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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Autoimmun. Author manuscript; available in PMC Dec 1, 2012.
Published in final edited form as:
PMCID: PMC3254601
NIHMSID: NIHMS326669
Sgp3 and Sgp4 Control Expression of Distinct and Restricted Sets of Xenotropic Retroviruses Encoding Serum gp70 Implicated in Murine Lupus Nephritis
Maso Kihara,a Valérie Leroy,a Lucie Baudino,a Leonard H. Evans,b and Shozo Izuia*
aDepartment of Pathology and Immunology, University of Geneva, Geneva, Switzerland
bLaboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840
* Corresponding author. Tel.: +41-22-379-5741; Fax: +41-22-379-5746. Shozo.Izui/at/unige.ch (S. Izui)
The envelope glycoprotein gp70 of endogenous retroviruses implicated in murine lupus nephritis is secreted by hepatocytes and its expression is controlled by Sgp3 (serum gp70 production 3) and Sgp4 loci derived from lupusprone mice. Among three different endogenous retroviruses (ecotropic, xenotropic and polytropic), xenotropic viruses are considered to be the major source of serum gp70. Although the abundance of xenotropic viral gp70 RNA in livers was up-regulated by the presence of these two Sgp loci, it has not yet been clear whether Sgp3 and Sgp4 regulate the expression of a fraction or multiple xenotropic viruses present in mouse genome. To address this question, we determined the genetic origin of xenotropic viral sequences expressed in wildtype and two different Sgp congenic C57BL/6 mice. Among 14 xenotropic proviruses present in the C57BL/6 genome, only two proviruses (Xmv10 and Xmv14) were actively transcribed in wild-type C57BL/6 mice. In contrast, Sgp3 enhanced the transcription of Xmv10 and induced the transcription of three additional xenotropic viruses (Xmv15, Xmv17 and Xmv18), while Sgp4 induced the expression of a different xenotropic virus (Xmv13). Notably, stimulation of TLR7 in Sgp3 congenic C57BL/6 mice led to a highly enhanced expression of potentially replication-competent Xmv18. These results indicated that Sgp3 and Sgp4 independently regulated the transcription of distinct and restricted sets of xenotropic viruses in trans, thereby promoting the production of nephritogenic gp70 autoantigens. Furthermore, the induced expression of potentially replication-competent xenotropic viruses by Sgp3 may contribute to the development of autoimmune responses against gp70 through the activation of TLR7.
Keywords: Systemic lupus erythematosus, Endogenous retrovirus, Toll-like receptor
The expression of the envelope glycoprotein gp70, encoded by the retroviral env gene, is linked to the differentiation state of the cells [1]. Substantial amounts of gp70, free from any association with viral particles, are present in sera of virtually all strains of mice [13]. More significantly, only lupus-prone (NZB × NZW)F1, MRL and BXSB mice spontaneously develop autoantibodies against serum retroviral gp70 [4]. gp70-anti-gp70 immune complexes (gp70 IC) are detected close to the onset of renal disease in the circulation and found as immune deposits within diseased glomeruli of lupus mice [46]. In addition, a remarkable correlation of serum levels of gp70 IC with the development of lupus nephritis revealed by several genetic studies [711] indicates the pathogenic role of gp70 IC in murine systemic lupus erythematosus (SLE).
Endogenous retroviruses are classified by the host range dictated by the gp70 protein as follows: ecotropic, xenotropic and polytropic retroviruses [12]. Based on differences in their gp70 nucleotide sequence, the xenotropic viruses has been divided into four subgroups, Xeno-I, Xeno-II, Xeno-III and Xeno-IV, [13, 14] and the polytropic viruses into two subgroups, polytropic (PT) and modified PT (mPT) [15]. Earlier serological and tryptic peptide mapping analysis showed that serum gp70 most closely resembles the gp70 protein of xenotropic viruses isolated from NZB mice [3, 16, 17]. Moreover, our recent analysis of the abundance of retroviral gp70 RNAs in different strains of mice suggested that PT and mPT proviruses that encode gp70s closely related to xenotropic gp70 could be additional sources of serum gp70 [14].
