Download Gene expression analysis of murine cells producing amphotropic

Journal of General Virology (2003), 84, 1677–1686
DOI 10.1099/vir.0.18871-0
Gene expression analysis of murine cells
producing amphotropic mouse leukaemia virus at a
cultivation temperature of 32 and 37 ˚C
Christiane Beer,1 Petra Buhr,1 Heidi Hahn,2 Daniela Laubner3 and
Manfred Wirth1
1
Molecular Biotechnology, German Research Centre for Biotechnology, GBF,
Mascheroder Weg 1, D-38124 Braunschweig, Germany
Correspondence
Manfred Wirth
2
[email protected]
Human Genetics, Georg August University, Göttingen, Germany
3
Institute for Experimental Genetics, GSF-National Research Centre for Environment and
Health, Neuherberg, Germany
Received 1 October 2002
Accepted 26 February 2003
Cultivation of retrovirus packaging cells at 32 ˚C represents a common procedure to achieve high
titres in mouse retrovirus production. Gene expression profiling of mouse NIH 3T3 cells producing
amphotropic mouse leukaemia virus 4070A revealed that 10 % of the 1176 cellular genes
investigated were regulated by temperature shift (37/32 ˚C), while 5 % were affected by retrovirus
infection. Strikingly, retrovirus production at 32 ˚C activated the cholesterol biosynthesis/transport
pathway and caused an increase in plasma membrane cholesterol levels. Furthermore, these
conditions resulted in transcriptional activation of smoothened (smo), patched (ptc) and gli-1; Smo,
Ptc and Gli-1, as well as cholesterol, are components of the Sonic hedgehog (Shh) signalling
pathway, which directs pattern formation, diversification and tumourigenesis in mammalian cells.
These findings suggest a link between cultivation at 32 ˚C, production of MLV-A and the Shh
signalling pathway.
INTRODUCTION
Retrovirus vector systems are involved in more than 50 %
of clinical protocols for gene transfer (Mountain, 2000).
Retrovirus vectors based on amphotropic mouse leukaemia
virus (MLV-A) are common vehicles for efficient gene
transfer into a variety of cells (Wu & Ataai, 2000). These
viruses are well characterized, exhibit high transduction
efficiencies and integrate stably into the genome of the host
cell. The problem of in vivo instability in primates, which
concerns viruses decorated with a-1,3-Gal epitopes and
which results in virus inactivation via the human complement system, has been minimized, for example, by the
development of non-mouse-derived packaging cells deficient in a-1,3-Gal glycosylation and by enzyme competition
(Cosset et al., 1995; unpublished data). To date, low titres
and physico-chemical instability still represent limitations
in the use of retrovirus packaging cell lines. Efforts have
been made to improve productivity of the packaging cell
lines and to optimize cultivation conditions and downstream processing (Kaptein et al., 1997; Kotani et al., 1994).
For example, several reports support the findings that
shifting the cultivation temperature from 37 to 32 uC leads
to an increase in virus titre by 4- to 15-fold after extended
cultivation. These higher ‘endpoint titres’ are thought to
be due to the increased thermal stability of retroviruses at
32 uC, while cellular productivity for retroviruses is affected
0001-8871 G 2003 SGM
only marginally at the reduced temperature (Le Doux et al.,
1999). However, recent reports and our own investigations
could not confirm these initial data (Forestell et al., 1995;
Cruz et al., 2000; Beer et al., 2003). Thus, additional factors
must contribute to explain these observations. To investigate the molecular basis of temperature shift and virus
production on cellular gene expression, we have analysed
the respective cellular transcription profiles in detail. DNA
microarrays are powerful tools to screen for alterations in
transcription levels of cellular genes. Therefore, microarray
analysis was used to investigate the effect of temperature
reduction on the host cell during virus production.
METHODS
Cells and retrovirus vectors. Cells were grown at 37 or 32 uC,
5 % CO2 and 95 % humidity in DMEM (Gibco-BRL) supplemented
with antibiotics and 10 % FCS. For the generation of wt retrovirus
MLV vectors, a helper virus approach was used to follow the infection process. Briefly, 5 mg p4070AMLV, which encodes a wt MLV-A
provirus (a kind gift from O. Merten and O. Danos, Genethon,
France), and 0?5 mg of the retrovirus vector pLEIN (Clontech),
which contains the EGFP (enhanced green fluorescent protein) and
the G418-resistance gene, were co-transfected into mouse NIH 3T3
cells (ATCC, CRL1658) using the calcium–phosphate DNA transfection protocol, as described previously (Wirth et al., 1988). Stably
transfected cells were selected with 1000 mg G418 ml21. TE-Fly is a
packaging cell line derived from the human rhabdomyosarcoma cell
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C. Beer and others
line TE671 (ATCC HTB-139, CRL 8805). TE-Fly A7 (a kind gift
from J.-L. Cosset, Lyon, France, via O. Merten) is derived from
TE-Fly stably transfected with pMFGNLSlacZ (Ferry et al., 1991)
and releases a replication-defective amphotropic MLV vector (Cosset
et al., 1995). Virus G418 and LacZ titre assays were performed as
described previously (Wirth et al., 1994).
Gene expression analysis. To study the effect of a 37 to 32 uC
temperature shift on gene expression, non-producing and wt MLVA-producing NIH 3T3 cells were incubated for 24 h at 32 or 37 uC
in DMEM supplemented with 10 % FCS and 2 mM L-glutamine.
Total RNA from these cells was isolated and the Atlas Mouse 1.2
Array II and data analyses were performed as described previously
(Lechner et al., 2001). Total RNA isolation was performed using
TriFast FL (Peqlab), as described by the manufacturer. A second
RNA extraction step and DNase I digestion was included to avoid
DNA contamination. cDNA used for hybridization was synthesized
with 3?5 mg RNA in the presence of 32P-labelled nucleotides.
