Selective Up-Regulation of Intact, but Not Defective env RNAs of Endogenous Modified Polytropic Retrovirus by the Sgp3 Locus of Lupus-Prone Mice

Kumiko Yoshinobu, Lucie Baudino, Marie-Laure Santiago-Raber, Naoki Morito, Isabelle Dunand-Sauthier, Bernard J. Morley, Leonard H. Evans and Shozo Izui
J Immunol June 15, 2009, 182 (12) 8094-8103; DOI: https://doi.org/10.4049/jimmunol.0900263

Abstract

Endogenous retroviruses are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Because four different classes of endogenous retroviruses, i.e., ecotropic, xenotropic, polytropic, or modified polytropic (mPT), are expressed in mice, we investigated the possibility that a particular class of endogenous retroviruses is associated with the development of murine SLE. We observed >15-fold increased expression of mPT env (envelope) RNA in livers of all four lupus-prone mice, as compared with those of nine nonautoimmune strains of mice. This was not the case for the three other classes of retroviruses. Furthermore, we found that in addition to intact mPT transcripts, many strains of mice expressed two defective mPT env transcripts which carry a deletion in the env sequence of the 3′ portion of the gp70 surface protein and the 5′ portion of the p15E transmembrane protein, respectively. Remarkably, in contrast to nonautoimmune strains of mice, all four lupus-prone mice expressed abundant levels of intact mPT env transcripts, but only low or nondetectable levels of the mutant env transcripts. The Sgp3 (serum gp70 production 3) locus derived from lupus-prone mice was responsible for the selective up-regulation of the intact mPT env RNA. Finally, we observed that single-stranded RNA-specific TLR7 played a critical role in the production of anti-gp70 autoantibodies. These data suggest that lupus-prone mice may possess a unique genetic mechanism responsible for the expression of mPT retroviruses, which could act as a triggering factor through activating TLR7 for the development of autoimmune responses in mice predisposed to SLE.

Endogenous retroviruses have been implicated in the pathogenesis of murine systemic lupus erythematosus (SLE).4 Relatively large amounts of the retroviral envelope glycoprotein, gp70, were found in sera and in glomerular immune deposits of lupus-prone (NZB × NZW)F1, MRL, and BXSB mice; Refs. 1 and 2). Furthermore, gp70-anti-gp70 immune complexes (gp70 IC) are detectable in the circulation close to the onset of renal disease, and their concentrations rise with the progression of lupus nephritis (3). Several genetic studies of murine lupus have revealed a remarkable correlation of serum levels of gp70 IC with the development of severe lupus nephritis (4, 5, 6, 7), further underlining the pathogenic role of gp70 IC in murine SLE.

The retroviral env gene encodes a precursor polyprotein, which is cleaved to produce two subunits; a surface gp70 protein and a membrane-anchored p15E protein. Both subunits remain associated with each other through intersubunit disulfide linkage on the intact virions (8). Serum gp70 is secreted by hepatocytes in the blood circulation and behaves like an acute-phase protein (9). Its expression is controlled by multiple structural and regulatory genes (10), and its concentrations are highly variable among different strains of mice (1, 2, 3). All SLE-prone strains of mice have high concentrations of gp70 in their sera (>15 μg/ml), whereas C57BL/6 (B6), C57BL/10 (B10), and BALB/c mice produce low levels of serum gp70 (<5 μg/ml). Recent genetic studies identified at least two loci, Sgp3 (serum gp70 production 3) on mid-chromosome 13 and Sgp4 on distal chromosome 4, which controlled basal serum levels of gp70 (7, 11, 12, 13, 14, 15, 16) through the regulation of the abundance of multiple endogenous retroviral gp70 transcripts in trans (10).

Endogenous retroviruses are classified as ecotropic, xenotropic, or polytropic according to the host range dictated by their respective gp70 proteins (17). Furthermore, based on differences in their gp70 nucleotide sequences (17, 18), the xenotropic proviruses have been divided into four subgroups, Xeno-I, Xeno-II, Xeno-III, and Xeno-IV, and the polytropic (PT) proviruses into two subgroups, termed PT and modified PT (mPT). NZB mice spontaneously produce a very high titer of replication-competent xenotropic viruses (19), but they fail to express ecotropic viruses because of the lack of ecotropic sequences in their genome (20). Although endogenous PT and mPT viruses are likely to be replication defective, replication-competent and infectious recombinant viruses containing the PT or mPT gp70 sequences can be generated. These recombinant viruses utilize the Xpr1 cell surface receptor for infection of mice as well as other species (21, 22, 23). Xenotropic viruses also utilize the Xpr1 cell surface receptor for infection of species other than mice; however, the polymorphic form of Xpr1 in laboratory mouse strains renders these strains resistant to infection by xenotropic viruses (24). It has been reported that an infectious murine leukemia virus has been isolated from NZB mice and that neonatal infection with this virus induced a lupus-like autoimmune syndrome in (BALB/c × NZB)F1 mice (25).