Serum retroviral gp70 is secreted by hepatocytes [18] and their concentrations are highly variable among different strains of mice [2, 46]. All SLE-prone strains (NZB, NZW, MRL and BXSB) have relatively high concentrations of gp70 in their sera (>15 μg/ml), whereas C57BL/6 (B6), C57BL/10 (B10) and BALB/c mice produce low serum levels of gp70 (<5 μg/ml). Genetic analysis of the progeny of crosses of lupus-prone mice with nonautoimmune B6 or B10 mice identified two loci in lupus-prone mice, Sgp3 (serum gp70 production 3) on mid chromosome 13 and Sgp4 on distal chromosome 4, which control basal-level expression of serum gp70 [11, 14, 1924]. In addition, the expression of serum gp70 in lupus-prone mice is enhanced by different inducers of acute phase proteins, including TLR7 and TLR9 agonists [18, 25]. However, unlike conventional acute phase proteins, the serum gp70 response is strain-dependent, in which only mice having high basal levels of serum gp70 such as lupus-prone mice displayed an up-regulated expression of serum gp70 in response to LPS [26, 27]. Notably, studies on Sgp congenic mice revealed that the Sgp loci contribute to the acute phase expression of serum gp70 [14, 22, 25].
The precise genetic mechanisms responsible for the expression of serum gp70 in livers remain poorly understood. The analysis of B6 and B10 mice congenic for the Sgp3 locus showed marked increases in levels of xenotropic, PT and mPT gp70 RNAs in livers [14, 28]. Thus, it has been speculated that Sgp3 regulates the expression of serum gp70 by controlling the transcription of multiple endogenous retroviral genomes in trans. In contrast, the effect of the Sgp4 locus was restricted to xenotropic viruses [14]. Notably, the presence of the Sgp4 locus derived from NZB mice resulted in a selectively up-regulated expression of Xeno-I gp70 RNA but a suppressed expression of Xeno-II/III gp70 RNAs. These data suggest that Sgp3 and Sgp4 enhance or induce the transcription of different sets of endogenous xenotropic retroviral sequences, thereby promoting the production of nephritogenic gp70 autoantigens implicated in murine SLE. To address this issue, we conducted a clonal analysis of xenotropic viral sequences expressed in wild-type (WT) B6 mice and those congenic for either the Sgp3 or Sgp4 locus derived from lupus-prone NZB mice. Results obtained from the present study indicated that the Sgp3 and Sgp4 loci independently regulate the transcription of distinct and restricted subpopulations of xenotropic proviruses in trans, thereby contributing to the increased production of serum gp70.
2.1 Mice
B6.Sgp3 congenic mice carrying an NZB interval flanked by markers D13Mit283 (63.4 Mb from the centromere) and D13Mit254 (76.1 Mb) and B6.Sgp4 congenic mice carrying an NZB interval flanked by markers D4Mit11 (122.9 Mb) and D4Mit33 (150.0 Mb) were generated by backcross procedures using marker-assisted selection, as described previously [14, 29]. All studies presented were carried out in male mice. Animal studies described in the present study have been approved by the Ethics Committee for Animal Experimentation of the Faculty of Medicine, University of Geneva (authorization number: 31.1.1005/3049/2-R).
2.2. RT-PCR cloning
Xenotropic viral cDNA spanning the env gene and the U3 region of the long terminal repeat (LTR) was amplified with Xeno1098F forward primer and Uniltr-4R reverse primer to determine the U3 sequence of the LTR, as described [28]. The amplified fragments were purified on a 2% agarose gel and ligated into pBluescript-SK+ plasmid.
2.3. Quantitative real-time PCR
RNA from livers was purified with TRIzol reagent (Invitrogen AG, Basel, Switzerland) and treated with DNase I (Amersham Biosciences Corp., Piscataway, NJ). The abundance of xenotropic gp70 RNAs (genomic RNA and mRNA) was quantified by real-time PCR, as described previously [14, 29]. Levels of G protein-coupled receptor kinase 5 (Grk5), nucleoporin like 2 (Nupl2), potassium voltage-gated channel, subfamily H (eag-related), member 2 (Kcnh2), coronin, actin binding protein, 2B (Coro2b) and 4921517L17Rik mRNAs were quantified with the following primers: Grk5 forward primer (5′-CTGACTGCCGACTGTCAATG-3′) and reverse primer (5′-CACTGGCTGATGTGAGGAAAC-3′); Nupl2 forward primer (5′-GCGCTACCATGACCATATGC-3′) and reverse primer (5′-GCTGGATGACGTTGGAGTACC-3′); Kcnh2 forward primer (5′-CCAGTGACCGGGAGATTATAGC-3′) and reverse primer (5′-ATGGTCCAGCGGTGGATTCTTG-3′); Coro2b forward primer (5′-CGCCCGCTTTCTGTATGAC-3′) and reverse primer (5′-TGGCCGCTTTCCCATACAC-3′); 4921517L17Rik forward primer (5′-AGATAAAGGCGGAGACACCC-3′) and reverse primer (5′-GCCTTGACGGAACATCTTACAC-3′). PCR was performed using the iCycler iQ Real-Time PCR Detection System (Bio-Rad, Philadelphia, PA) and iQ SYBR green Supermix (Bio-Rad). Results were quantified using a standard curve generated with serial dilutions of a reference cDNA preparation from NZB livers and normalized using TATA-binding protein (TBP) mRNA. PCR products were visualized after electrophoresis on 2% agarose gels by staining with ethidium bromide.
