Gene expression analysis of murine cells producing amphotropic mouse leukaemia virus at a cultivation temperature of 32 and 37 °C

Christiane Beer1, Petra Buhr1, Heidi Hahn2, Daniela Laubner3 and Manfred Wirth1

1 Molecular Biotechnology, German Research Centre for Biotechnology, GBF, Mascheroder Weg 1, D-38124 Braunschweig, Germany
2 Human Genetics, Georg August University, Göttingen, Germany
3 Institute for Experimental Genetics, GSF-National Research Centre for Environment and Health, Neuherberg, Germany

Correspondence
Manfred Wirth
mwi{at}gbf.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 {alpha}-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 {alpha}-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 °C 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 °C, while cellular productivity for retroviruses is affected 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and retrovirus vectors.
Cells were grown at 37 or 32 °C, 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 µg p4070AMLV, which encodes a wt MLV-A provirus (a kind gift from O. Merten and O. Danos, Genethon, France), and 0·5 µg 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 µg G418 ml-1. TE-Fly is a packaging cell line derived from the human rhabdomyosarcoma cell 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 °C temperature shift on gene expression, non-producing and wt MLV-A-producing NIH 3T3 cells were incubated for 24 h at 32 or 37 °C 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 µg 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 µg) from non-producing and wt MLV-A-producing NIH 3T3 cells cultivated at 32 or 37 °C 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 °C, 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 concentrations, NIH 3T3 cells were plated onto 96-well plates (4x103 cells per well) and cultivated for 24 h at 37 or 32 °C. 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 p11xGli1-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 µg ml-1) and cultivated at 32 or 37 °C 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 p11xGli1-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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 °C 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 °C 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 °C 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 °C (Table 1). While virus titres from TE-Fly packaging cells remained the same upon cultivation of cells at 32 °C, virus titres from NIH 3T3 host cells decreased more than 10-fold compared to 37 °C 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 °C propagation of virus-releasing host cells. Shifting the cultivation temperature to 32 °C, a ‘moderate cold-shock’, 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 °C (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 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 °C. Total RNA was isolated and reverse-transcribed with gene-specific primers for the microarray in the presence of 32P-labelled dATP. 32P-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 virus-producing NIH 3T3 cells. Obviously, cultivation of NIH 3T3 cells at 32 °C causes multiple changes in cellular gene expression. Approximately 10 % of the genes investigated were regulated due to the temperature shift to 32 °C. 125 genes (MLV-A-producing NIH 3T3 cells) and 117 (non-producing 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.


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Table 1. Effect of 32 °C cultivation of host cells on MLV-A titres

 

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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

 
Virus production itself affected the expression of nearly 5 % of the genes investigated and virus propagation caused altered expression of 56 (at 37 °C) and 89 (at 32 °C) 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, 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.


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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

 
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 °C induces an increase in the production of cellular cholesterol, we used filipin to quantify the cholesterol content of the plasma membrane of wt MLV-A-producing NIH 3T3 cells cultivated at 32 and 37 °C. 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.



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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, x1000). (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 filipin-stained NIH 3T3 cells grown in 96-well plates (statistic significance by Student's t-test, P=0·05).

 
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 cholesterol-modified 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).



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Fig. 2. Shh signalling pathway. Cholesterol-modified 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.

 
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 Shh signalling pathway. For smo, ptc and gli-1 gene expression profiling, total RNA was isolated from non- and wt MLV-A-producing NIH 3T3 cells cultivated at either 32 or 37 °C. In contrast to the 1.2K arrays (Clontech), the oligonucleotides for the customized array encountered specific regions of the 5'UTR, coding region and 3'UTR of the genes. To exclude the different biases in labelling and hybridization of Cy-labelled dNTPs, the RNA was reverse-transcribed with poly(dT) primers in the presence of biotin- or fluorescein-labelled dCTP. Labelled cDNAs 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. 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 °C. 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 °C (Fig 3a, b).



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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 (-). Based on the maximum variation in the expression of nine housekeeping genes, the threshold value for gene regulation was defined for each array.

 
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 p11xGli-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 cultivated at 32 or 37 °C 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 °C (Fig. 4, lanes 1 and 2). However, cultivation of mock- or wt MLV-A-infected NIH 3T3 cells at 32 °C 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 °C. This suggests that the Shh signalling pathway has been activated by cultivation of NIH 3T3 cells at 32 °C.



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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, p11xGli1-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.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 °C cultivation and MLV-A infection on cellular gene expression.

Formerly, it has been reported that cultivation of retrovirus packaging cells at 32 °C 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 °C 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 °C. 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 °C 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 °C 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 °C 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 °C, cell proliferation gradually decreases (for example, in mouse leukaemia cells) (Fujita, 1999) and is reduced after the temperature shift from 37 to 32 °C in NIH 3T3 cells (data not shown). Remarkably, the FIN13 gene is overexpressed more than 3-fold in 32 °C-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 °C 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 °C, cultivation of non-producing as well as MLV-A-producing cells at 32 °C 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-A-producing NIH 3T3 cells at 32 °C resulted in the production of phenotypically altered viruses. The investigation revealed that cultivation of MLV-A producing NIH 3T3 cells at 32 °C 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 °C, 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).

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 °C, 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 °C. 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.


   ACKNOWLEDGEMENTS
 
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.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Beer, C., Meyer, A., Müller, K. & Wirth, M. (2003). The temperature stability of mouse retroviruses depends on the cholesterol levels of viral lipid shell and cellular plasma membrane. Virology (in press).

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Received 1 October 2002; accepted 26 February 2003.



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