Quantification (spot densities, local background) was performed
using a PhosphorImager (Fuji) and Array-Vision software, version
5.1. After background subtraction, a threshold level of 1?4 was determined. For normalization of individual spot intensities, the average
of the total of all spot intensities was used.
Customized HPSF OligoChip arrays were delivered by MWG-Biotech.
For each gene, three oligonucleotides, each derived from different
regions of the genome, were spotted in duplicate. Total RNA (10 mg)
from non-producing and wt MLV-A-producing NIH 3T3 cells
cultivated at 32 or 37 uC was labelled using the Micromax TSA
Labelling and Detection kit (Perkin Elmer), according to the
manufacturer’s instructions. cDNA hybridization on glass arrays was
carried out at 42 uC, according to the instructions of the manufacturer. Spot intensities were determined using the EasyWin32 software
(Herolab), after scanning with an Affymetrix 418 Array Scanner. Based
on the signals derived from nine housekeeping genes (internal control),
a threshold for gene regulation was defined for each array and errors
were calculated from variation in signal intensities. Processed array
data is available online at http://www.gbf.de/mbio/mwi.
Cholesterol assays. To determine cellular cholesterol concentra-
tions, NIH 3T3 cells were plated onto 96-well plates (46103 cells
per well) and cultivated for 24 h at 37 or 32 uC. Filipin staining was
performed as described previously (Gu et al., 1997). For fluorometry, a Bio-Tek MikroTek microplate reader was used (excitation
wavelength, 360 nm; emission wavelength, 460 nm). Filipin-labelled
NIH 3T3 cells were photographed using a Photometrics Coolsnap
Colour-CCD camera (filter set XF113; excitation wavelength,
387 nm; emission wavelength, 450 nm; Omegafilters).
Gli-1 reporter assays. To perform the Gli-1 reporter assay, the
plasmid p116Gli1-BS, which contains Gli-1-binding sites in front
of a herpes simplex virus thymidine kinase (HSV-TK) promoter and
firefly luciferase was stably transfected into NIH 3T3 cells. To investigate the effect of the temperature shift and/or virus infection on
gli-1 expression, the cells were mock- or wt MLV-A-infected (m.o.i.
of 10) in the presence of polybrene (8 mg ml21) and cultivated at 32
or 37 uC for 0, 24 and 72 h. After the time intervals indicated, luciferase expression was determined (Luciferase Assay system, Promega)
and normalized to protein content (BCA Protein Assay kit, Pierce).
The Gli-1-encoding plasmid, pFLAG-Gli1, was co-transfected with
p116Gli1-BS and used as a positive control. The vector pGL-TK,
containing the luciferase gene driven by the HSV-TK promoter, was
used as a negative control.
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RESULTS
MLV-A production and downshift of cultivation
temperature to 32 ˚C results in complex
alterations in cellular gene expression
The temperature shift of MLV-A-producing cells to 32 uC
has been reported to result in high-yield vector production
(Kaptein et al., 1997; Kotani et al., 1994). It was assumed that
increased accumulative titres resulted from an increase in
the half-life of the virus during the 32 uC production period,
while at the same time, cellular productivity for viruses was
altered only marginally (Le Doux et al., 1999). Surprisingly,
in our hands, neither cultivation at 32 uC of mouse NIH 3T3
cells releasing amphotropic MLV hybrid viruses generated
by a helper virus approach, nor human TE-Fly packaging
cells synthesizing a replication-defective MLV-A vector,
resulted in increased virus yield compared to that observed
at 37 uC (Table 1). While virus titres from TE-Fly packaging
cells remained the same upon cultivation of cells at 32 uC,
virus titres from NIH 3T3 host cells decreased more than
10-fold compared to 37 uC propagation of host cells.
Interestingly, the total numbers of virus particles released
from NIH 3T3 and TE-Fly host cells were not, or only
slightly, altered (C. Beer, A. Meyer, K. Müller and M. Wirth,
unpublished data). This suggests that additional parameters
must contribute to the virus titres observed upon 32 uC
propagation of virus-releasing host cells. Shifting the
cultivation temperature to 32 uC, a ‘moderate coldshock’, is known to influence cellular gene expression but
does not inhibit proliferation (Fujita, 1999; Sonna et al.,
2002). Additionally, it may also affect viral gene expression.
Changes in cellular and/or viral gene expression may result
in viruses with altered properties. In a recent investigation,
we noticed no effect on viral gene expression when the total
yield of env and gag gene products in MVL-A was compared
in Western blots from NIH 3T3 cells, cultivated at either 37
or 32 uC (Beer et al., 2003; data not shown). Thus, we
focussed our investigations on alterations in cellular gene
expression using DNA microarrays. Experiments were
designed and the results were evaluated in such a manner
that virally induced effects could be discerned from
Table 1. Effect of 32 ˚C cultivation of host cells on MLV-A
titres
Mean titre (SD)*
Host cell
32 ˚C
37 ˚C
NIH 3T3D
TE-Flyd
5?96106 (6?66105)
4?16105 (2?16105)
6?76107 (1?96107)
4?66105 (3?46105)
*Cells were cultivated for 24 h. Mean titres (from 106 cells per day)
were determined from independent experiments.
DReplication competent, hybrid MLV-A generated by a helper virus
approach.
dMLV-A generated from stably transfected packaging cells.
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Journal of General Virology 84
Gene expression upon 32 uC cultivation
alterations in gene expression caused by the shift in
cultivation temperature. cDNA array hybridization was
performed from both wt MLV-A and non-producing NIH
3T3 cells, cultivated at either 32 or 37 uC. Total RNA was
isolated and reverse-transcribed with gene-specific primers
for the microarray in the presence of 32P-labelled dATP.
