1 Department of Laboratory Medicine, Children's and Women's Health, Faculty of Medicine, Norwegian University of Science and Technology, MTFS Olav Kyrresg. 3, N-7489 Trondheim, Norway
2 St Olavs Hospital HF, Trondheim, Norway
Correspondence
Torunn Bruland
Torunn.Bruland{at}medisin.ntnu.no
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ABSTRACT |
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INTRODUCTION |
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The nucleotide sequence of the FIS-2 LTR shows high identity with that of the F-MuLV LTR, except for the deletion of one of the two direct repeats (DRs) in F-MuLV LTR and a few point mutations (Dai et al., 1994). The deletion of the second copy of the DR in the FIS-2 LTR has been associated with the generation of a binding site for the glucocorticoid receptor (GR), 5'-AGAACAGATGG-3', at the 3' end of the remaining DR (Dai et al., 1994
). This glucocorticoid response element (GRE) contains a conserved hexanucleotide, 5'-AGAACA-3', defined as the GRE core sequence (DeFranco & Yamamoto, 1986
; Miksicek et al., 1986
). The core motif also has affinity for the progesterone receptor and androgen receptor (Nelson et al., 1999
, and references therein). A glucocorticoid-regulated transcription of provirus through the binding of a GR to the GRE has been described for both simple retroviruses (Beato et al., 1989
; Celander et al., 1988
; DeFranco & Yamamoto, 1986
; Miksicek et al., 1986
) and complex retroviruses such as bovine leukaemia virus (Niermann & Buehring, 1997
) and human immunodeficiency virus (HIV) (Kolesnitchenko & Snart, 1992
; Mitra et al., 1995
; Russo et al., 1999
; Soudeyns et al., 1993
). The best-studied steroid-inducible retrovirus is mouse mammary tumour virus (MMTV), in which the promoter is silent in the absence of hormones. The MMTV provirus is rapidly induced to high transcription levels by glucocorticoid hormones as well as progesterins and androgens (Beato et al., 1989
; Cato et al., 1986
; Darbre et al., 1986
; Otten et al., 1988
; Schüle et al., 1988
). We have previously proposed that the presence of the GRE in the FIS-2 LTR could contribute to the significant sex difference in early FIS-2 replication (Bruland et al., 2001
). The present study was undertaken to investigate this hypothesis further.
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METHODS |
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Viruses.
The viruses used in this study included a molecular clone of an immunosuppressive variant of Friend murine leukaemia virus, the N-tropic FIS-2 (Dai et al., 1994) and the FIS-2 GRE mutant (see above). Virus stocks were prepared and titrated from supernatants collected from transfected cells as described previously (Dai et al., 1998
). The titres of the FIS-2 wt and FIS-2 GRE mutant stocks were 2·2x106 and 2·65x106 infectious units (IU) ml-1, respectively. RT-PCR and subsequent sequencing verified the presence of the mutated GRE in the prepared virus stock. The reverse primer corresponded to a sequence in the LTR region (nt 80758095) and the forward primer corresponded to a sequence at the end of the env gene (nt 76307650), producing a fragment of 466 bp. Amplification was confirmed by electrophoresis and the RT-PCR product was purified (Qiagen PCR purification kit) and sequenced as described above. The stability of the single nucleotide exchange in the GRE core sequence after in vitro and in vivo replication was confirmed by RT-PCR and subsequent sequencing of the virus genome isolated from in vitro-infected NIH 3T3 cell culture supernatants and serum from infected mice, respectively.
RT assay.
NIH 3T3 cells (1·5x104) were seeded into each well of 24-well plates. After attachment for 24 h, the medium was changed and the cells were incubated for 24 h with 5000 IU (0·3 IU per cell) of either wt FIS-2 or FIS-2 GRE mutant in fresh medium supplemented with polybrene and with different concentrations of dexamethasone or hydrocortisone and RU486. The medium was then replaced with a virus- and polybrene-free medium and the cells were further incubated with or without freshly added hormones and anti-hormone. RT activity of extracellular particles was used as a measure of virion production at different times post-infection (p.i.). RT activity was determined using a standard technique based on that of Goff et al. (1981). In brief, 800 µl cell-free culture supernatant was centrifuged at 15 000 g for 60 min and the pellet was resuspended in 25 µl dH2O before 25 µl reaction cocktail was added. The reaction mixture was incubated at 37 °C for 1 h. Radioactivity was counted in a
-scintillation counter.
MTT assay.