2.4. Genomic PCR
The presence of Xmv13 and Xmv14 proviruses in mice was determined by genomic PCR analysis in the following combinations of primers for 5′- and 3′-flanking sequences and those for the U3 sequence: 5′-Xmv13 franking forward primer (5′-CATGCACAGCACTGAAGTCTAC-3′), 3′-Xmv13 flanking reverse primer (5′-TTAGGCGGAAGTGAGTGTC-3′), 5′-Xmv14 flanking forward primer (5′-TCACTTCCACTGCCCAAATCAC-3′), 3′-Xmv14 flanking reverse primer (5′-CTTGTCTTTTCCACTGGGTCAC-3′), U3 forward (5′-AAGCTAGCTGCAGTAACGCCATTTTGC-3′) and reverse (5′-AGCTTGCTAAGCCTTATGGTGG-3′) primers. PCR products were visualized by staining with ethidium bromide after electrophoresis on a 2% agarose gel.
2.5. Serological assay
Serum levels of retroviral gp70 in 2–3 mo-old male mice were determined by ELISA as described previously [30]. Results are expressed as μg/ml of gp70 by referring to a standard curve obtained from a serum pool of NZB mice.
2.6. Injection of TLR7 agonist
TLR7 agonist, 1V136 [31] (TLR7 Ligand II, Calbiochem, EMD Chemicals Inc., Darmstadt, Germany) was diluted with sterile PBS and i.p injected into 2–3 mo-old male mice. Livers and sera were collected 9 h after injection of 1V136 (100 μg).
2.7. Statistical analysis
Unpaired comparison for frequencies of clones expressing different Xmv env RNAs in livers and for levels of different mRNA and xenotropic retroviral RNAs in livers and paired comparison for serum levels of gp70 before and after injection of 1V136 were analyzed by Student’s t test. Unpaired comparison for levels of hepatic gp70 and serum gp70 among B6.Sgp3, WT B6 and their F1 mice was analyzed by one-way ANOVA test. Probability values <5% were considered significant.
3.1. Highly heterogeneous structures of the U3 regions of LTR of endogenous xenotropic proviruses in B6 mice
Previous restriction fragment length polymorphism (RFLP) analysis with the use of xenotropic-specific env probe in B6 and recombinant inbred mice reported the presence and chromosomal localization of 19 Xmv xenotropic proviral loci in the B6 mouse genome [32]. BLAST search analysis, according to gp70 sequences specific for four different subgroups of xenotropic proviruses [14], led us to identify in B6 mice 14 Xmv loci carrying the intact gp70-encoding sequence, which belong to 4 Xeno-I, 3 Xeno-II, 5 Xeno-III and 2 Xeno-IV subgroups (Table 1). In contrast to three Xmv loci (Xmv7, Xmv11 and Xmv40) reported to be present in the Y chromosome [32], BLAST search revealed the presence of a single copy of xenotropic provirus in the Y chromosome. Thus, this provirus is tentatively named XmvY. Notably, among 15 xenotropic proviruses detected by RFLP analysis in NZB mice [33], only five Xmv loci (Xmv10, Xmv15, Xmv17, Xmv45 and XmvY) are shared with B6 mice (Table 1). Furthermore, Xmv13 and Xmv14 are respectively located within the Sgp3 and Sgp4 intervals of B6 mice, but absent in NZB mice. The inspection of the coding sequences of the gag, pol and env genes indicated that only 5 proviruses (Xmv12, Xmv13, Xmv14, Xmv18 and Xmv45) present in B6 mice are potentially replication competent.
