf, M.,
G. Tanigawa, and P. L. McCarthy, Jr.
1990.
A DNA signal from the Thy-1 gene defines de novo methylation patterns in embryonic stem cells.
Mol. Cell. Biol.
10:4396-4400[Abstract/Free Full Text].
|
| 57.
|
Teyssier, J. R.,
J. Benard,
D. Ferre,
J. Da Silva, and L. Renaud.
1989.
Drug-related chromosomal changes in chemoresistant human ovarian carcinoma cells.
Cancer Genet. Cytogenet.
39:35-43[CrossRef][Medline].
|
| 58.
|
Vanin, E. F.,
M. Kaloss,
C. Broscius, and A. W. Nienhuis.
1994.
Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis.
J. Virol.
68:4241-4250[Abstract/Free Full Text].
|
| 59.
|
Varmus, H. E.,
N. Quintrell, and S. Ortiz.
1981.
Retroviruses as mutagens: insertion and excision of a nontransforming provirus alter expression of a resident transforming provirus.
Cell
25:23-36[CrossRef][Medline].
|
| 60.
|
Weiss, R. A., and C. S. Tailor.
1995.
Retrovirus receptors.
Cell
82:531-533[CrossRef][Medline].
|
| 61.
|
Wilson, C.,
M. S. Reitz,
H. Okayama, and M. V. Eiden.
1989.
Formation of infectious hybrid virions with gibbon ape leukemia virus and human T-cell leukemia virus retroviral envelope glycoproteins and the gag and pol proteins of Moloney murine leukemia virus.
J. Virol.
63:2374-2378[Abstract/Free Full Text].
|
| 62.
| Young, W.-B., E. J. Beecham, G. L. Lindberg, and C. J. Link, Jr. Restriction mapping of
retroviral vector episomal DNA. BioTechniques, in press.
|
Journal of Virology, April 2000, p. 3177-3187, Vol. 74, No. 7
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