32
P-labelled cDNAs were hybridized onto identical replicas
of Atlas Mouse 1.2 Array II, which includes 1176 genes,
and analysed by PhosphorImager quantification. Based on
normalization over the expression of all genes, a factor of
1-4 was considered as a threshold value for gene regulation (Lechner et al., 2001). To distinguish effects of the
temperature shift from the effects caused by the virus–host
interaction to evaluations of data from the different experimental settings were performed concerning the effects of
either virus infection or temperature shift. A selection of
genes in NIH 3T3 cells and wt MLV-A-producing NIH 3T3
cells affected in expression by the temperature shift is
shown in Table 2. The gene listing is derived from two
arrays and encompasses genes that exhibited regulation
upon temperature shift in both mock-infected and virusproducing NIH 3T3 cells. Obviously, cultivation of NIH
3T3 cells at 32 uC causes multiple changes in cellular gene
expression. Approximately 10 % of the genes investigated
were regulated due to the temperature shift to 32 uC. 125
genes (MLV-A-producing NIH 3T3 cells) and 117 (nonproducing NIH 3T3 cells) genes were affected in expression;
of the latter, 59 were upregulated and 58 were downregulated. The affected genes are involved in processes
concerning, for example, transcription, signal transduction,
apoptosis and lipid metabolism. Gene expression of ST2
and FIN13, genes involved in cell proliferation, was altered
by a factor of 3?3 and 5, respectively. ST2 is a response gene
acting at the initial phase of cell proliferation, whereas the
protein FIN13 encodes a serine/threonine phosphatase that
causes G1/S arrest of the cell upon overexpression
(Tominaga, 1989; Guthridge et al., 1997). Interestingly,
gene expression of cellular nucleic acid-binding protein
CNBP, a key factor of cholesterol homeostasis, was
upregulated 2?4-fold.
Virus production itself affected the expression of nearly
5 % of the genes investigated and virus propagation caused
altered expression of 56 (at 37 uC) and 89 (at 32 uC) genes,
respectively. Upon virus propagation, the ratio of up/
downregulation changed, as more genes were upregulated
upon virus propagation compared to mock infection.
Table 3 depicts a selection of genes that exhibited regulated
expression upon virus production, irrespective of the
cultivation temperature. Again, the affected genes are
involved in a variety of cellular processes, like apoptosis,
transport and metabolism of RNA, signal transduction,
transcription, translation and development of tumours. The
most strongly upregulated gene was the smoothened (smo)
gene, which is an important component of the Sonic
hedgehog (Shh) signalling pathway. This pathway plays a
fundamental role in early embryonic patterning of the
neural tube, axial skeleton, limbs and lungs. In addition,
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patched (ptc) has been reported to act as a tumour repressor
gene, whereas shh and smo are proto-oncogenes (Hahn et al.,
1999). The 20?5-fold increase in smo gene expression is
not only due to the temperature shift but is caused by the
combination of temperature shift and wt MLV-A production. Strikingly, the expression of a huge number of
genes of the cytoskeleton gene family is affected by virus
interaction. This is indicative for a role of the cytoskeleton
in the virus life cycle.
Effect of the cultivation of cells at 32 ˚C on
plasma membrane cholesterol levels
Upregulation of the CNBP gene was striking and attracted
our interest. The CNBP gene is upregulated by sterols like
cholesterol and, thus, may indicate increased levels of
cellular cholesterol (Rajavashisth et al., 1989). Cholesterol
exhibits numerous cellular functions. Cholesterol determines the fluidity and physical state of the cellular plasma
membrane. To investigate whether the cultivation of NIH
3T3 cells at 32 uC induces an increase in the production
of cellular cholesterol, we used filipin to quantify the
cholesterol content of the plasma membrane of wt MLV-Aproducing NIH 3T3 cells cultivated at 32 and 37 uC. Filipin
is a fluorescent polyene macrolide which binds specifically
to cholesterol and is used for staining the cholesterol of
biological membranes (Gu et al., 1997). Quantification of
the fluorescence using a fluorescence microplate reader
showed a 1?4-fold increase in cholesterol concentration
(statistically significant by Student’s t-test, probability of
99?5 %). This finding could be confirmed by fluorescence
microscopy and evaluation of the digitized images of the
stained cells (Fig. 1). Control NIH 3T3 cells exhibited a
comparable effect (data not shown). Thus, CNBP upregulation coincides with increased levels of plasma membrane
cholesterol.
Interestingly, cholesterol is a key compound in the activation of the Shh signalling pathway (Fig. 2). Increased levels
of cholesterol are associated with smo gene upregulation.
Shh has cholesterol covalently attached to its N-terminal
end. It is tempting to speculate that the sterol-sensing
domain (SSD) of Ptc has a role in targeting cholesterolmodified Shh, which then triggers the activation of the
signalling pathway. However, although the SSD of Ptc is
essential for its activity, recent reports show that the SSD
of Ptc is not involved in the interaction with Shh (Martin
et al., 2001; Strutt et al., 2001). Rather, the SSD might be
important for inhibition of Smo (Martin et al., 2001). In
addition, a role for Ptc in cholesterol transport and homeostasis is currently under discussion (Hahn et al., 1999).
Induction of the Shh signalling pathway
To confirm the influence of temperature shift and virus
production on the Shh signalling pathway, we used
customized HPSF OligoChip arrays containing oligonucleotides for the smo, ptc and gli-1 genes. Ptc, a Shh receptor,
and Gli-1, a transcription factor, are key elements of the
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C. Beer and others
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Table 2. Effect of temperature on cellular gene expression in wt MLV-A-producing and non-producing NIH 3T3 cells propagated at 37 or 32 ˚C
Position on Atlas
Mouse 1.2 Array II
Induced/downregulated
gene
Ratio
C32 ˚C/C37 ˚C*
Ratio
V32 ˚C/V37 ˚C*
A03j
ST2
3?1
3?3
E10k
E12d
FIN13
ALG2
1?6
1?5
5?0
2?2
B03i
PBR
21?8
21?8
D01l
F04i
C14h
CNBP
CTSL
PTB
1?9
1?5
1?4
2?4
2?0
2?1
B04m
4F2/CD98 lc
1?5
1?8
B13m
D01f
E07g
E09e
G6PD
HMGI-C
smo
snk
1?6
3?0
1?0
1?4
2?9
3?2
20?5
1?9
Putative functions of gene products (accession no.)