Table 1
Table 1
Chromosomal localization of xenotropic proviruses and their subgroups in B6 and NZB mice
The U3 regions of LTR of 14 xenotropic proviruses present in B6 mice carry a deletion of 190 nucleotides characteristic of xenotropic viruses [34, 35], with the exception of Xmv42, the U3 structure of which is identical to that of PT viruses (Fig. 1 and S1). The structure of the U3 regions of xenotropic proviruses in B6 mice displayed remarkable heterogeneity. According to the classification of the U3 structure reported by Tomonaga and Coffin [35], Xmv43, Xmv45 and XmvY are assigned to the X-I subgroup, Xmv8, Xmv13 and Xmv14 to the X-II subgroup and the rest of the Xmv proviruses to the X-III subgroup, except Xmv42 which is assigned to the P-I subgroup corresponding to PT viruses (Table 1 and Fig. 1).
Fig. 1
Fig. 1
Schematic representation of the U3 structures of 14 different Xmv proviruses present in the B6 genome. The U3 structure of these Xmv proviruses are assigned to the X-I, X-II and X-III subgroups, except Xmv42, which is assigned to the PT virus (P-I), according (more ...)
3.2. Selective transcription of Xmv10 and Xmv14 proviruses in B6 mice
Considerable heterogeneity of the U3 sequences of these 14 xenotropic proviruses could allow us to identify the genetic origin of xenotropic viruses expressed in WT and Sgp congenic B6 mice. To this end, we amplified a region spanning the xenotropic env gene and the U3 region of xenotropic viral cDNA derived from livers, cloned them and determined which Xmv proviruses were expressed in B6 mice through the nucleotide sequence analysis of the U3 region. We observed that livers of B6 mice expressed xenotropic env RNAs derived from only two xenotropic proviruses, Xmv10 and Xmv14, belonging to the Xeno-I and Xeno-III subgroups, respectively. Quantitative analysis of ~20 clones from each of four individual mice revealed that the mean frequencies (± SEM) of Xmv10 and Xmv14 transcripts were 67.4 ± 4.1% and 32.7 ± 4.1%, respectively (Fig. 2).
Fig. 2
Fig. 2
Frequencies of different Xmv transcripts in livers from B6, B6.Sgp3 and B6.Sgp4 mice. A region spanning the xenotropic env gene and the U3 region of xenotropic viral cDNA derived from livers was amplified, and amplicons were cloned and sequenced to identify (more ...)
The transcription of endogenous xenotropic viruses in B6 mice can be influenced by the presence, absence or enrichment of regulatory sequences present in the U3 region. Enhancer elements are enriched in the Xmv14 provirus due to the duplication of the box 4 which carries three different enhancer elements (E-Box1, NF1 and GRE) (Fig. 1 and and3).3). However, this enrichment is not unique for Xmv14. More strikingly, because of a deletion of the region spanning the box 2 and box 4, the Xmv10 provirus lacks several enhancer elements including CArG, LVb, Core, E-Box1 and NF1 sites, unlike other xenotropic proviruses (Fig. 1 and and3).3). Thus, it is unlikely that the selective expression of Xmv10 and Xmv14 in B6 mice can be attributed to enhancer elements within their U3 sequences. Some studies indicated that the tRNA primer-binding site (PBS) sequence can trigger silencing of integrated infectious retroviruses in embryonic cells [3638]. However, such a mechanism is unlikely for the selective expression of Xmv10 and Xmv14 in B6 mice, since the tRNA PBS nucleotide sequences of these two proviruses are identical to those of 5 and 3 other xenotropic proviruses, respectively, which are not actively transcribed (Fig. 4).
Fig. 3
Fig. 3
Potential enhancer sequence regions and transcriptional factor binding sites in the U3 region of Xmv14 and Xmv10 proviruses. Upper panel: Schematic representation of the U3 structures of Xmv14 and Xmv10 proviruses. Lower panel: Transcriptional factor (more ...)
Fig. 4
Fig. 4
The nucleotide sequences of the tRNA PBS of 14 different Xmv proviruses present in B6 mice. Dots indicate nucleotide identity. Note the PBS nucleotide sequence of Xmv10 is identical to that of Xmv8, Xmv12, Xmv17, Xmv18 and Xmv19, and the PBS sequence (more ...)
