UNIGEN Center for Molecular Biology/Institute of Laboratory Medicine, Norwegian University of Science and Technology, N-7489 Trondheim, Norway1
Department of Microbiology/Institute of Laboratory Medicine, Trondheim Regional Hospital/NTNU, N-7489 Trondheim, Norway2
Author for correspondence: Are Dalen. Fax +47 73 867765. e-mail Are.Dalen{at}medisin.ntnu.no
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Abstract |
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The viruses used in this study included a molecular clone of an immunosuppressive and low leukaemogenic variant of F-MuLV, FIS-2 (Dai et al., 1994 ; Faxvaag et al., 1993
), the prototype F-MuLV, clone 57 (Troxler et al., 1980
), and two chimeras, RE3 and RE4. Chimera RE3 contains FIS-2 LTR in an F-MuLV clone 57 background and chimera RE4 contains F-MuLV clone 57 LTR in an FIS-2 background (Dai et al., 1998
). In all experiments we used 6- to 10-week-old male and female NMRI mice (an outbred strain where all mice have the same genotype of MHC, H-2q) purchased from Bomholt Gaard Breeding Research Center, Rye, Denmark, and kept in groups (<10) under conditions of controlled temperature and 12:12 h light/dark cycle, with food pellets and water ad libitum.
In the first experiment we inoculated male and female mice intraperitoneally with various doses of FIS-2. At 6, 8, 10 and 14 days post-infection (p.i.) groups of mice were bled from the cervical vein under anaesthesia (1:1 mixture of intraperitoneally or subcutaneously administrated hypnorm:dormicum) and then sacrificed by cervical dislocation. Virus in serum was detected by co-culturing serum with subconfluent monolayers of NIH 3T3 cells cultured in 24-well plates. The cultures were passed (trypsinated and diluted 1:10) three times before positive cells were detected by indirect membrane immunofluorescence using a mix of monoclonal antibody against gag-encoded MA protein (antibody 34) and monoclonal antibody reactive with F-MuLV gp70 (antibody 48) (Chesebro et al., 1981 ). The assay detection sensitivity was 50 infectious units (IU)/ml serum (20 µl serum per well) and has been described previously (Dai et al., 1998
).
We observed that a much lower infective dose was required to obtain the same incidence of early viraemia in male compared to female mice (Fig. 1, upper panels). Viraemia in male mice was initially detected at 6 days p.i. with 640 IU. A similar incidence of detectable viraemia in female mice emerged first between days 8 and 10 p.i. with 6400 IU. Since infection in the spleen indicates a successful establishment of primary infection, we prepared single cell suspensions of splenocytes and co-cultivated them with NIH 3T3 cells (2x105 splenocytes per well) to test for productive infection by indirect immunofluorescence detection. The gender difference in initial viraemia was mirrored in the fractions of mice with detectable infection in the spleen (Fig. 1
, lower panels).
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These results indicated that gender-related factors could have a more direct effect on the activity of FIS-2 LTR than on the activity of F-MuLV LTR. The nucleotide sequence of FIS-2 LTR shows high homology with that of F-MuLV LTR except for the deletion of one direct repeat and a few point mutations, including generation of a glucocorticoid response element, GRE (Dai et al., 1994 ). GRE mediates gene induction by glucocorticoids, progesterone and androgens, but not by oestrogens (Beato et al., 1989
; Cato et al., 1986
; Darbre et al., 1986
; Otten et al., 1988
; Schüle et al., 1988
). It has been suggested that incorporation of a GRE within the LTR represents a common strategy among retroviruses that could serve to increase the transcription activity of their promoters (Beato et al., 1989
; Miksicek et al., 1986
). Several studies have reported regulation of gene activity by GRE in vitro by a broad spectrum of viruses including hepatitis B virus (Tur-Kaspa et al., 1986
), human polyomavirus BK (Moens et al., 1994
) and complex retroviruses such as bovine leukaemia virus (Niermann et al., 1997
) and human immunodeficiency virus (Kolesnitchenko & Snart, 1992
; Mitra et al., 1995
; Soudeyns et al., 1993
). Incorporation of a GRE in FIS-2 LTR might contribute to the significant gender difference in FIS-2 infection.
It is also possible that the number of target cells or the receptor level can be affected by the gender-related hormonal environment. It has been shown in rat quiescent liver cells that type C ecotropic retrovirus receptor can be upregulated by some hormones, including dexamethasone and insulin (Wu et al., 1994 ). Vassiliadou et al. (1999)
showed in another study that progesterone-induced inhibition of chemokine receptor expression on peripheral blood mononuclear cells correlated with reduced infectiousness of human immunodeficiency virus type 1 in vitro. This study also suggested that progesterone could have negative effects on chemokine-mediated recruitment of lymphocytes and monocytes to mucosal epithelia. In the present study NMRI mice were inoculated intraperitoneally with different virus clones, and one potential explanation for the observed general gender difference in susceptibility might be that sex hormones induce dimorphism in the recruitment of target cells at this site of infection.
In conclusion, we have shown that gender clearly is of importance in primary FIS-2 infection. The time from virus inoculation to detectable infection was shorter for male mice than for female mice and this gender-related sensitivity, probably due to high initial replication of FIS-2 in male mice, resulted in higher male susceptibility to FIS-2-induced immunosuppression. Studies with recombinant viruses between FIS-2 and the prototype F-MuLV, clone 57, revealed that the FIS-2 LTR was one major factor contributing to the observed gender difference, and we speculate that this might, at least in part, be due to the presence of a GRE in FIS-2 LTR. We now focus on identifying the cell types infected by FIS-2 during primary infection in male and female mice and investigate possible direct activation of FIS-2 LTR by sex hormones in these cells.
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References |
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Received 5 February 2001;
accepted 26 March 2001.