Response gene at initial stage of cell proliferation; ST2 gene products
are thought to have functions as signal transducers (Y07519)
Encodes a type 2C serine/threonine phosphatase; overexpression causes G1/S arrest (U42383)
Ca2+-binding protein; required for T-cell receptor-, Fas- and glucocorticoid-induced apoptosis,
apoptotic function is regulated by Ca2+ binding of the protein (U49112)
Involved in regulation of cellular proliferation, immunomodulation, steroidogenesis, apoptosis;
mediates cholesterol delivery from the outer to the inner mitochondrial membrane (D21207)
Binds to the SRE; has a role in sterol-mediated control of transcription (L12693)
Lysosomal cysteine proteinase, which is activated by growth factors and oncogenes (X06086)
Plays a role in tissue-specific regulation of alternative splicing, control
of polyadenylation and mRNA localization (X52101)
Functions in lymphocyte activation, integrin signalling,
membrane fusion, tumour progression and amino acid transport (AB017189)
Catalyses the first step of the hexose monophosphate pathway, essential for cell division (Z11911)
Transcription factor, involvement in cell cycle, overexpression in tumours (X58380)
Transmembrane protein involved in the Shh/Ptc/Smo signalling pathway (AF089721)
Polo-like kinase, serine/threonine-specific protein kinase, involved in cell proliferation,
possibly mediating signalling in response to Ca2+ influx (M96163)
Journal of General Virology 84
*The ratio is calculated by dividing the spot densities of cells cultivated at 32 uC by the spot densities of cells cultivated at 37 uC. Downregulated genes are indicated by a negative ratio. C32 uC,
control NIH 3T3 cells cultivated at 32 uC; C37 uC, control NIH 3T3 cells cultivated at 37 uC; V32 uC, wt MLV-A-producing NIH 3T3 cells cultivated at 32 uC; V37 uC, wt MLV-A-producing NIH
3T3 cells cultivated at 37 uC.
FIN13, Fibroblast growth factor-inducible gene 13; ALG2, apoptosis-linked gene 2; PBR, peripheral benzodiazepine receptor; CNBP, cellular nucleic acid-binding protein; CTSL, cathepsin L
precursor; PTB, polypyrimidine tract-binding protein; 4F2/CD98 lc, CD98 light chain; G6PD, X-linked glucose-6-phosphate dehydrogenase; HMGI-C, high-mobility group protein I isoform C;
smo, smoothened; Snk, serum-inducible kinase; SRE, sterol regulatory element.
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Table 3. Effect of infection on gene expression in wt MLV-A-producing and non-producing NIH 3T3 cells due to virus production at 37 or 32 ˚C
Position on Atlas
Mouse 1.2 Array II
Induced/downregulated
gene
Ratio
V32 ˚C/C32 ˚C*
Ratio
V37 ˚C/C37 ˚C*
E12d
ALG2
2?4
1?6
F08a
F09m
Fascin homologue 1
Non-muscle myosin MYL6
2?7
1?8
1?7
2?1
F09l
E12a
Myosin isoform MLC1EMB
Annexin A2
4?4
2?1
5?1
2?0
A12h
H60
28?3
25?9
C14b
hnRNP A1
4?2
2?4
D01d
HMG 2
1?7
3?1
B08e
Fibronectin 1
27?3
27?5
A01j
CD37 antigen
4?8
11?2
Putative functions of gene products (accession no.)
2+
Ca -binding protein; required for T-cell receptor-, Fas- and glucocorticoid-induced
apoptosis, apoptotic function is regulated by Ca2+ binding of the protein (U49112)
Actin cross-linking protein; has a role in cell motility and cell adhesion (L33726)
Actin-based motor protein; important for different aspects of muscle and
non-muscle cell motility, thought to play a role in cancer cell metastasis (U04443)
Actin-based motor protein (M20772)
Calcium-dependent, phospholipid-binding protein; extracellular annexin II is thought to play
a role in tumour metastasis and in cell–cell adherence of non-metastatic cells (D10024)
MHC class I-like protein; recognition by NKG2D of H60-related proteins
thought to be involved in anti-viral and anti-tumour immune response (AF084643)
Involved in different RNA-related processes, including alternative RNA splicing and
mRNA transport; thought to recruit telomerase to telomeres (D86728)
Binds to DNA without sequence specificity; plays a role in recombination,
initiation of transcription and DNA repair (Z46757)
Component of the extracellular matrix of the cell; mediates cell adhesion,
migration and signal transduction (X93167)
CD antigen expressed on chronic lymphocytic leukaemia (U18372)
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Gene expression upon 32 uC cultivation
*C32 uC, Control NIH 3T3 cells cultivated at 32 uC; C37 uC, control NIH 3T3 cells cultivated at 37 uC; V32 uC, wt MLV-A-producing NIH 3T3 cells cultivated at 32 uC; V37 uC, wt MLV-Aproducing NIH 3T3 cells cultivated at 37 uC.
ALG2, Apoptosis-linked gene 2; H60, histocompatability antigen 60; hnRNP A1, heterogeneous nuclear ribonucleoprotein A1; HMG 2, high-mobility group protein 2; NKG2D, natural killer cell
receptor.
C. Beer and others
were hybridized onto identical replicas of the arrays,
detected and analysed as described in Methods. Based on
nine housekeeping genes, an appropriate threshold value
for gene regulation was defined for each array.