High-level transcription of Xmv10 can be due to a unique integration site of this provirus, as it is integrated in the opposite transcription direction within the 9th intron of the 4921517L17Rik gene encoding a hypothetical protein, LOC70873. Indeed, this gene is actively transcribed in livers of B6 mice, as determined by RT-PCR (data not shown). However, it is noted that the transcripts of Xmv18, integrated in the opposite transcription direction within the 2nd intron of the Grk5 gene, were not detectable in livers of B6 mice, despite active transcription of the Grk5 gene (data not shown). That Xmv18 is not transcribed may reflect differences in the transcriptional control mechanisms of LOC70873 and Grk5.
3.3. Induced expression of Xmv15, Xmv17 and Xmv18 proviruses by the presence of the Sgp3 locus in B6 mice
The previous analysis of the abundance of four different subgroups of xenotropic gp70 RNAs in livers of B6.Sgp3 congenic mice indicated that Sgp3 up-regulates the transcription of xenotropic viruses belonging to Xeno-I, Xeno- II and Xeno-III subgroups [14]. Therefore, we investigated whether Sgp3 derived from NZB mice is able to enhance the transcription of Xmv10 (Xeno-I) and Xmv14 (Xeno-III) sequences expressed in B6 mice and also induce the transcription of multiple other xenotropic proviruses which were not expressed in B6 mice. Clonal analysis of xenotropic env sequences expressed in livers of B6.Sgp3 mice revealed the presence of transcripts derived from not only Xmv10 and Xmv14 but also three additional proviruses, Xmv15, Xmv17 and Xmv18, belonging to either Xeno-II or Xeno-III subgroup (Fig. 2). Notably, among the three xenotropic viruses induced by Sgp3, Xmv18 is potentially replication competent. The expression of Xmv10 was most frequent (mean of 5 mice ± SEM: 63.3 ± 2.9%), followed by that of Xmv18 (19.4 ± 3.0%), Xmv17 (11.2 ± 2.9%), Xmv14 (4.1 ± 2.5%) and Xmv15 (2.0 ± 1.2%). Notably, the expression frequency of Xmv10 in B6.Sgp3 mice was comparable to that in WT B6 mice, while the frequency of Xmv14 transcripts was 8 times lower than that of B6 mice (P < 0.0005). It should be stressed that the frequencies of the xenotropic transcripts do not indicate the level of transcription. In this regard, the findings that Xmv10 was the only Xeno-I family highly expressed in B6.Sgp3 and WT B6 mice and that B6.Sgp3 mice displayed higher levels in Xeno-I gp70 RNA than WT B6 mice [14] indicated that the transcription of the Xmv10 provirus was up-regulated by Sgp3.
The Xmv10, Xmv15 and Xmv18 proviruses are integrated in the opposite transcription direction within the intron of the 4921517L17Rik, Coro2b and Grk5 genes, respectively. However, the levels of 4921517L17Rik and Grk5 mRNAs were not elevated in livers of B6.Sgp3 mice (mean of 6 mice ± SEM: 4921517L17Rik mRNA: B6.Sgp3, 1.1 ± 0.2; B6, 1.0 ± 0.2; Grk5 mRNA: B6.Sgp3, 1.1 ± 0.1; B6, 1.1 ± 0.2), nor was Coro2b mRNA which was undetectable in livers of B6 and B6.Sgp3 mice. These results thus argued against the possibility that the enhanced and induced expression of Xmv10, Xmv15 and Xmv18 in B6.Sgp3 mice was a result of co-regulated transcription of the respective host gene, in which Xmv10, Xmv15 and Xmv18 are integrated. In addition, the lack of detection of transcripts of two neighboring genes, Nupl2 and Kcnh2, located to ~30 and ~100 Kb upstream and downstream of Xmv17, respectively, in livers of B6.Sgp3 mice do not support the possibility that the induced expression of Xmv17 in B6.Sgp3 mice was influenced by enhancer elements of host genes flanking Xmv17.
We have recently shown that injection of TLR7 or TLR9 agonists, in addition to LPS (TLR4 agonist), induced the acute phase expression of serum gp70 in NZB, but not B6 mice [25]. Since Sgp3 contributed to LPS-mediated acute phase response of serum gp70 [14, 22], we investigated whether injection of a TLR7 agonist, 1V136, could alter the expression profile of Xmv proviruses in B6.Sgp3 congenic mice. B6.Sgp3 mice displayed 2-fold increases in serum concentrations of gp70 (P < 0.01), in parallel to more than 4-fold up-regulation of xenotropic gp70 RNA levels in livers (P < 0.005), 9h after injection of 1V136 (Fig. 5A and B). Although no induced expression of additional xenotropic proviruses was observed, the expression pattern of xenotropic proviruses in B6.Sgp3 mice was altered after injection of 1V136 in which the frequency of Xmv17 and potentially replication-competent Xmv18 transcripts was highly enhanced (mean of 3 mice ± SEM: 44.9 ± 2.5% and 44.8 ± 4.6%, respectively; P < 0.005) (Fig. 5C).
Fig. 5
Fig. 5
Levels of serum gp70 and hepatic xenotropic gp70 RNA as well as frequencies of different Xmv transcripts in livers in B6.Sgp3 mice injected with 1V136.
3.4. Induced expression of the Xmv13 provirus and lack of expression of the Xmv14 provirus in B6.Sgp4 mice
In contrast to Sgp3, we have previously shown that up-regulated and suppressed expression of Xeno-I and Xeno-III gp70 RNAs, respectively, in B6.Sgp4 mice. It was speculated that the Sgp4 locus carries several regulatory elements, which positively and negatively control the transcription of different subgroups of xenotropic proviruses. However, the present clonal analysis of the expression of xenotropic viruses revealed that the Xmv14 provirus expressed in B6 mice is the only xenotropic virus belonging to the Xeno-III subgroup and located within the Sgp4 interval of B6 mice, while it is absent in NZB mice (Table 1). Thus, the absence of Xmv14 in B6.Sgp4 mice results from its deletion upon the introduction of the NZB-derived Sgp4 interval into B6 mice. This was confirmed by genomic PCR analysis using primers for 5′- and 3′-flanking sequences coupled with primers for the U3 region (data not shown) as well as the finding that Xmv14 transcripts were undetectable in B6.Sgp4 mice (Fig. 2). In contrast, B6.Sgp4 mice expressed Xmv10 and Xmv13 sequences, both of which belong to the Xeno-I subgroup, at respective frequencies (mean of 4 mice ± SEM) of 59.6 ± 12.8% and 40.4 ± 12.8% (Fig. 2). These results were consistent with our previous demonstration of a selectively up-regulated expression of Xeno-I gp70 RNA in B6.Sgp4 mice [14].
3.5. No expression of the Xmv13 provirus in (B6.Sgp3 x B6)F1 mice
Xmv13 expressed in B6.Sgp4 mice is located within the Sgp3 interval of B6 mice, but absent in NZB mice (Table 1), which was confirmed by genomic PCR analysis (data not shown). Because of the deletion of Xmv13 in B6.Sgp3 mice, we could not determine whether Sgp3 could also induce the transcription of Xmv13. To address this question, the expression profiles of Xmv proviruses was assessed in (B6.Sgp3 × B6)F1 mice, which carry a single copy of Xmv13 derived from the B6 genome. Notably, the analysis of the abundance of xenotropic gp70 RNA showed that Sgp3 heterozygous mice displayed intermediate levels between B6.Sgp3 homozygous and WT B6 mice (fold increases relative to WT B6 mice: B6.Sgp3 mice, 7.9 ± 1.0; F1 mice, 2.4 ± 0.4; B6 mice; 1.0 ± 0.2; P < 0.0001) (Fig. 6A). In agreement with these results, serum levels of gp70 in F1 mice were intermediate (B6.Sgp3 mice, 19.8 ± 1.0 μg/ml; F1 mice, 8.0 ± 0.8 μg/ml; B6 mice; 2.4 ± 0.2 μg/ml; P < 0.0001) (Fig. 6B). The expression pattern of different Xmv proviruses in Sgp3 heterozygous mice was essentially identical to that of B6.Sgp3 homozygous mice (Fig. 2 and and6C).6C). Notably, despite the presence of Xmv15, Xmv17 and Xmv18 transcripts, the absence of Xmv13 transcripts in Sgp3 heterozygous mice indicated that Sgp3 was unable to induce the transcription of Xmv13 proviral sequences.
Fig. 6
Fig. 6
Levels of hepatic xenotropic gp70 RNA and serum gp70 in B6.Sgp3, (B6.Sgp3 × B6)F1 and WT B6 mice, and frequencies of different Xmv transcripts in livers of (B6.Sgp3 × B6)F1 mice.
The envelope glycoprotein, gp70, of endogenous retroviruses secreted by hepatocytes represents one of the major nephritogenic autoantigens implicated in murine SLE. We have previously shown that the transcription of endogenous xenotropic viruses is promoted by at least two different loci, Sgp3 on chromosome 13 and Sgp4 on chromosome 4, derived from lupus-prone mice and that Sgp3 up-regulates the transcription of xenotropic viruses belonging to Xeno-I, Xeno-II and Xeno-III subgroups, while Sgp4 selectively enhances the level of Xeno-I gp70 RNA [14]. The present clonal analysis of xenotropic env transcripts in livers of B6 mice demonstrates that the transcription of distinct and restricted sets of endogenous xenotropic viruses among 14 Xmv proviruses present in the B6 genome is independently regulated by Sgp3 and Sgp4 derived from lupus-prone NZB mice. Moreover, our analysis revealed the induced expression by Sgp3 and Sgp4 of two potentially replication-competent xenotropic viruses (Xmv13 and Xmv18), which could act as a triggering factor for the development of autoimmune responses via TLR7.
Among 14 Xmv proviruses present in the B6 genome, Xmv10 and Xmv14 were selectively expressed in B6 mice, while the transcription of only a few xenotropic proviruses was induced by Sgp3 or Sgp4. Sgp3 enhanced or induced the expression of Xmv10, Xmv15, Xmv17 and Xmv18, which are not located within the Sgp3 interval, and Sgp4 induced the transcription of Xmv13 located within the Sgp3 interval. This indicates that Sgp3 and Sgp4 are indeed loci that act in trans to control the expression of endogenous xenotropic viruses. Because of the integration of Xmv10 and Xmv18 within the intron of host genes, we explored the possible co-regulation of host and retroviral genes by Sgp3. However, the lack of up-regulated expression of the corresponding host genes argues against this possibility. Moreover, the lack of enhanced expression of host neighbor genes in B6.Sgp3 mice also argues against the Sgp3-mediated coregulation of host and Xmv17 genes.
Our on-going analysis of Sgp3 subcongenic lines has narrowed the Sgp3 locus down to a 5.5 Mb interval between 64.5 and 70.0 Mb of chromosome 13. There are 30 genes mapped to this region in the NCBI database, and significantly, a cluster of 21 KRAB (Krüppel-associated box)-ZFPs (zinc finger protein) has been identified in this region, although the target genes regulated by most of these Zfp genes are still unknown [39, 40]. Several studies reported the contribution of ZFP to the expression of endogenous retroviruses. First, a KRAB transcription repressor domain suppressed lentivirus proviral transcription by inducing heterochromatization in the lentiviral integration sites [41]; second, ZFP809 silenced integrated retroviral DNA through the recruitment of KRAB-associated protein 1 (KAP1, also known as tripartite motif-containing protein 28, TRIM28) in embryonic stem cells [38]; and third, KAP1 negatively controlled the expression of endogenous retroelements during early embryonic development [42]. Thus, it may be possible that several of the Zfp genes present in the Sgp3 locus are involved in the regulation of the expression of different endogenous retroviruses. Considering that ZFP809 suppresses the transcription of a retroviral gene through recognition of a single base pair mutation in the tRNA PBS [38], it might not be surprising that a subtle difference, even single nucleotide polymorphism, in the U3 sequence could be the target of different KRAB-ZFPs.
We have recently shown that the production of gp70 IC implicated in murine lupus nephritis was dependent on TLR7 [28, 43] and that the Sgp3 locus contributes to the development of anti-gp70 autoimmune responses [23]. Since it is unlikely that virion-free serum gp70 is able to trigger TLR7, Sgp3 could enhance the production of endogenous retroviral virions carrying singlestranded RNA, which would then promote the development of autoimmune responses against serum retroviral gp70 through the activation of TLR7. Indeed, we observed that the Sgp3 locus could induce the generation of replicationcompetent xenotropic viruses, as the Xmv18 provirus expressed in B6.Sgp3 mice appears to carry the intact gag, pol and env sequences. Moreover, the expression of Xmv18 in them was strongly up-regulated following stimulation of TLR7. Although the xenotropic and polytropic retrovirus receptor (XPR1) expressed in laboratory strains of mice does not confer susceptibility to xenotropic viruses, PT and mPT retroviruses are able to utilize XPR1 for infectious entry [44]. In this regard, it is noted that Sgp3 is responsible for more than 100-fold increased transcription of mPT proviruses bearing the intact gp70 protein in lupus-prone mice, as compared with non-autoimmune strains of mice [28]. Although it is unlikely that replication-competent endogenous mPT viruses are abundantly expressed in lupus-prone mice, one can speculate that the remarkably enhanced expression of mPT proviruses in lupus-prone mice could results in the utilization of mPT gp70 for infectious retrovirus spread. This could occur through the generation of chimeric virions containing mPT gp70 proteins and xenotropic genomes [45] or possibly by recombination with replicationcompetent xenotropic viruses. Such infectious retroviruses could enter plasmacytoid DC (pDC), a subset of DC which highly expresses TLR7 resulting in their activation. Excessive activation of pDC by endogenous retroviruses could play an important role in the accelerated development of SLE in that activated pDC secrete robust amounts of IFNα, a cytokine that plays a substantial role in the development of SLE [46, 47].
It is also possible that the activation of gp70-specific B cells does not necessarily require infectious retroviruses. Analogous to a model proposed for the activation of anti-nuclear autoreactive B cells through BCR/TLR engagement [48], anti-gp70 autoreactive B cells could recognize and internalize endogenous retroviruses by BCR and subsequently be activated through TLR7. Thus, the high levels of endogenous retroviruses produced in the presence of the Sgp3 locus might be sufficient to trigger anti-gp70 autoimmune responses in mice predisposed to SLE. These autoimmune responses could be further accentuated during the course of SLE as a result of activation of pDC in response to IgG IC containing nuclear antigens and endogenous retroviruses. This mechanism can sustain the production of IFN-α, thereby establishing a vicious cycle not only aggravating the autoimmune process but also promoting the development of autoimmune responses against a wide array of autoantigens that do not engage TLR7. Notably, endogenous retroviruses could elicit autoimmune responses against not only retroviral gp70 but also nuclear autoantigens in mice predisposed to SLE [49], and Sgp3 congenic mice produced significant titers of anti-DNA autoantibodies [22, 23].
In addition to the contribution of TLR7 to the development of autoimmune responses against retroviral gp70, we have shown that the stimulation of TLR7 enhanced serum levels of gp70 and the generation of potentially replication-competent Xmv18 virus in B6.Sgp3 mice. Notably, activation of TLR7 in monocytes/macrophages induced the secretion of IL-6 and TNF [50, 51], both of which efficiently induce the acute phase expression of serum gp70 [25]. Thus, RNA-containing IgG IC could efficiently activate monocytes/macrophages through interaction with IgG Fc receptors and subsequently with TLR7 to induce secretion of these cytokines. This would act as a positive feedback on the production of serum gp70 and endogenous retroviruses, providing an additional source for antigenic stimulation and nephritogenic IC formation. This is consistent with the observation of increased levels of serum retroviral gp70 during the course of SLE, in association with increases in serum levels of gp70 IC and accelerated development of lupus nephritis [52]. Thus, TLR7 displays dual effects on the induction and effector phases of the development of SLE which further highlights the importance of this innate receptor in the pathogenesis of SLE.
The possible contribution of endogenous retroviruses to the development of murine and human SLE is a long-standing story [5355]. Clearly, it is of importance to define the role of endogenous retroviruses for autoimmune responses in SLE and to identify the molecular basis responsible for the increased expression of endogenous retroviruses implicated in murine SLE. These experiments will enable us to address the relevance of their human counterparts, and has obvious and promising implications for diagnostic, prognostic and therapeutic approaches in SLE and related autoimmune diseases.
Research Highlights
Endogenous xenotropic retroviruses are implicated in murine systemic lupus. Their expression is controlled by Sgp3 and Sgp4 loci derived from lupus-prone mice. Each locus promotes expression of distinct and restricted sets of xenotropic viruses. Xenotropic viruses can contribute to autoimmune responses through TLR7 activation.
Supplementary Material
Acknowledgments
This work was supported by the Swiss National Foundation for Scientific Research (grant 310030_127644). L.H.E. was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. We thank Mr. G. Brighouse for his excellent technical assistance.
Footnotes
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