An experimental in vitro design similar to the one described above was performed with cells grown in 96-well plates. The cell number, virus concentration and dexamethasone concentration were adjusted to those of the 24-well plates and the two cell cultures were run in parallel. The growth of cells was assessed by the MTT reduction assay, essentially as described by Mosmann (1983).
Flow cytometric quantification of virus-infected NIH 3T3 cells.
All steps were performed at 04 °C. Adherent NIH 3T3 cells were incubated in PBS containing 9 g EDTA l-1 for 2 min before being harvested by gentle scraping and low-speed centrifugation. A mix of hybridoma supernatants 34 and 48 (described above) was added. The cell suspensions were transferred to a 96-well plate where the cells were incubated for 2 h. After three washes in PBS containing 0·1 % BSA, the cells were further incubated for 1 h with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG (Dako). The cells were then washed three times, resuspended in PBS and analysed with a FACScan flow cytometer (Becton Dickinson). Five-thousand cells were analysed per sample on a single-cell basis and displayed as frequency distribution histograms. Dead cells and debris were gated out of analysis on the basis of a forward light scatter signal. The results were expressed as the percentage of cells that stained positive after subtraction of the stained cells in the control samples. The number of positive cells per well was calculated from the average number of cells in the corresponding wells.
Animals.
Seven-week-old male and female NMRI mice were purchased from the Bomholdt Gaard Breeding Research Center, Rye, Denmark. The mice were kept in groups of five under conditions of controlled temperature and a 12 : 12 h light/dark cycle, with food pellets and water ad libitum. The handling and treatment of the experimental animals were performed according to the regulations of the Norwegian Law on the Care and Use of Laboratory Animals.
Determination of virus titres in serum and spleen.
All mice were bled from the cervical vein under anaesthesia (1 : 1 mixture of hypnorm : dormicum) for viraemia analysis and subsequently sacrificed by cervical dislocation. Viraemia titres were determined by end-point dilution (Grist et al., 1990), as described previously (Bruland et al., 2001
). Preparation of single-cell suspensions of splenocytes and detection of virus-positive cells by indirect immunofluorescence were performed as described by Dai et al. (1998)
. The virus load in spleens was determined by end-point dilution of single-cell suspensions: 10-fold dilutions were transferred into four wells (1·5 cm2) of subconfluent monolayers of NIH 3T3 cells of up to 107 spleen cells per well. After two rounds of trypsination and 1 : 10 dilution, the cells were prepared for immunofluorescent detection of positive cells. The number of productively infected nucleated splenocytes was calculated from the lowest dilution with three or more positive wells.
Determination of antibodies directed against viral membrane proteins on FIS-2-infected cells.
Subconfluent monolayers of NIH 3T3 cells transfected by FIS-2 clones were grown in 96-well plates. A twofold dilution of sera from infected and uninfected (control) mice was used as a source of primary antibody. The cells were prepared for immunofluorescence as described by Dai et al. (1998). The antibody titres were given as the highest dilution giving immunofluorescent signals on the infected cells.
Statistical methods.
Student's t-test was used to determine the level of significance between the means of samples in the in vitro experiments. Statistical analysis of viraemia titres and number of infected splenocytes were performed on log10-transformed data with the two-tailed MannWhitney U rank sum test for independent samples, using the exact test option (corrected for ties). A value of P0·05 was considered significant. The rank correlation between viraemia titres and the number of productively infected cells in the spleen at 8 days p.i. was analysed by calculating Spearman's rho. Correlation was significant at the 0·01 level (two-tailed). The SPSS version 10.07 software was utilized for all statistical analyses.
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RESULTS |
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More virus is produced from wt FIS-2-infected cells than from FIS-2 GRE mutant-infected cells, and wt FIS-2 production can be further augmented by dexamethasone
Subsequently, we examined whether more virus was produced from FIS-2-infected cells compared with FIS-2 GRE mutant-infected cells and whether wt FIS-2 production could be augmented by glucocorticoid. Based on the results of Fig. 2, a near-physiological concentration of dexamethasone (0·02 µM) was chosen for further in vitro experiments. NIH 3T3 cells were cultured and incubated with and without virus and dexamethasone in 24 well plates as described in Methods. After 72 h, the cells were harvested and prepared for flow cytometric quantification of virus-positive cells per well. There was no significant difference (P=0·3) between the number of wt FIS-2-positive cells and FIS-2 GRE mutant-positive cells when the cells were grown in the absence of dexamethasone (Fig. 4
A). However, relatively higher levels of RT activity were detected in cultures infected with wt FIS-2 than with the FIS-2 GRE mutant: the RT activity per well was normalized relative to the value for FIS-2 GRE mutant-infected cells grown without dexamethasone in the same experiment (assigned a value of 1). An average 3·75±0·33-fold higher RT activity was detected in wt FIS-2-infected cultures compared with FIS-2 GRE mutant-infected cultures (Fig. 4B
). This observation indicated that more virus was produced from wt FIS-2-infected cells than from FIS-2 GRE mutant-infected cells.