Fig. 1. Cultivation of NIH 3T3 cells at 32 ˚C causes an
increase in the cholesterol concentration of the plasma membrane. To study the effect of the temperature shift from 37 to
32 ˚C on NIH 3T3 cells, wt MLV-A-producing cells were incubated at 37 and 32 ˚C for 24 h and stained for cholesterol
with filipin (magnification, 61000). (a) NIH 3T3 cells cultivated
at 32 ˚C; (b) NIH 3T3 cells cultivated at 37 ˚C. (c) Quantification of the cholesterol levels of NIH 3T3 cells cultivated at 32
or 37 ˚C for 24 h. Data were obtained by fluorimetry using filipinstained NIH 3T3 cells grown in 96-well plates (statistic significance by Student’s t-test, P=0?05).
Shh signalling pathway. For smo, ptc and gli-1 gene
expression profiling, total RNA was isolated from nonand wt MLV-A-producing NIH 3T3 cells cultivated at either
32 or 37 uC. In contrast to the 1.2K arrays (Clontech), the
oligonucleotides for the customized array encountered
specific regions of the 59UTR, coding region and 39UTR of
the genes. To exclude the different biases in labelling and
hybridization of Cy-labelled dNTPs, the RNA was reversetranscribed with poly(dT) primers in the presence of
biotin- or fluorescein-labelled dCTP. Labelled cDNAs
Fig. 3 shows the results of the array experiments. Upregulation of smo gene expression was confirmed using a
customized array. The induction level of 2?65-fold was not
as high as that determined by the 1.2K array, which is due to
the different experimental settings, such as oligonucleotide
sets and cDNA labelling (radioactive versus fluorescence).
Induction of smo gene expression only occurred under the
condition of temperature shift and wt MLV-A infection.
Besides smo, ptc gene expression is also activated when
retroviruses are propagated in NIH 3T3 cells at 32 uC.
However, induction of gli-1 was detected in both temperature-shifted wt MLV-A and non-producing NIH 3T3 cells.
Wt MLV-A propagation in NIH 3T3 cells alone did not
cause any change in the gene expression of smo, ptc and gli-1
at either 32 or 37 uC (Fig 3a, b).
Since transcriptional upregulation of genes does not
necessarily reflect the activity of the respective gene
products, we performed an assay to measure directly the
activation of the Shh/Ptc/Smo signalling pathway. For that
purpose, a firefly luciferase (Photinus pyralis) reporter gene
assay for Gli-1 has been developed that allows the quantification of Gli-1 activity. Gli-1 plays a central role in the
Shh signalling pathway and the induction of this pathway
results in the activation of Gli-1 (Hahn et al., 1999).
To investigate the effect of the temperature shift and/or
virus production on the activity of Gli-1, the plasmid
p116Gli-BS was stably transfected in NIH 3T3 cells. This
plasmid contains nine binding sites for the Gli-1 protein
upstream of the luciferase gene driven by a HSV-TK
promoter and binding results in luciferase expression. NIH
3T3 cells stably transfected with a plasmid containing only
the HSV-TK promoter upstream of the luciferase gene were
used to determine background values. Transfected NIH 3T3
cells were mock-infected or infected with wt MLV-A and
Sonic
Hedgehog
Cholesterol
Sonic
Hedgehog
Patched
Sonic
Hedgehog
Smoothened
Patched
Smoothened
Gli-1
Targets
Nucleus
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cell membrane
Fig. 2. Shh signalling pathway. Cholesterolmodified Sonic hedgehog binds to Patched.
Binding results in the dissociation and activation of Smoothened. Active Smoothened
triggers the activation of transcription factor
Gli-1 and subsequent factors.
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Journal of General Virology 84
Gene expression upon 32 uC cultivation
(a)
threshold 1.80
1.50
1.00
0.50
1.42
0.00
gli 1
_0.50
_1.00
patched
_1.52
_1.50
threshold _1.60
_2.00
(c)
non-producing NIH3T3
3.00
2.50
2.00
ratio 32˚C/37˚C
smoothened
_1.12
2.50
ratio wt MLV-A prod./nonprod. NIH3T3
2.00
cultivation at 37˚C
3.00
2.00
1.50
0.50
0.00
_0.50
_1.00
2.31
0.75
0.50
0.00
_0.50
_1.00
_1.50
gli 1
patched
smoothened
threshold _1.60
smoothened
_1.39
threshold 2.00
wt MLV-A producing NIN3T3
3.00
2.50
2.00
1.25
_1.73
_2.00
threshold 1.80
1.00
patched
gli 1
_1.09
_1.50
(d)
1.50
threshold 2.00
1.00
ratio 32˚C/37˚C
ratio wt MLV-A prod./nonprod. NIH3T3
2.50
(b)
cultivation at 32˚C
3.00
1.50
1.00
threshold 1.60
2.49
2.65
1.82
0.50
0.00
_0.50
gli 1
_1.00
_1.50
patched
smoothened
threshold _2.00
_2.00
_2.00
Fig. 3. Effects of temperature shift or wt MLV-A production in NIH 3T3 cells on gli-1, ptc and smo gene expression. Effect of
wt MLV-A production on gli-1, ptc and smo gene expression of NIH 3T3 cells cultivated at 32 (a) and 37 ˚C (b). Effect of the
temperature shift on gli-1, ptc and smo gene expression upon cultivation of NIH 3T3 cells (c) or of wt MLV-A-producing NIH
3T3 cells (d) for 24 h at 37 or 32 ˚C. Expression analyses were done on customized HPSF OligoChip arrays. Ratios were
calculated by the division of the spot densities of the three oligonucleotides per gene (average error rate, 23 %).