Our previous analysis of the abundance of xenotropic, PT, and mPT gp70 RNAs in livers of different strains of mice suggested that the expression level of mPT gp70 RNAs was selectively increased in all four lupus-prone mice, relative to the expression levels in several other strains of mice that are not predisposed to autoimmune diseases (10). However, this conclusion is considered to be preliminary because the number of nonautoimmune strains analyzed was limited. Furthermore, our quantification based on real-time RT-PCR covering a limited region of the gp70 may have included both intact mPT env RNA and defective env transcripts. In this study, we have conducted a more extended RT-PCR analysis covering the additional regions of the gp70 sequence to further explore a possible association of mPT proviral expression with murine SLE. Our results demonstrated that lupus-prone mice expressed abundant levels of mPT env RNA compared with nonautoimmune strains of mice. Furthermore, many strains of mice expressed not only wild-type (WT) mPT env transcripts but also two defective mPT env transcripts, whereas lupus-prone mice expressed predominantly the WT mPT env RNA at the near exclusion of the defective transcripts. This specific pattern of expression was regulated by the Sgp3 locus derived from lupus-prone mice.

Materials and Methods

Mice

NZB, NZW, BXSB, MRL, 129, AKR, DBA/2, BALB/c, CBA, C3H/He, and SB/Le mice were purchased from The Jackson Laboratory. NFS mice have been maintained at Rocky Mountain Laboratories, Veterinary Branch (Helena, MT). B6.NZB-Sgp3, B10.BXSB-Sgp3, and B6.NZB-Sgp4 congenic mice were generated by backcross procedures using marker-assisted selection, as described previously (10, 12, 15). The generation of TLR7-sufficient or -deficient B6.Nba2-congenic mice homozygous for the NZB-derived Nba2 lupus susceptibility locus (flanked by markers D1Mit47 and D1Mit461 in chromosome 1) has been described previously (26). Animal studies described in the present study have been approved by the Ethical Committee for Animal Experimentation of the Faculty of Medicine, University of Geneva, Geneva, Switzerland).

Quantitative real-time RT-PCR

RNA from livers was purified with TRIzol reagent (Invitrogen) and treated with DNase I (Amersham Biosciences). The abundance of ecotropic, xenotropic, PT, and mPT env RNAs (genomic RNA and mRNA) was quantified by real-time RT-PCR. For ecotropic viral gp70 cDNA, a forward primer (5′-AGGCTGTTCCAGAGATTGTG-3′) and a reverse primer (5′-TTCTGGACCACCACATGAC-3′) were used. For the amplification of all four different subgroups of xenotropic viral gp70 cDNA, a common Xeno1098F forward primer (5′-TCTATGGTACCTGGGGCTC-3′) and a common Xeno1298R reverse primer (5′-GGCAGAGGTATGGTTGGAGTAG-3′) were used. For PT and mPT viral gp70 cDNA, a common PT/mPT730F forward primer (5′-CCGCCAGGTCCTCAATATAG-3′) and reverse primers specific for PT (PT892R: 5′-AGAAGGTGGGGCAGTCT-3′) and mPT (mPT880R: 5′-CGTCCCAGGTTGATAGAGG-3′) viruses were used. The two different env cDNAs of the deletion mutants D1 and D2 were amplified by the following primers: mPT1115F forward (5′-GTTCCCAAAACCCATCAGGC-3′) and D1-R reverse (5′-GGCTCTCTTTTATAGGCAGG-3′) primers for the D1 mutant; mPT1317F forward (5′-ATGTTTATGGCCAGTTTGAGAG-3′) and D2-R reverse (5′-CAGTGCATGAAGCTGCTGAG-3′) primers for the D2 mutant. PCR was performed using the iCycler iQ Real-Time PCR Detection System (Bio-Rad) and iQ SYBR green Supermix (Bio-Rad). Results were quantified using a standard curve generated with serial dilutions of a reference cDNA preparation from AKR, NZB, or BXSB liver and normalized using TATA-binding protein (TBP) mRNA.

RT-PCR and genomic PCR

The 5′ portions of xenotropic, PT, or mPT gp70 cDNAs were amplified with the following primers: Xeno277F forward (5′-CCAGCCGGAACAGCATGGAAG-3′) and Xeno400R reverse primers (5′-CTGTCACGTTGTACCGAGG-3′) for xenotropic gp70; PT/mPT46F common forward (5′-CCGCCAGGTCCTCAATATAG-3′) and PT892R reverse primers for PT gp70; PT/mPT46F forward and mPT880R reverse primers for mPT gp70. The gp70-p15E junction region of env cDNAs was amplified with xenotropic (Xeno1098F)-, PT (PT863F: 5′-TCTATAGTCCCTGAGACTG-3′)- and mPT-specific (mPT858F: 5′-CAGCCTCTAACAACCTGG-3′) gp70 forward primers and a common p15E-R reverse primer (5′-ATCTAATCCTCTCCGGTTCT-3′). For the amplification of WT and two deletion mutants (D1 and D2) of mPT env RNAs, an mPT858F forward primer and reverse primers specific for WT (mPT1447R: 5′-TAGGGCTGTAGTCCCTGTTCC-3′) or deletion mutants (D1-R and D2-R) were used. Using these sets of primers, the abundance of three different species of mPT env RNAs was semiquantified with 5-fold serially diluted cDNA templates. As a control, the abundance of GAPDH cDNA was semiquantified in a parallel assay. The presence of mPT proviruses carrying the WT and mutant env genes in the genome was determined by PCR on genomic DNA prepared from livers, using mPT858F forward primer and mPT1447R, D1-R or D2-R reverse primers. PCR products were visualized by staining with ethidium bromide after electrophoresis on 3.5% polyacrylamide or 2% agarose gels.

RT-PCR cloning

mPT cDNA spanning the env gene and the U3 regions of the long terminal repeat (LTR) was amplified with mPT858F forward primer and p15E-R or Uniltr-4R (5′-GGGCGACTCAGTCTATCGG-3′) reverse primer. The amplified fragments were purified on a 2% agarose gel and ligated into pBluescript-SK+ plasmid.

Serological assays

Serum levels of gp70 IC were quantified by ELISA combined with the treatment of sera with 10% polyethylene glycol (average molecular mass 6000), which precipitates only antibody-bound gp70, but not free gp70, as described (27). Results are expressed as micrograms per milliliter of gp70 by referring to a standard curve obtained from a serum pool of NZB mice.

Statistical analysis

Unpaired comparison for env RNA expression was analyzed by Student’s t test. Analysis for serum levels of gp70 IC was performed with the Mann-Whitney U test. A value of p < 5% were considered significant.

Results

Selectively increased expression of mPT gp70 RNA in livers of lupus-prone, but not other strains of mice

Our previous analysis of four lupus-prone mice (NZB, NZW, BXSB, and MRL) and four other nonautoimmune strains of mice (NFS, 129, B6, and B10) showed that the abundance of mPT gp70 RNA was consistently higher in lupus-prone mice than in other strains of mice that are not predisposed to SLE, whereas this was not the case for the levels of xenotropic and PT gp70 RNAs (10). To further address a possible association of high-level expression of mPT gp70 RNA with murine SLE, the abundance of ecotropic, xenotropic, PT, and mPT gp70 RNAs in livers of five additional nonautoimmune strains of mice (DBA/2, AKR, BALB/c, CBA, and C3H/He) was assessed and compared with that in lupus-prone mice. Levels of ecotropic, xenotropic and PT gp70 RNAs were highly heterogeneous among different strains, and not consistently high in lupus-prone mice (Fig. 1⇓). In contrast, all four lupus-prone mice displayed markedly increased levels of mPT gp70 RNA, as compared with nine other non-lupus-prone strains of mice (Fig. 1⇓). Indeed, the levels of mPT gp70 RNA in lupus-prone strains were 15- to 39-fold higher than that of NFS mice, which exhibited the highest value among mouse strains not predisposed to SLE (p < 0.01).

FIGURE 1.
FIGURE 1.

Expression levels of different retroviral gp70 RNAs in livers of various strains of mice. Levels of ecotropic (Eco), xenotropic (Xeno), PT, and mPT gp70 RNAs in livers of 2- to 3-mo-old male mice were quantified by real-time RT-PCR using a standard curve generated with serial dilutions of a reference cDNA preparation from AKR liver (for ecotropic gp70) or NZB liver (for xenotropic, PT, and mPT gp70s) and normalized using TBP mRNA. Results are expressed as relative abundance of each gp70 RNA vs TBP mRNA. Each symbol represents an individual animal; horizontal line, mean values.

Presence of three different species of env transcripts derived from endogenous mPT retroviruses

To explore the presence of the possible heterogeneity of endogenous retroviral env transcripts in livers, we developed RT-PCRs with xenotropic, PT-specific, or mPT-specific forward primers and a common reverse primer covering the 5′ portion of gp70 or the gp70-p15E junction region. The analysis of RNA from livers of B6 mice revealed the presence of three species of mPT-specific env amplicons spanning the gp70-p15E junction. One of the amplicons corresponded to the expected size (727 bp) derived from an intact WT form of mPT env transcript, whereas two of the RT-PCR products were smaller, indicative of mPT env deletion mutants (Fig. 2⇓A). In contrast, such heterogeneity was not observed with xenotropic or PT amplicons covering the gp70-p15E junction region. Furthermore, the analysis of the 5′ portion of gp70 cDNAs did not reveal heterogeneity for any of the three classes of retroviral gp70 transcripts (data not shown).

FIGURE 2.
FIGURE 2.

RT-PCR analysis for xenotropic, PT, and mPT env RNAs and for WT, D1, and D2 mPT env genes in different strains of mice. A, The presence of xenotropic, PT, and mPT retroviral env RNAs in livers of 2- to 3-mo-old male NZB and B6 mice was determined by RT-PCR with xenotropic and PT- and mPT-specific gp70 forward primers and a common p15E-R reverse primer. Note the presence of mPT WT and two deletion mutants (D1 and D2) in B6 mice. B, The presence of three different species of WT, D1, and D2 mPT env RNAs in livers of 2- to 3-mo-old male mice of different strains was determined by RT-PCR with mPT858F forward and p15E-R reverse primers. Representative results of three individual animals were analyzed from lupus-prone NZB, NZW, BXSB, and MRL mice; nonautoimmune NFS, 129, AKR, and DBA/2 mice with high serum concentrations of gp70 (>15 μg/ml); and nonautoimmune B6, B10, BALB/c, CBA, and C3H/He mice with low serum concentrations of gp70 (<5 μg/ml). As a control, a mixture of three different plasmids containing WT, D1, and D2 clones obtained from B6 mice was included (Ctl). C, The presence of WT and D1- and D2-specific mPT env transcripts and respective proviral DNA sequences in four different lupus-prone mice and B6 mice was analyzed by RT-PCR and genomic PCR with reverse primers specific for the three different mPT env genes (mPT1447R, D1-R, and D2-R) and a common forward mPT-specific primer (mPT858F). The amplification of only a single fragment of an expected size of corresponding mPT env sequences confirmed the specificity of these PCR analyses. Lane 1, NZB; lane 2, NZW; lane 3, BXSB, lane 4, MRL; lane 5, B6.

To define the possible presence of deletions in two aberrant mPT transcripts obtained with B6 mice, different RT-PCR fragments were cloned. The nucleotide sequence analysis identified the presence of two deletion mutants, designated D1 and D2. The D1 mutant contains a 116-nucleotide deletion in the 3′ portion of the gp70-encoding sequence (Fig. 3⇓). This results in the creation of a premature translation stop codon, thus generating a truncated form (390-aa length) of a gp70 protein, in contrast to the WT gp70 protein of 431 aa (Fig. 4⇓). The D2 mutant has a 99-nucleotide deletion and a 30-nucleotide insertion in the 5′ portion of the transmembrane p15E-encoding sequence (Fig. 3⇓). The deduced amino acid sequence of the env protein indicated that this mutant provirus could generate a defective p15E transmembrane protein with a deletion of 33-aa residues and an insertion of 10-aa residues in its extracellular domain. The gp70 protein-encoding region of the D2 mutant is intact (Fig. 4⇓). BLAST search analysis confirmed the presence in the B6 mouse genome of these two mPT mutant proviruses in chromosomes 3 and 5, respectively. Indeed, an mPT provirus, NL1, corresponding to the D1 mutant was previously cloned from NFS mice (28).

FIGURE 3.
FIGURE 3.

Alignment of partial nucleotide sequences of env regions of D1 and D2 mPT retroviruses in B6 mice. mPT env cDNA spanning the gp70-p15E junction region was amplified with mPT858F forward and p15E-R reverse primers. As a comparison, WT mPT sequence derived from MX33 provirus (GenBank accession number: M17327; http://www.ncbi.nlm.nih.gov/ GenBank) is shown. The sequences of different primers used in the present study are underlined, and the names of the primers are indicated. Dashes indicate nucleotide identity, and dots indicate absence of a nucleotide. A premature translation stop codon (TAA), as a result of a 116-nucleotide deletion, in the 3′ end of the gp70 region of the D1 mutant is shown in bold. The D1 sequence is identical with that of the NL1 mPT provirus derived from NFS mice (GenBank accession number: AY219562; http://www.ncbi.nlm.nih.gov/GenBank). The GenBank accession number for the D2 sequence is FJ182230 (http://www.ncbi.nlm.nih.gov/ GenBank).

FIGURE 4.
FIGURE 4.

Alignment of predicted amino acid sequences of WT, D1, and D2 env polyproteins. Nucleotide sequences of the env genes of the D1 and D2 mutant proviruses in B6 mice were derived from RP24-548E8 and RP23-229N3 BAC clones, respectively, and their predicted amino acid sequences are shown. The WT mPT env sequence is derived from MX33 provirus. Dashes indicate amino acid identity, and dots indicate absence of an amino acid residue. A 10-aa insertion in the D2 mutant is indicated as a box. The predicted proteolytic cleavage sites for the gp70 surface protein and the p15E transmembrane protein are indicated by the arrow and a hydrophobic stretch of 29 aa’s presumed to span the membrane is underlined. Numbers indicate amino acid positions of the WT mPT env polyprotein.

Predominant expression of the WT mPT env RNA in livers of lupus-prone, but not other strains of mice

Because NZB mice selectively expressed the WT mPT env transcript in contrast to the predominant expression of the D2 mutant env RNA in B6 mice, we compared the expression patterns of the three different species of mPT env transcripts in livers of various strains of mice by using the above-described mPT-specific RT-PCR spanning the gp70-P15E junction region. The analysis of all four different lupus-prone strains of mice (NZB, NZW, BXSB, and MRL) revealed that they predominantly expressed the WT mPT env transcript (Fig. 2⇑B). In contrast, despite the presence of relatively high serum concentrations of gp70 (>15 μg/ml), NFS, 129, AKR, and DBA/2 mice displayed more heterogeneous expression patterns of the different mPT env transcripts without apparent predominance of the WT transcript. This was also the case in B6, B10, BALB/c, CBA, and C3H/He mice, which exhibit low serum levels of gp70 (<5 μg/ml).

The predominant expression of the WT mPT env RNA in lupus-prone mice could be due to the absence of D1 and D2 mutant proviruses in their genomes or, alternatively, to control mechanisms governing RNA levels in these mice. To examine these possibilities, we developed PCRs to distinguish the mPT env transcripts using reverse primers specific for the three different mPT env sequences and a common forward mPT-specific primer (the locations of which are shown in Fig. 3⇑). We then determined the presence of the three different species of mPT proviruses in the genomic DNA of four different lupus-prone mouse strains and compared the results with the env RNA expression patterns in livers of the respective strains. As shown in Fig. 2⇑C, D1 env RNA was expressed in all four lupus-prone mice, while D2 env RNA was found only in BXSB mice among them. Analysis of the genomic DNA revealed a complete correlation between the presence of different mPT env genes and the expression of respective env RNAs in livers. Thus, the lack of the expression of the D2 env RNA in NZB, NZW, and MRL mice was due to the absence of the D2 provirus. This was also the case in several nonautoimmune strains tested (129, DBA/2, CBA, and C3H/He; data not shown). Quantitative genomic PCR analysis showed no apparent differences in the copy number of WT mPT proviruses between NZB and B6 mice (data not shown), suggesting differences in transcriptional control mechanisms of WT mPT env genes in these mice.

Predominant expression of WT mPT env RNA in B6- or B10-congenic mice bearing the NZB- or BXSB-Sgp3 allele but not in B6-congenic mice bearing the NZB-Sgp4 allele

Recent analysis of B6- and B10-congenic mice bearing the Sgp3 or Sgp4 locus derived from NZB or BXSB mice demonstrated a substantial role of both Sgp3 and Sgp4 loci in the increase of serum gp70 and various gp70 RNAs in livers (10, 12, 15). To investigate the role of the Sgp3 and Sgp4 loci in the predominant expression of WT mPT env transcripts in lupus-prone mice, we assessed the relative expression of the three different species of mPT env transcripts in Sgp3 and Sgp4 congenic mice. As shown in Fig. 5⇓A, B6 mice carrying the NZB-Sgp3 locus and B10 mice carrying the BXSB-Sgp3 locus displayed a predominant expression of the WT mPT env RNA, similar to the parental NZB and BXSB strains of mice. In contrast, the expression pattern was unchanged in B6 mice bearing the NZB-Sgp4 locus.

FIGURE 5.
FIGURE 5.

RT-PCR analysis for WT, D1, and D2 mPT env RNAs in Sgp3- and Sgp4-congenic mice. A, The abundance of three different species of WT, D1, and D2 mPT env RNAs in livers of 2- to 3-mo-old male B6- and B10-congenic mice bearing the NZB- or BXSB-derived Sgp3 or Sgp4 locus and from the parental strains of male mice was determined by RT-PCR with mPT858F forward and p15E-R reverse primers. Results are representative of three individual mice analyzed. As a control, a mixture of three different plasmids containing WT, D1, and D2 clones obtained from B6 mice was included (Ctl). B, Semiquantitative RT-PCR analysis for WT, D1, and D2 mPT env RNAs with reverse primers specific for the three different mPT env genes (mPT1447R, D1-R, and D2-R) and a common forward mPT-specific primer (mPT858F) was conducted with 5-fold serially diluted cDNAs from Sgp3- or Sgp4-congenic male mice and from the parental strains of male mice. As a control, the abundance of GAPDH mRNA was assessed in parallel. Four 5-fold dilutions of cDNAs were examined for WT mPT env RNA, whereas three 5-fold dilutions of cDNAs were examined for D1/D2 mPT env RNAs and GAPDH mRNA. Results are representative of three individual mice analyzed. C, The abundance of three different species of mPT env RNAs (WT, D1, and D2) in livers of 2- to 3-mo-old male BXSB, SB/Le, and B6 mice was determined by RT-PCR with mPT858F forward and p15E-R reverse primers.

The levels of three different mPT env RNAs in livers were semiquantified by RT-PCR using a common forward mPT-specific primer and the reverse primers specific for the three different mPT env sequences. This approach was necessary due to the remarkable homology in the gp70-p15E junction region between mPT and PT env genes which precluded the design of a specific mPT WT primer suitable for real-time RT-PCR. The analysis with 5-fold serially diluted cDNA samples from B6.NZB-Sgp3 and B10.BXSB-Sgp3 congenic mice showed marked and selective increases (∼100- and ∼25-fold, respectively) in WT mPT env transcripts, as compared with B6 and B10 mice, respectively, whereas no appreciable increases in D1 and D2 mPT env RNAs were observed (Fig. 5⇑B). In addition, >100-fold higher levels of WT mPT env RNAs were expressed in NZB and BXSB mice carrying Sgp3, compared with B6 and B10 mice lacking Sgp3. In contrast to B6 and B10 mice, the great majority of the mPT env transcripts in lupus-prone NZB and BXSB mice were indeed derived from WT mPT proviruses rather than the defective mPT proviruses. None of the three mPT env transcripts was increased in B6.NZB-Sgp4 congenic mice (Fig. 5⇑B). Real-time RT-PCR analyses indicated that the abundance of total mPT env RNA (WT, D1 and D2) was increased 19- and 6-fold in B6.NZB-Sgp3 and B10.BXSB-Sgp3 congenic mice, respectively (p < 0.01), while defective mPT env RNAs were not significantly elevated in either Sgp3 and Sgp4 congenic mice (Table I⇓). These results corroborated the RT-PCR results and indicated that the observed increases in total mPT env RNA in Sgp3 congenic mice were due to elevated WT mPT env RNA.

View this table:
Table I.

Fold changes of mPT env RNAs in livers of Sgp3 and Sgp4 congenic mice relative to those in B6 micea

The selectively up-regulated expression of WT mPT env RNA in lupus-prone mice and in Sgp3 congenic mice could be the result of the presence in the Sgp3 region of an mPT provirus carrying the intact env gene, which may be especially highly expressed because of its particular integration site. If this was the case, one could expect that the expression pattern of the three different mPT env RNAs in SB/Le mice should be similar to that of BXSB mice, since BXSB is a recombinant strain derived from a cross of B6 and SB/Le mice. However, this possibility was unlikely, since we found that livers of SB/Le mice failed to display the predominant expression of the intact mPT env transcripts (Fig. 5⇑C). More significantly, these results indicate that the BXSB-Sgp3 allele responsible for the predominant and abundant expression of intact mPT env transcripts is inherited neither from the B6 strain nor from the SB/Le strain. This suggested that during the establishment of the BXSB strain, Sgp3 must have mutated and become defective in these mice.

Mutations in the U3 regulatory region of the LTR in the D1 mutant

Differentially regulated expression of three different mPT env RNAs by Sgp3 could be related to the possible differences in the U3 regulatory regions of the LTR (29) among these three different mPT retroviruses. To this end, we amplified mPT env cDNA spanning the env gene and the U3 region and then isolated three different species of cDNA clones from livers of NZB, B6.NZB-Sgp3, and B6 mice. The nucleotide sequence analysis of at least three independent clones revealed an addition of T (thymine) in the upstream conserved region (UCR) core sequence, CGCCATTTT (30), and a substitution of G (guanine) with A (adenine) in an SV40 core-like motif, GTGGTAA (31, 32), in the U3 region of the D1 mPT mutant (Fig. 6⇓). These two mutations could have significant consequences on the expression of the D1 provirus, given that UCR and SV40 core-like elements likely regulate the expression of endogenous retroviruses. In contrast, the U3 region of the D2 mutant was identical with that of WT mPT.

FIGURE 6.
FIGURE 6.

Alignment of nucleotide sequences of the U3 region of the LTR of WT, D1, and D2 mPT retroviruses in B6 mice. mPT cDNA spanning the U3 region was amplified with mPT858F forward and UnitlrR reverse primers. Dashes, nucleotide identity; dots, absence of a nucleotide. Direct repeats and unique sequences present in the retroviruses are boxed (29 ). The UCR and SV40 core-like motif, as negative and positive regulatory elements, respectively, are underlined. The U3 sequence of the WT and D1 mutant is identical with that of the MX33 and NL1 mPT proviruses, respectively.

Critical role of TLR7 in the formation of gp70 IC

Because TLR7 is an innate immune receptor specific for single-stranded RNA and plays a significant role in the development of murine SLE (33), we explored the possible contribution of TLR7 to autoimmune responses against serum retroviral gp70 Ags in B6 mice congenic for the Nba2 locus, which regulates overall production of lupus autoantibodies (12, 34). At 8 mo of age, female B6.Nba2 mice displayed significantly elevated levels of gp70 IC in sera (0.79 ± 0.44 μg/ml), as compared with control female B6 mice (0.27 ± 0.11 μg/ml; p < 0.02; Fig. 7⇓). In contrast, the formation of gp70 IC was almost completely suppressed in TLR7-deficient B6.Nba2 female mice (0.10 ± 0.07 μg/ml; p < 0.0001 vs B6.Nba2 mice), and their levels were even significantly lower than those in control B6 mice (p = 0.001). These data clearly underlined the critical role of TLR7 for autoimmune responses against retroviral gp70 Ags.

FIGURE 7.
FIGURE 7.

Serum levels of gp70 IC in TLR7-sufficient or -deficient female B6.Nba2 mice and control female B6 (WT) mice at 8 mo of age. Each symbol represents an individual animal. Results are expressed as micrograms per milliliter for gp70 IC. Horizontal line, mean values.

Discussion

In the present study, we explored a possible association of mPT viral expression with murine SLE by assessing expression levels of different endogenous retroviral env RNAs in livers from lupus-prone and nonautoimmune strains of mice. Our results demonstrate that: 1) lupus-prone mice displayed a selectively up-regulated expression of mPT gp70 RNA; 2) many strains of mice expressed not only intact but also two defective mPT env transcripts; 3) lupus-prone mice predominantly expressed abundant levels of intact mPT env transcripts but only low or nondetectable levels of the mutant env transcripts; and 4), the Sgp3 locus derived from lupus-prone mice was responsible for the selective expression of the WT mPT env RNA. Our data suggest that lupus-prone mice could have a unique genetic mechanism responsible for the expression of mPT retroviruses.

The deletions of two different mPT env mutants isolated in the present study are located in the gp70-p15E junction region. Because of the deletion in the extracellular domain of the p15E transmembrane protein in the D2 mutant, it is possible that this aberrant p15E protein cannot efficiently form disulfide-linked envelope protein complexes with the intact form of gp70 generated by this mutant. Thus, the expression of this mutant could promote a release of free gp70 into the circulating blood. However, the lack of up-regulated expression of the D2 env RNA in Sgp3-congenic mice and its poor or absent expression in mice having high serum levels of gp70 clearly indicate that the D2 mutant is not an important source for serum gp70. The generation of a small truncated form of gp70 by the D1 mutant also excluded this mutant protein as a source of serum gp70.

The expression patterns of three different species (WT and two mutants) of mPT env RNAs in livers were highly variable among various strains of mice. Part of this variation is due to the absence of the mPT D2 provirus in several strains; however, much of the variation appears to be the result of control mechanisms. The most striking observation in the present study was that all four lupus-prone mouse strains exhibited very high transcriptional levels of the WT mPT env gene when compared with the transcriptional levels of nine additional strains of mice tested, all of which are not predisposed to autoimmune diseases. This was clearly not due to differences in the copy number of mPT proviruses in the genomes between lupus-prone and other strains of mice. Indeed, the reported number of mPT proviral loci in NZB mice was not higher than that of B6, AKR and DBA/2 mice (35, 36, 37), and this was consistent with the results obtained with quantitative PCR analysis for WT mPT env genes between NZB and B6 mice. Instead, the analysis of B6 and B10 mice congenic for the Sgp3 locus derived from the NZB and BXSB strains clearly demonstrated that the Sgp3 locus is responsible for the selective expression of the WT mPT proviruses in lupus-prone mice. Since our previous analysis suggested that Sgp3 regulates the expression of multiple endogenous retroviral transcripts in trans, it was surprising that the expression of the WT mPT env RNA, but not mutant env RNAs, was predominantly up-regulated in the two Sgp3-congenic mice. Because both B6 and B10 mice expressed significant levels of D1 and D2 env RNAs, the lack of an enhanced transcription of these env genes in Sgp3-congenic mice indicate that the possible function of Sgp3 as a trans-activating factor may be somehow more selective for a subset of the mPT proviruses encoding the WT env gene. Consistent with our previous demonstration that Sgp4 controlled the expression of xenotropic gp70 RNA, but not PT and mPT gp70 RNAs (10), the presence of the Sgp4 locus failed to up-modulate the expression of any mPT env transcripts.

The selective effect of Sgp3 on the expression of WT mPT env genes suggests that the regulatory elements modulated by Sgp3 may be less efficient in the D1 and D2 mutants. Indeed, the analysis of the U3 regulatory region of the LTR identified the presence of two mutations in the D1 mutant. A substitution of G (guanine) with A (adenine) in an SV40 core-like motif (GTGATCA instead of GTGGTCA) could markedly reduce its enhancer activity, as it has previously been shown that a G to A substitution in this motif (GTGGTAA to GTAGTAA) abolished the enhancer function (31). The second mutation, i.e., an insertion of T (thymine) in the UCR, may alter a binding affinity of the UCR-binding protein, which negatively regulates the expression of endogenous retroviruses (30). Thus, the presence of these two mutations could at least partly explain the lack of an up-regulated transcription of the D1 env gene in Sgp3-congenic mice and its poor expression in lupus-prone mice. In contrast, we did not find any mutations in the U3 sequence of the D2 mutant. At present, we cannot offer a straightforward explanation for a limited expression of the D2 env RNA in Sgp3-congenic mice. Because the expression of retroviral sequences is strongly affected by the state of DNA methylation (38, 39, 40), one plausible explanation is that the enhancer element(s) of the U3 region, implicated in the increased expression by the presence of Sgp3, may be selectively methylated in this particular mutant. Another conceivable explanation is that Sgp3 may influence degradation of the mPT RNAs rather than transcription of the mPT proviruses, thus enhancing stabilization of a particular secondary structure which may not form with mutant mPT env RNAs.