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The glucocorticoid antagonist RU468 inhibits virus production in a dose-dependent manner
The type II glucocorticoid antagonist RU486GR complex has been shown to bind weakly to the MMTV GRE and inhibit an agonist-induced transcription of the virus (Belikov et al., 2001). We therefore used this antagonist to examine the involvement of GR and glucocorticoids in FIS-2 transcription. In addition to the synthetic glucocorticoid dexamethasone, we also stimulated the NIH 3T3 cells (cultured with 10 % serum) with the natural agonist hydrocortisone (cortisol). An initial hormone titration experiment showed that 0·2 µM hydrocortisone or dexamethasone augmented wt FIS-2 production on average 1·26±0·2-fold and 1·33±0·1-fold, respectively (72 h p.i., n=3). This concentration was therefore used for both agonists in subsequent experiments. The antagonist RU486 was added in 100-fold excess (20 µM) in order to display the agonists from GR.
In the first experiment, RT activity was assayed in the cell culture supernatant from wt FIS-2-infected NIH 3T3 cell cultures treated with different combinations of hormones and RU486. Cell cultures infected with the FIS-2 GRE mutant served as a reference. Concomitant addition of hormones and a 100-fold excess of RU486 resulted in a significant depression of wt FIS-2 production (Fig. 5A). When 20 µM RU486 was added alone, both wt FIS and the FIS-2 GRE mutant production were almost totally inhibited. However, when the cells were counted in a haemocytometer at the end of the experiment (116 h p.i.), we found that there were significantly lower cell numbers in RU486-treated cell cultures compared with cultures grown without the glucocorticoid antagonist (P<0·01). We then performed a second experiment where we assayed the effect of different RU486 concentrations (020 µM) on both cell proliferation and RT activity at 80 h p.i. with wt FIS-2 or the FIS-2 GRE mutant. The cells were grown with or without 0·2 µM hydrocortisone. In this experiment, the 20 µM RU496 dose also reduced the cell numbers significantly (P<0·01) (Fig. 5B
). When we assayed the RT activity in the culture supernatants, we found that RU486 depressed virus production in a dose-dependent manner, also when the glucocorticoid antagonist did not inhibit cell proliferation significantly (Fig. 5C
). Overall, these experiments indicated an involvement of GR in FIS-2 transcription.
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The difference in viraemia titres and numbers of virus-producing cells in the spleen between male mice inoculated with the wt FIS-2 and the other groups were most notable at 8 days p.i. The virus titres in serum and spleen at 8 days p.i. obtained in several experiments were therefore pooled and analysed statistically. Both viraemia titres (n=1422 per group, a total of 71 mice) and the number of infected cells in the spleen (n=910 per group, a total of 39 mice) were significantly higher (P<0·05) in male mice infected with wt FIS-2 than in the three other groups at 8 days p.i. There was a strong correlation (Rs=0·707, P<0·0001) between viraemia titres and the number of virus-producing cells in the spleen (Fig. 6C). We found no significant difference in viraemia (P=0·370) or number of virus-positive cells in the spleen (P=0·831) between male and female mice infected by the FIS-2 GRE mutant. Hence, the observed sex difference in early FIS-2 dissemination was only significant when an intact GRE was present.
Viraemia titres were markedly reduced in all groups at 14 days p.i. (Fig. 6A). When we analysed for antibodies directed against viral proteins on FIS-2-infected cells, we found that the decline in viraemia titres at 14 days p.i. coincided with the onset of a detectable anti-FIS-2 response (Fig. 6D
). The average anti-FIS-2 response in female mice was stronger than the average response in male mice at 14 days p.i. This is consistent with our previously reported observation that male mice are more immunosuppressed than female mice at 2 weeks p.i. when inoculated with equal doses of wt FIS-2 (Bruland et al., 2001
). The levels of anti-FIS-2 response in both male and female mice inoculated with the FIS-2 GRE mutant were low, although female mice seemed to have slightly higher levels (P=0·05).
