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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
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
).
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 hostpathogen 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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cicala, C., Arthos, J., Selig, S. M. & 11 other authors (2002). HIV envelope induces a cascade of cell signals in non-proliferating target cell that favor virus replication. Proc Natl Acad Sci U S A 99, 93809385.
Cosset, F. L., Takeuchi, Y., Battini, J. L., Weiss, R. A. & Collins, M. K. L. (1995). High-titer packaging cells producing recombinant retroviruses resistant to human serum. J Virol 69, 74307436.[Abstract]
Cossins, A. R. (editor) (1994). Temperature Adaptation of Biological Membranes. Colchester: Portland Press.
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, 213221.[CrossRef][Medline]
Cudmore, S., Reckmann, I. & Way, M. (1997). Viral manipulations of the actin cytoskeleton. Trends Microbiol 5, 142148.[CrossRef][Medline]
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, 83778381.[Abstract]
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, 723730.[Medline]
Fujita, J. (1999). Cold shock response in mammalian cells. J Mol Microbiol Biotechnol 1, 243255.[Medline]
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, 816.[CrossRef][Medline]
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, 73787383.
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, 54855498.[Abstract]
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, 459468.[CrossRef][Medline]
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, 172176.[CrossRef][Medline]
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, 1950319511.
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, 1928.[Medline]
Lechner, O., Lauber, J., Franzke, A., Sarukhan, A., von Boehmer, H. & Buer, J. (2001). Fingerprints of anergic T cells. Curr Biol 11, 587595.[CrossRef][Medline]
Le Doux, J. M., Davis, H. E., Morgan, J. R. & Yarmush, M. L. (1999). Kinetics of retrovirus production and decay. Biotechnol Bioeng 63, 654662.[CrossRef][Medline]
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, 601607.[CrossRef][Medline]
Mountain, A. (2000). Gene therapy: the first decade. Trends Biotech 18, 119128.[CrossRef][Medline]
Nishiyama, H., Itoh, K., Kaneko, Y., Kishishita, M., Yoshida, O. & Fujita, J. (1997). A glycine-rich RNA-binding protein mediating cold-inducible suppression of mammalian cell growth. J Cell Biol 137, 899908.
Okamoto, T., Schlegel, A., Scherer, P. E. & Lisanti, M. P. (1998). Caveolins, a family of scaffolding proteins for organizing preassembled signaling complexes' at the plasma membrane. J Biol Chem 273, 54195422.
Pietiäinen, V., Huttunen, P. & Hyypiä, T. (2000). Effects of echovirus 1 infection on cellular gene expression. Virology 276, 243250.[CrossRef][Medline]
Rajavashisth, T. B., Taylor, A. K., Andalibi, A., Svenson, K. L. & Lusis, A. J. (1989). Identification of a zinc finger protein that binds to the sterol regulatory element. Science 245, 640643.[Medline]
Ryo, A., Suzuki, Y., Ichiyama, K., Wakatsuki, T., Kondoh, N., Hada, A., Yamamoto, M. & Yamamoto, N. (1999). Serial analysis of gene expression in HIV-1-infected T cell lines. FEBS Lett 462, 182186.[CrossRef][Medline]
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, 9951005.[CrossRef][Medline]
Sodeik, B. (2000). Mechanisms of viral transport in the cytoplasm. Trends Microbiol 8, 465472.[CrossRef][Medline]
Sonna, L. A., Fujita, J., Gaffin, S. L. & Lilly, C. M. (2002). Effects of heat and cold stress on mammalian gene expression. J Appl Physiol 92, 17251742.
Strutt, H., Thomas, C., Nakano, Y., Stark, D., Neave, B., Taylor, A. M. & Ingham, P. W. (2001). Mutations in the sterol-sensing domain of Patched suggest a role for vesicular trafficking in Smoothened regulation. Curr Biol 11, 608613.[CrossRef][Medline]
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, 301304.[CrossRef][Medline]
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, 419426.[CrossRef][Medline]
Wirth, M., Grannemann, R., Klehr, D. & Hauser, H. (1994). Screening retroviral packaging cells for highly efficient virus production by using a combined selection procedure. J Virol 68, 566569.[Abstract]
Wu, N. & Ataai, M. M. (2000). Production of viral vectors for gene therapy applications. Curr Opin Biotechnol 11, 205208.[CrossRef][Medline]
Received 1 October 2002;
accepted 26 February 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |