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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Autoimmun. Author manuscript; available in PMC 2013 June 1.
Published in final edited form as:
PMCID: PMC3358543
NIHMSID: NIHMS367173

Sgp3 and TLR7 Stimulation Differentially Alter the Expression Profile of Modified Polytropic Retroviruses Implicated in Murine Systemic Lupus

Abstract

The envelope glycoprotein, gp70, of endogenous retroviruses represents one of the major nephritogenic autoantigens implicated in murine systemic lupus erythematosus. Among different endogenous retroviruses (ecotropic, xenotropic and polytropic), lupus-prone mice express remarkably high levels of modified polytropic (mPT) retroviruses, which are controlled by the Sgp3 (serum gp70 production) locus. To define the ontribution of the Sgp3 locus derived from lupus-prone mice to the expression of the specific mPT proviruses, the genetic origin of different mPT viruses expressed in livers and thymi of wild-type and Sgp3 congenic C57BL/6 mice was determined through clonal analysis of their transcripts. Among 13 mPT proviruses present in the C57BL/6 genome, only 3 proviruses (Mpmv6, Mpmv10 and Mpmv13) were selectively but differentially expressed in livers and thymi. This was likely a result of co-regulated expression with host genes because of their integration in the same transcriptional direction. In contrast, Sgp3 induced the steady-state expression of an additional select group of mPT proviruses and, after stimulation of TLR7, the highly upregulated expression of a potentially replication-competent mPT virus Mpmv4. These results indicated that the expression of distinct subpopulations of mPT retroviruses was regulated by Sgp3- and TLR7-dependent mechanisms. The induction of potentially replication-competent mPT viruses and the upregulation of one such virus after stimulation with TLR7 in Sgp3 congenic mice further highlight the implication of Sgp3 in autoimmune responses against nephritogenic serum gp70 through the activation of TLR7.

Keywords: Systemic lupus erythematosus, Endogenous retrovirus, Toll-like receptor

1. Introduction

Endogenous retroviruses are classified as ecotropic, xenotropic or polytropic according to the host range defined by their respective surface gp70 proteins. Furthermore, based on differences in their gp70 nucleotide sequences [1], the polytropic proviruses have been divided into two subgroups, termed polytropic (PT) and modified PT (mPT). The retroviral env (envelope) gene encodes a precursor polyprotein, which is cleaved to produce two subunits: a surface gp70 protein and a membrane-anchored p15E protein. The expression of gp70 is modulated during embryonic development and is linked to the differentiation state of the cells [2]. Indeed, retroviral gp70 is a constituent of the surface of various epithelia, thymocytes and peripheral lymphocytes [25] and also secreted by hepatocytes as a free protein into the circulation [6]. Significantly, only lupus-prone (NZB × NZW)F1, MRL and BXSB mice spontaneously develop autoantibodies against serum gp70, detected as gp70-anti-gp70 immune complexes (gp70 IC), and display deposits of these IC in diseased glomeruli [79]. A remarkable correlation of serum levels of gp70 IC with the development of severe lupus nephritis underlines the pathogenic role of gp70 IC in murine SLE [1013].

Serum gp70 has been considered to be a product of xenotropic, PT and mPT retroviruses [14, 15]. Its concentrations are highly variable among different strains of mice [79] and largely regulated by the Sgp3 (serum gp70 production 3) locus, which was identified in lupus-prone mice and controls the transcription of xenotropic, PT and mPT proviruses in livers [1518]. Moreover, gp70 expression in lupus-prone mice is enhanced by inducers of acute phase proteins, including TLR7 and TLR9 agonists, but unlike conventional acute phase proteins, the acute phase expression of serum gp70 is regulated by the Sgp3 locus [6, 15, 17, 19].

Analysis of the abundance of different retroviral gp70 RNAs demonstrated that the expression level of mPT gp70 RNAs was selectively increased in lupus-prone mice, as compared with other strains of mice which are not predisposed to autoimmune diseases [20]. Furthermore, many strains of mice expressed not only low levels of the intact wild-type (WT) form of mPT env transcripts but also two different env deletion mutants, designated D1 and D2, while lupus-prone mice expressed predominantly the WT mPT env RNA at the near exclusion of the defective transcripts. This specific expression pattern was regulated by the Sgp3 locus which is responsible for the more than 100-fold increased transcription of mPT proviruses bearing the intact env sequence in lupus-prone mice, as compared with non-autoimmune strains of mice [20].

We have previously shown that the production of gp70 IC implicated in murine lupus nephritis was dependent on the single-stranded RNA-specific innate receptor TLR7 [20, 21] and that the Sgp3 locus contributed to the development of anti-gp70 autoimmune responses [18]. Thus, Sgp3 may enhance the production of endogenous retroviral virions carrying single-stranded RNA, which would then promote the development of autoimmune responses against serum gp70 through the activation of TLR7. Since the Sgp3 locus is responsible for the abundant and predominant expression of the WT mPT env transcripts in lupus-prone mice, we investigated the possibility that Sgp3 is able to induce or enhance the expression of a unique subpopulation(s) of mPT viruses with a pathogenic potential among a number of endogenous mPT proviruses present in the mouse genome. To address this question, we conducted a clonal analysis of mPT viral sequences expressed in C57BL/6 (B6) mice and those congenic for the Sgp3 locus derived from lupus-prone mice to determine the genetic origin of mPT viral sequences expressed in them. Results obtained from the present study indicate that only 3 of the 13 mPT proviruses present in the B6 genome are actively transcribed in WT mice, likely due to their unique localization within the host genes. In contrast, Sgp3 congenic mice transcribed multiple but select mPT proviruses, including potentially replication-competent mPT viruses, and stimulation of TLR7 led to a highly enhanced transcription of one potentially replication-competent mPT virus.

2. Materials and methods

2.1 Mice

B6 and C57BL/10 (B10) mice congenic for the NZB- and BXSB-derived Sgp3 locus (B6.Sgp3 and B10.Sgp3), respectively, were generated by backcross procedures, as described [16, 18]. All studies presented were carried out in 2–3 mo-old female 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

RNA from livers was purified with TRIzol reagent (Invitrogen AG, Basel, Switzerland) and treated with DNase I (Amersham Biosciences Corp., Piscataway, NJ). mPT viral cDNA spanning the env gene and the U3 region of the long terminal repeat (LTR) was amplified with mPT858F forward primer and Uniltr-4R reverse primer, as described [20]. The amplified fragments were purified and ligated into the pBluescript-SK+ plasmid. Clones containing the WT form of mPT env transcripts were selected by PCR with the use of mPT1115F (5’-GTTCCCAAAACCCATCAGGC-3’) and mPT1585R (5’-ATCTAATCCTCTCCGGTTCT-3’) primers, which give different sizes of amplicons (471, 355 and 402 bps for WT, D1 mutant and D2 mutant, respectively; Figure 1A). To enrich the intact WT form of mPT amplicons in livers of B6 mice, in which D2 transcripts are predominant [20], amplified fragments derived from WT and D1 mutant were digested with BglI, which is unable to cleave D2 transcripts into two fragments, and cloned into the pBluescript-SK+ plasmid.

Fig. 1
Expression of different Mpmv proviruses in livers and thymi of B6 mice

2.3. Quantitative real-time PCR

The abundance of xenotropic, mPT and PT env RNAs (genomic RNA and mRNA) was quantified by real-time PCR, as described [15, 22]. Levels of cathepsin E (Ctse), arginine/serine-rich coiled-coil 1 (Rsrc1), UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 11 (Galnt11), ADP-ribosylation factor 6 (Arf6) and methyltransferase like 21D (Mettl21d) mRNAs were quantified with the following primers: Ctse forward primer (5’-GCCCAACCCATCCACTTAC-3’) and reverse primer (5’-TCTCCCTTCCACCCTTCTC-3’); Rsrc1 forward primer (5’-GCCACCCTGGTAGAACAAGTC-3’) and reverse primer (5’-GCACTTCACTTGGTTCTACTGC-3’); Galnt11 forward primer (5’-GTGGATGACAGCAGTGACTTTG-3’) and reverse primer (5’-TCCCTCGCGCTTCATATTTC-3’); Arf6 forward primer (5’-CCAATCGGTGACCACCATCC-3’) and reverse primer (5’-GGTCCCGGTGTAGTAATGCC-3’); Mettl21d forward primer (5’-CTCCTGCAGCTAGACTTTGAC-3’) and reverse primer (5’-CTTGACCTTACCCAGGATGTTG-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.

2.4. Genomic PCR

The presence of the Mpmv11 provirus in mice was determined by genomic PCR analysis with combinations of primers for the U3 sequence and those for Mpmv11 5’- and 3’-flanking sequences: U3 forward (5’-AAGCTAGCTGCAGTAACGCCATTTTGC-3’) and reverse (5’-AGCTTGCTAAGCCTTATGGTGG-3’) primers; Mpmv11 5’-flanking forward (5’-CTATGCTTAGCAGGCAGGTATC-3’) and 3’-flanking reverse (5’-AAGGTTGTCTGGCTTGAAGG-3’) primers.

2.5. Injection of TLR7 agonist

The TLR7 agonist 1V136 (TLR7 Ligand II, Calbiochem, EMD Chemicals Inc., Darmstadt, Germany) [23] was diluted with sterile PBS and i.p injected into mice. Livers were collected 9 h after injection of 1V136 (100 µg).

2.6. Statistical analysis

Unpaired comparison for frequencies of clones expressing different Mpmv env RNAs and for levels of different mRNAs and retroviral gp70 RNAs was analyzed by Student's t test. Probability values <5% were considered significant.

3. Results

3.1. The presence of 13 mPT proviruses in the B6 mouse genome

Previous restriction fragment length polymorphism (RFLP) analysis with the use of an mPT-specific env probe showed the presence and chromosomal localization of 13 mPT proviral Mpmv loci in the B6 mouse genome [24]. This was confirmed by BLAST search analysis, according to gp70 sequences specific for mPT viruses (Table 1). The Mpmv10 and Mpmv13 proviruses correspond to D1 and D2 mutants which contain a deletion in the env sequence of the 3’ portion of the gp70 protein and the 5’ portion of the p15E protein, respectively [20], while 11 other mPT poroviruses carry the full-length WT form of env sequence. The inspection of the coding sequences of the gag, pol and env genes indicated that 6 proviruses (Mpmv1, Mpmv2, Mpmv4, Mpmv7, Mpmv9 and Mpmv12) are potentially replication-competent (Table 1). According to the classification reported by Tomonaga et al. [25], the U3 structure of LTR of all 13 mPT proviruses is assigned to the P-II subgroup characteristics of mPT viruses (Fig. S1). NZB mice carrying 10 mPT proviruses detected by RFLP analysis [26] share 6 Mpmv loci (Mpmv3, Mpmv6, Mpmv8, Mpmv9, Mpmv10 and Mpmv11) with B6 mice.

Table 1
Chromosomal localization of mPT proviruses in B6 mice.

3.2. Predominant transcription of the Mpmv13 provirus in livers of B6 mice

Our previous RT-PCR analysis of RNA from livers of B6 mice revealed the presence of full-length mPT env RNAs as well as two env deletion mutants, Mpmv10 (D1) and Mpmv13 (D2) [20]. To determine the heterogeneity of the full-length WT mPT env transcripts, we amplified, cloned, selected clones containing the full-length env transcripts by PCR (Fig. 1A), and sequenced a region spanning the env gene and the U3 of the LTR. The nucleotide sequences in this region are highly heterogeneous (Fig. S2) and allow the precise determination of the genetic origin of the transcripts. We observed that livers of B6 mice expressed, in addition to Mpmv10 (D1) and Mpmv13 (D2), only a single full-length mPT provirus, Mpmv6. Quantitative analysis of ~30 clones from each of three individual mice revealed that, as expected, the expression of Mpmv13 (D2) was most frequent (mean ± SEM: 72.2 ± 1.8%), followed by that of Mpmv10 (D1) (15.3 ± 3.1%) and Mpmv6 (12.5 ± 3.8%; Fig. 1B). Since the number of WT mPT clones analyzed was limited, we enriched WT mPT amplicons by treating them with BglI, which facilitates the purification of digested WT- and Mpmv10 (D1)-derived fragments. Analysis of additionally obtained 7–10 WT mPT clones from each of three individual mice confirmed that they were all derived from the Mpmv6 provirus.

The transcription of mPT viruses in livers of B6 mice could be influenced by the regulatory sequences present in the U3 region. However, this is not the case for the selective expression of Mpmv6, Mpmv10 and Mpmv13 in B6 mice, as the U3 nucleotide sequence of Mpmv13 is identical to that of Mpmv1 and Mpmv11, which are not actively transcribed in B6 mice (Fig. S1 and S2). Studies reported that the tRNA primer-binding site sequence can trigger silencing of integrated infectious retroviruses in embryonic cells [2729]. However, such a mechanism is also excluded for the selective expression of Mpmv6, Mpmv10 and Mpmv13, since the nucleotide sequences of the tRNA primer-binding site are different from each other and are themselves shared with other mPT viruses (Fig. S3).