Downregulation is indicated by a minus sign (2). Based on the maximum variation in the expression of nine housekeeping
genes, the threshold value for gene regulation was defined for each array.
cultivated at 32 or 37 uC for 24, 48 or 72 h. Luminescence
was quantified to determine Gli-1 activity. The reporter
gene assay confirmed the results derived from microarray
experiments (Fig. 4). Infection of NIH 3T3 cells with wt
MLV-A and release of virions within 24 h after infection
did not activate the Shh signalling pathway at either 37 or
32 uC (Fig. 4, lanes 1 and 2). However, cultivation of
mock- or wt MLV-A-infected NIH 3T3 cells at 32 uC
resulted in activation of Gli-1, as has been suggested by
microarray data. The activity of Gli-1 increases up to more
than 5-fold after 72 h of cultivation at 32 uC. This suggests
that the Shh signalling pathway has been activated by
cultivation of NIH 3T3 cells at 32 uC.
DISCUSSION
Gene expression profiling using cDNA microarrays is a
potent means to elucidate host–virus vector interactions
and the effect of cultivation conditions on these interactions. Using DNA microarray technology, we have investigated the influence of 32 uC cultivation and MLV-A
infection on cellular gene expression.
Formerly, it has been reported that cultivation of retrovirus
http://vir.sgmjournals.org
Fig. 4. Effect of temperature shift or wt MLV-A infection of
NIH 3T3 cells on Gli-1 activity. NIH 3T3 cells transfected with
the Gli-1 reporter plasmid, p116Gli1-BS, were infected with
wt MLV-A or mock-infected and cultivated at 32 or 37 ˚C for
24, 48 or 72 h. After the indicated periods, luciferase expression was determined. Values are normalized to the cellular protein content. C32 ˚C, control NIH 3T3 cells cultivated at
32 ˚C; C37 ˚C, control NIH 3T3 cells cultivated at 37 ˚C;
V32 ˚C, wt MLV-A-infected NIH 3T3 cells cultivated at 32 ˚C;
V37 ˚C, wt MLV-A-infected NIH 3T3 cells cultivated at 37 ˚C.
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C. Beer and others
packaging cells at 32 uC increases the yield of infectious
virus particles by up to 4- to 15-fold (Kaptein et al., 1997;
Kotani et al., 1994). However, we and others could not
confirm these initial findings (Forestell et al., 1995; Cruz
et al., 2000; Beer et al., 2003; this investigation). Generally,
retrovirus yields are dependent on the cultivation time and
temperature. While productivity of cells at 32 uC is only
reduced marginally, temperature stability has been shown
to be increased (Le Doux et al., 1999). This may explain
previous results showing a favourable effect of retrovirus
production at 32 uC. The controversial results suggest that
additional factors contribute to the different effects that
have been noticed. Generally, multiple changes in gene
expression are observed during adaptation to temperatures
lower than 37 uC or virus infection. Growth at low temperatures is known to cause changes in lipid composition,
including increases in fatty acid unsaturation, alterations
in the proportion of lipid classes and lipid : protein ratio
(Cossins, 1994) and causes the induction of expression of
certain genes (Fujita, 1999; Sonna et al., 2002). To delineate
the molecular basis of the temperature shift on cellular
gene expression and MLV-A production, we have investigated the effects of the shift to 32 uC and virus production on
host cell expression and virus replication. Whereas no
differences in virus replication (virus titre, particle numbers
and Env and Gag proteins) were noticed (Table 1; Beer et al.,
2003; data not shown), cellular gene expression was affected
considerably, as determined by DNA microarray techniques.
Of the genes investigated, 10 or 5 % exhibited altered
expression due to the temperature shift and wt MLV-A
production, respectively. Cultivation of NIH 3T3 cells at
32 uC resulted in similar numbers of up- and downregulated
genes; approximately 80 % of the genes regulated during
virus production with or without temperature shifts were
upregulated. This remarkable shift to the upregulation of
gene expression is in accordance with a report of Pietiäinen
et al. (2000), who investigated gene expression during
echovirus type 1 infection. Pietiäinen et al. (2000) found that
only in 20 % of the affected genes was gene expression
downregulated (0?5 % downregulation and 2 % upregulation). However, our data do not reflect results found upon
infection of cells with human immunodeficiency virus type
1 (HIV-1) (Ryo et al., 1999, 2000; Geiss et al., 2000), where
downregulated genes and upregulated genes were equal in
number. This emphasizes that considerable differences exist
in host–pathogen interactions upon lentivirus and oncoretrovirus infection. It is of special interest to elucidate the
kinetics of induction and to identify the virus components
that are responsible for the alterations in cellular gene
expression, as has been investigated for the envelope of
HIV-1 and its interaction with T-cells (Cicala et al., 2002).
We have identified several pathways that respond to cultivation of cells at the reduced temperature. As expected, gene
expression of components of the cell proliferation or cell
cycle machinery were altered (ST2, FIN13, HMGI-C and
snk). This is not surprising, since with decreasing temperature, for example, from 37 to 28 uC, cell proliferation
1684
gradually decreases (for example, in mouse leukaemia cells)
(Fujita, 1999) and is reduced after the temperature shift
from 37 to 32 uC in NIH 3T3 cells (data not shown).
Remarkably, the FIN13 gene is overexpressed more than
3-fold in 32 uC-shifted NIH 3T3 cells. FIN13 overexpression
is known to result in G1/S arrest of the cell cycle (Guthridge
et al., 1997). It has been noticed earlier that the G1 phase
seems to be the most severely affected of the four phases
of the cell cycle (Fujita, 1999). For example, it has been
shown by flow cytometry that mouse BALB 3T3 fibroblasts
cultured at 32 uC exhibited a considerably prolonged G1
phase (Nishiyama et al., 1997). Thus, during mild
hypothermia of NIH 3T3 cells, FIN13 may cause a phase
extension or cell cycle arrest, thereby reducing cell growth.