It has previously been shown that like Sgp3, the Gv1 (Gross virus Ag 1) locus controls the levels of endogenous retroviral sequences in different tissues, including the liver (41), and regulates the abundance of thymocyte differentiation GIX gp70 Ag (42), the expression of which is closely correlated to serum levels of gp70 (43, 44). Because the Gv1 locus, identified in the 129 strain (45), directly overlaps with the Sgp3 locus (12, 14, 15, 46), it has been speculated that Gv1 and Sgp3 are identical or related genes regulating the transcription of retroviral sequences, and that the GIX+ 129 strain shares the Sgp3 allele with lupus-prone mice. However, we observed that the expression pattern of mPT env transcripts in 129 mice is clearly different from that of B6 and B10 mice bearing the Sgp3 locus derived from lupus-prone mice. This was also the case for other GIX+ strains of mice, such as AKR, DBA/2, and C3H/He (43). In this regard, it is worth noting that the Gv1 locus derived from the 129 strain regulated the transcription of PT proviruses, but not mPT proviruses (46). Accordingly, it is possible that lupus-prone mice carry different regulatory elements in the Sgp3 interval, which independently control the levels of mPT, PT and possibly xenotropic proviral sequences, and that the presence of the regulatory element controlling the mPT proviral expression may be unique in lupus-prone mice. A recent study has identified two Rsl (regulator of sex limitation) genes in this region, Rsl1 and Rsl2, which encode Krüppel-associated box zinc-finger proteins and control sexually dimorphic gene expression in liver (47). In addition to the two Rsl genes, there exist in this region 20 more Rsl candidate genes, the function of which is still unknown. Because the Krüppel-associated transcription repressor domain has been shown to suppress lentivirus proviral transcription by inducing heterochromatization in the lentiviral integration sites (48), it will be of interest to explore whether any of the Rsl or Rsl candidate genes are indeed Sgp3 or Gv1.

A substantial role of TLR7, an innate immune receptor specific for single-stranded RNA, for the development of autoimmune responses against RNA-related nuclear autoantigens in murine SLE has recently emerged (26, 49, 50, 51, 52). In this study, we have demonstrated that TLR7 was also critically involved in the production of anti-gp70 autoantibodies. This raises an attractive hypothesis that endogenous retroviruses contribute to the development of this autoimmune response, possibly as a result of the activation of dendritic cells and anti-gp70-specific autoreactive B cells through interaction with TLR7. One could speculate that abundant and preferential expression of mPT proviruses possessing an intact env gene could facilitate the generation of replication-competent mPT-derived infectious viruses which may act as a triggering factor for the development of murine SLE. It has been reported that among various strains of mice, an 8.4-kb transcript corresponding to the full-length size of mPT retroviruses was expressed uniquely in thymi of NZB, BXSB, and MRL mice, whereas the expression of full-length transcripts of xenotropic and PT retroviruses was not limited to lupus-prone mice (53). Although it was not defined whether these mPT-derived transcripts correspond to the genomic RNA encoding replication-competent infectious viruses, these findings may be related to our current observations. The development of a lupus-like autoimmune syndrome was promoted by neonatal infection with a murine leukemia virus from NZB mice (25), and spontaneous production of antinuclear and anti-gp70 autoantibodies was also found in Sgp3-congenic mice (14, 15). Considering the remarkable role of TLR7 in the development of SLE, it is of importance to reassess the implication of endogenous retroviruses as a triggering factor in murine SLE.

A possible contribution of endogenous retroviruses to the development of human SLE has long been suspected. With the use of polyclonal Abs raised against murine and feline leukemia viruses, the presence of an Ag related to mammalian retroviral core protein p30 was reported in immune deposits of glomerular lesions from human SLE (54). In addition, increased titers of Abs reactive with peptides derived from the env and gag genes of human endogenous retroviruses or human T cell leukemia virus-1-related endogenous sequences have also been described in sera from human SLE patients (55, 56, 57, 58). However, the search for the presence of serum retroviral gp70 and its immune complex in human SLE has not been successful. One possible explanation for this failure may be a lack of appropriate Abs to specifically detect retroviral gp70 Ags implicated in human SLE. Clearly, the eventual identification of replication-competent endogenous retroviruses implicated in murine SLE and of mouse genes regulating their production could provide a clue for the potential role of endogenous retroviruses in human SLE.

Acknowledgments

We thank Guy Brighouse and Giuseppe Celetta for their excellent technical assistance.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by a grant from the Swiss National Foundation for Scientific Research. L.H.E. was supported by the intramural research program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

  • ↵2 K.Y. and L.B. contributed equally to this work.

  • ↵3 Address correspondence and reprint requests to Dr. Shozo Izui, Department of Pathology and Immunology, Centre Médical Universitaire, 1211 Geneva 4, Switzerland. E-mail address: Shozo.Izui{at}unige.ch

  • ↵4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; gp70 IC, gp70-anti-gp70 immune complexes; PT, polytropic; mPT, modified PT; WT, wild type; TBP, TATA-binding protein; LTR, long terminal repeat; UCR, upstream conserved region.

  • Received January 23, 2009.
  • Accepted April 9, 2009.

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