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DISCUSSION |
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A number of possible mechanisms may explain these observations. First, it is well documented that an androgen receptor and a progesterone receptor can bind to and activate a hormone response element with the common high-affinity core motif 5'-AGAACA-3' found in the FIS-2 GRE (Fig. 1; Dai et al., 1994
). Nelson et al. (1999)
suggested that this one type fits all response element may be advantageous for extending the cellular host range of the virus to exploit a number of endocrine signalling pathways. This might be especially advantageous for the simple retroviruses whose host cells are limited to actively dividing cells (Lewis & Emerman, 1994
). Erythroid progenitor cells are among the first cells infected in an FIS-2 infection, and the proliferation and differentiation of these cells are highly influenced by androgens and glucocorticoids (Udupa et al., 1986
; von Lindern et al., 1999
). Thus, androgens might affect virus production, both indirectly, through growth promotion of erythroblasts, and directly, through androgen receptor binding to the GRE in the FIS-2 LTR. Secondly, GRs in association with a chromatin-remodelling complex can bind to target sites within promoter regions of genes assembled as chromatin. This interaction alters the nucleosome architecture to allow binding of other transcription factors, such as NF1 (Fig. 1
), that cannot bind an LTR assembled into chromatin (Archer et al., 1992
; for review on GR-mediated chromatin remodelling, see Deroo & Archer, 2001
). One single GR binding site is sufficient for the structural transition to occur (Belikov et al., 2000
). The glucocorticoid antagonist RU486 promotes GRGRE binding without inducing chromatin remodelling (Belikov et al., 2001
). In the present study we showed that RU486 inhibited FIS-2 production from in vitro-infected cells in a dose-dependent manner. This observation suggested that such a GR-induced chromatin remodelling might be important in FIS-2 transcription. It is possible that male mice have target cells with more or other factors that can influence a GRGRE-mediated virus gene activation than female mice. Thirdly, young male mice in our study were more frequently involved in aggressive behaviour than female and castrated male mice (data not shown). This observation is in accordance with findings made by others, and it has been shown that behavioural factors such as testosterone-dependent aggression can modify the expression of sex differences in infection (reviewed in Klein, 2000
; Azpiroz et al., 2002
). In our model system, we have observed that both male castrated mice and female mice are less susceptible to F-MuLV infection than non-castrated male mice (T. Bruland, L. A. S. Lavik, H. Y. Dai & A. Dalen, unpublished results). In mice, social conflict (i.e. inter-male aggression) increases the plasma corticosterone concentration (Dréau et al., 1999
; Padgett et al., 1998
). Since stress-induced hormones like cortisol bind to GR with high affinity, stress-induced elevating levels of circulating cortisol might have a stimulatory effect on FIS-2 production.
One should be cautious when drawing analogies from experiments with simple retroviruses like FIS-2 to complex retroviruses such as HIV, due to their production of accessory proteins and their ability to infect non-dividing cells (Coffin et al., 1997; Lewis & Emerman, 1994
). However, there are some interesting resemblances in relation to hormonal influence on virus production: the HIV provirus does contain an intact and functional GRE within the vif open reading frame (Soudeyns et al., 1993
). The HIV-1 virion-associated accessory protein, Vpr, has also been shown to be a co-activator of the human GR (Kino et al., 1999
; Refaeli et al., 1995
) and it has been suggested that an intact GRE in the HIV-1 LTR is critical for Vpr activity (Sherman et al., 2000
; Vanitharani et al., 2001
). Glucocorticoids have been shown to both activate (Kolesnitchenko & Snart, 1992
; Russo et al., 1999
) and suppress (Kino et al., 2000
; Mitra et al., 1995
; Russo et al., 1999
) HIV replication in vitro. The functional significance of the presence of the GRE in in vivo HIV replication is still uncertain. However, in the context of our findings, it is intriguing that several studies have shown that women have lower numbers of circulating HIV RNA copies than men (reviewed in Gandhi et al., 2002
), particularly at or near the time of seroconversion (Evans et al., 1997
; Lyles et al., 1999
; Sterling et al., 1999
).
In conclusion, our results in this report show that the presence of a single intact GRE in FIS-2 U3 can enhance virus production from in vitro-infected NIH 3T3 cells. In addition we have shown that sex-related factors can influence early FIS-2 replication in vivo via mechanisms involving this element. The exact mechanisms for sex-related virus production through the GRE require further examination. Work in progress is now focusing on possible stress- and/or androgen-induced virus gene activation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 30 May 2002;
accepted 13 November 2002.
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