The production of wt MLV-A resulted in notable changes
in the gene expression of members of the cytoskeleton and
cell adhesion gene families (for example, myosin, fascin,
annexin and fibronectin). Modification of the cytoskeleton
as a result of virus infection has been described for several
virus families (Cudmore et al., 1997; Ryo et al., 1999).
Interaction with the cytoskeleton is important for certain
steps in the life cycle of distinct viruses, for example, entry,
budding and motility (Cudmore et al., 1997), and supports
the efficient and directed transport of virus components
to the places of virus replication, assembly and budding
(Sodeik, 2000). Based on microarray results, further detailed
investigations concerning the role of the cytoskeleton in
MLV replication and infection are promising.
In comparison to cells grown at 37 uC, cultivation of nonproducing as well as MLV-A-producing cells at 32 uC
was accompanied by an activation of gene expression of
members of the cholesterol biosynthesis/transport pathway, resulting in an increase in cholesterol in the plasma
membrane concomitant with an increase in the transcription of genes involved in Shh/Ptc/Smo signalling.
As viral membranes are derived from the cellular plasma
membrane, an interesting consequence of activation of
cholesterol biosynthesis/transport and an increase in the
levels of cellular cholesterol could be an increase in the
cholesterol content of the outer viral shell. This issue was
part of a concomitant investigation that followed initial
microarray experiments (Beer et al., 2003). Here, we have
shown for the first time that cultivation of MLV-Aproducing NIH 3T3 cells at 32 uC resulted in the production of phenotypically altered viruses. The investigation
revealed that cultivation of MLV-A producing NIH 3T3
cells at 32 uC resulted in an increased incorporation of
cholesterol into the viral membrane and, moreover, in
phenotypically altered retroviruses. Interestingly, these
viruses exhibited stability at lower temperatures compared
to viruses released from cells propagated at 37 uC, which
manifests as a decrease in the virus half-life at a given
temperature. Furthermore, we could provide direct evidence for a link between retrovirus cholesterol levels and
MLV-A half-life (Beer et al., 2003).
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Journal of General Virology 84
Gene expression upon 32 uC cultivation
Activation of the Shh signalling pathway was indicated by
smo gene upregulation using microarrays and was confirmed by activation of Gli-1, which occupies a central
position in Shh signalling (using a luminescent promoter
activation assay). Although the signalling pathway is
induced by cultivation at 32 uC, virus infection alone does
not affect Gli-1 activity. MLV-A production of NIH 3T3
cells blocks the expression of members of the Shh/Ptc/Smo
signalling pathway at both temperatures and, when compared to non-producing NIH 3T3 cells, does not lead to
activation, as determined by Gli-1 activity. Therefore, one
may speculate that the activation of the signalling pathway
is due to the increase in the cholesterol concentration in
the plasma membrane rather than virus infection or virus
production. Cholesterol is a key player in the transport
and, most likely, the function of the Shh receptor complex
(Karpen et al., 2001). These authors reported an association
of Ptc with caveolin-1, which is the major coat protein of
caveolae. Caveolae are cholesterol-rich invaginations of the
cellular plasma membrane that are involved in endocytosis,
cholesterol trafficking and several signalling pathways
(Okamoto et al., 1998). It is, therefore, conceivable that
the increase in plasma membrane cholesterol has an effect
on the correct trafficking of the Shh receptor complex and,
thus, on the activation of the Shh signalling pathway.
Furthermore, activation by a conformational change of the
Shh receptor complex is an alternate mechanism to account
for our observations. Further experiments are necessary to
elucidate the role of Shh pathway activation upon temperature shift to 32 uC. As shh and smo are protooncogenes
and ptc is a tumour suppressor gene (Hahn et al., 1999), it is
conceivable that the conditions applied in our experiments
may predispose cells to tumour development. Therefore, it
is of considerable interest to confirm our results using
primary fibroblasts and to perform the respective experiments indicating a predisposition to tumourigenesis.
Cruz, P. E., Almeida, J. S., Murphy, P. N., Moreira, J. L. & Carrondo,
M. J. T. (2000). Modeling retrovirus production for gene therapy. I.
Determination of optimal bioreaction mode and harvest strategy.
Biotechnol Prog 16, 213–221.
Cudmore, S., Reckmann, I. & Way, M. (1997). Viral manipulations of
the actin cytoskeleton. Trends Microbiol 5, 142–148.
Ferry, N., Duplessis, O., Houssin, D., Danos, O. & Heard, J.-M.
(1991). Retroviral-mediated gene transfer into hepatocytes in vivo.
Proc Natl Acad Sci U S A 88, 8377–8381.
Forestell, S. P., Bohnlein, E. & Rigg, R. J. (1995). Retroviral end-
point titer is not predictive of gene transfer efficiency: implications
for vector production. Gene Ther 2, 723–730.
Fujita, J. (1999). Cold shock response in mammalian cells. J Mol
Microbiol Biotechnol 1, 243–255.
Geiss, G. K., Bumgarner, R. E., An, M. C. & 7 other authors (2000).
Large-scale monitoring of host cell gene expression during HIV-1
infection using cDNA microassays. Virology 266, 8–16.
Gu, J. Z., Carstea, E. D., Cummings, C. & 12 other authors (1997).
Substantial narrowing of the Niemann-Pick C candidate interval by
yeast artificial chromosome complementation. Proc Natl Acad Sci
U S A 94, 7378–7383.
Guthridge, M. A., Bellosta, P., Tavoloni, N. & Basilico, C. (1997).
FIN13, a novel growth factor-inducible serine-threonine phosphatase
which can inhibit cell cycle progression. Mol Cell Biol 17, 5485–5498.
Hahn, H., Wojnowski, L., Miller, G. & Zimmer, A. (1999). The patched
signaling pathway in tumorigenesis and development: lessons from
animal models. J Mol Med 77, 459–468.
Kaptein, L. C. M., Greijer, A. E., Valerio, D. & van Beusechem, V. W.
(1997). Optimized conditions for the production of recombinant
amphotropic retroviral vector preparations. Gene Ther 4, 172–176.
Karpen, H. E., Bukowski, J. T., Hughes, T., Gratton, J. P., Sessa,
W. C. & Gailani, M. R. (2001). The sonic hedgehog receptor patched
associates with caveolin-1 in cholesterol-rich microdomains of the
plasma membrane. J Biol Chem 276, 19503–19511.
Kotani, H., Newton, P. B, III, Zhang, S., Chiang, Y. L., Otto, E.,
Weaver, L., Blaese, R. M., Anderson, W. F. & McGarrity, G. J. (1994).
Improved methods of retroviral vector transduction and production
for gene therapy. Hum Gene Ther 5, 19–28.
Lechner, O., Lauber, J., Franzke, A., Sarukhan, A., von Boehmer, H.
& Buer, J. (2001). Fingerprints of anergic T cells. Curr Biol 11, 587–595.
ACKNOWLEDGEMENTS
Le Doux, J. M., Davis, H. E., Morgan, J. R. & Yarmush, M. L. (1999).
We are grateful to Jo Lauber (MI, GBF, Braunschweig) for help in
microarray analysis, Olivier Danos and Otto Merten (Genethon,
France) for supplying the MLV-A provirus vector and Dave Monner
(ZIB GBF, Braunschweig) for critically reading the manuscript.
Kinetics of retrovirus production and decay. Biotechnol Bioeng 63,
654–662.
Martin, V., Carrillo, G., Torroja, C. & Guerrero, I. (2001). The sterol-
sensing domain of Patched protein seems to control Smoothened
activity through Patched vesicular trafficking. Curr Biol 11, 601–607.
Mountain, A. (2000). Gene therapy: the first decade. Trends Biotech
18, 119–128.
REFERENCES
Beer, C., Meyer, A., Müller, K. & Wirth, M. (2003). The temperature
Nishiyama, H., Itoh, K., Kaneko, Y., Kishishita, M., Yoshida, O. &
Fujita, J. (1997). A glycine-rich RNA-binding protein mediating
stability of mouse retroviruses depends on the cholesterol levels of
viral lipid shell and cellular plasma membrane. Virology (in press).
cold-inducible suppression of mammalian cell growth. J Cell Biol
137, 899–908.
Cicala, C., Arthos, J., Selig, S. M. & 11 other authors (2002). HIV
Okamoto, T., Schlegel, A., Scherer, P. E. & Lisanti, M. P. (1998).
envelope induces a cascade of cell signals in non-proliferating
target cell that favor virus replication. Proc Natl Acad Sci U S A 99,
9380–9385.
Caveolins, a family of scaffolding proteins for organizing ‘preassembled signaling complexes’ at the plasma membrane. J Biol
Chem 273, 5419–5422.
Cosset, F. L., Takeuchi, Y., Battini, J. L., Weiss, R. A. & Collins, M. K. L.
(1995). High-titer packaging cells producing recombinant retroviruses
Pietiäinen, V., Huttunen, P. & Hyypiä, T. (2000). Effects of echovirus
resistant to human serum. J Virol 69, 7430–7436.
Cossins, A. R. (editor) (1994). Temperature Adaptation of Biological
Rajavashisth, T. B., Taylor, A. K., Andalibi, A., Svenson, K. L. &
Lusis, A. J. (1989). Identification of a zinc finger protein that binds
Membranes. Colchester: Portland Press.
to the sterol regulatory element. Science 245, 640–643.
http://vir.sgmjournals.org
1 infection on cellular gene expression. Virology 276, 243–250.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 09:38:25
1685
C. Beer and others
Ryo, A., Suzuki, Y., Ichiyama, K., Wakatsuki, T., Kondoh, N., Hada,
A., Yamamoto, M. & Yamamoto, N. (1999). Serial analysis of gene
Patched suggest a role for vesicular trafficking in Smoothened
regulation. Curr Biol 11, 608–613.
expression in HIV-1-infected T cell lines. FEBS Lett 462, 182–186.
Tominaga, S. (1989). A putative protein of a growth specific cDNA
from BALB/c-3T3 cells is highly similar to the extracellular portion
of mouse interleukin 1 receptor. FEBS Lett 258, 301–304.
Ryo, A., Suzuki, Y., Arai, M. & 8 other authors (2000). Identification
and characterization of differentially expressed mRNAs in HIV type
1-infected human T cells. AIDS Res Hum Retroviruses 16, 995–1005.
Trends Microbiol 8, 465–472.
Wirth, M., Bode, J., Zettlmeissl, G. & Hauser, H. (1988). Isolation of
overproducing recombinant mammalian cell lines by a fast and
simple selection procedure. Gene 73, 419–426.
Sonna, L. A., Fujita, J., Gaffin, S. L. & Lilly, C. M. (2002). Effects of
Wirth, M., Grannemann, R., Klehr, D. & Hauser, H. (1994). Screening
heat and cold stress on mammalian gene expression. J Appl Physiol
92, 1725–1742.
retroviral packaging cells for highly efficient virus production by
using a combined selection procedure. J Virol 68, 566–569.
Strutt, H., Thomas, C., Nakano, Y., Stark, D., Neave, B., Taylor, A. M.
& Ingham, P. W. (2001). Mutations in the sterol-sensing domain of
Wu, N. & Ataai, M. M. (2000). Production of viral vectors for gene
Sodeik, B. (2000). Mechanisms of viral transport in the cytoplasm.
1686
therapy applications. Curr Opin Biotechnol 11, 205–208.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 09:38:25
Journal of General Virology 84