3.3. Predominant transcription of the Mpmv6 provirus in thymi of B6 mice

Selective transcription of Mpmv6, Mpmv10 (D1) and Mpmv13 (D2) in B6 mice could be related to their unique integration sites. Indeed, these three proviruses are integrated in the same transcription direction as the host gene within the first intron of the Ctse gene, the 4th intron of the Rsrc1 gene and the 1st intron of the Galnt11 gene, respectively (Table 1). Thus, we hypothesized that the transcription of these three proviral sequences is favored by their local chromatin environment due to the expression of the corresponding host genes and that the expression levels of these genes in distinct tissues may differ. If so, the expression pattern of these three mPT proviruses may reflect the expression levels of the genes in which they are nested. In this regard, the Ctse gene has been reported to be highly expressed in lymphocytes [30] which is in agreement with our finding that the abundance of Ctse mRNA was approximately 10-fold higher in thymi than in livers (Table 2). In contrast, the levels of Rsrc1 and Galnt11 mRNAs were 3 times less in thymi than in livers (P < 0.0001). Analysis of the expression pattern of the three different species of mPT env transcripts by mPT-specific RT-PCR revealed the predominant abundance of the full-length form of mPT env RNA in thymi, which contrasted with that of the Mpmv13 (D2) env RNA in livers (Fig. 1C). Furthermore, clonal analysis of mPT sequences expressed in the thymi confirmed that the full-length mPT transcript was derived from the Mpmv6 provirus located within the Ctse gene, and its frequency was highest (mean of 4 mice ± SEM: 80.3 ± 4.3%), followed by that of Mpmv13 (16.0 ± 3.7%) and Mpmv10 (4.9 ± 1.9%; Fig. 1D). Notably, we were unable to detect transcripts of the Mpmv2, Mpmv8 and Mpmv9 proviruses, which are integrated in the opposite transcription direction within their respective host genes (Table 1), despite active transcription of host genes (Abr for Mpmv2 and Fam73a for Mpmv9) in livers and thymi (data not shown). This could be due to possible steric hindrance or interference caused by annealing of the complementary RNAs of the host genes, leading to degradation of corresponding Mpmv transcripts [31].

Table 2
Relative abundance of Ctse, Rsrc1 and Galnt11 mRNA in thymus and liver of B6 mice.a

3.4. Predominant transcription of the Mpmv11 provirus induced by the presence of the Sgp3 locus in livers of B6 mice

Previous analyses in livers of B6.Sgp3 congenic mice indicated that the Sgp3 locus derived from lupus-prone mice selectively upregulates the level of the full-length mPT env RNA [20]. Therefore, we investigated whether the upregulation by Sgp3 derived from lupus-prone NZB mice resulted in the enhanced transcription of the Mpmv6 provirus or the expression of other mPT proviruses carrying the intact env gene. Clonal analysis of mPT sequences expressed in livers of B6.Sgp3 mice revealed the presence of transcripts derived from not only Mpmv6, Mpmv10 (D1) and Mpmv13 (D2) but also from multiple other proviruses (Fig. 2A). Strikingly, Mpmv11 was the most highly expressed provirus, as its frequency reached 62.0 ± 5.3% (mean of 5 mice ± SEM). Accordingly, the relative expression frequencies of Mpmv6, Mpmv10 (D1) and Mpmv13 (D2) were significantly reduced in B6.Sgp3 mice (Mpmv6: 3.4 ± 1.4%; Mpmv10: 5.6 ± 2.5%; Mpmv13: 6.7 ± 2.1%; P < 0.05, P < 0.05 and P < 0.0001, respectively), as compared with those in WT B6 mice (Fig. 1B). Notably, the previous quantitative PCR analysis showed the lack of increased expression of Mpmv10 (D1) and Mpmv13 (D2) in B6.Sgp3 mice [20]. Collectively, these results suggest that Sgp3 did not upregulate the expression of the three mPT proviruses expressed in B6 mice. In addition, it was noted that the expression of potentially replication-competent mPT viruses such as Mpmv4, Mpmv7 and Mpmv9 was induced by Sgp3, although their frequencies were low (Fig. 2A).

Fig. 2
Expression of different Mpmv proviruses in livers of C57BL mice congenic for the Sgp3 locus derived from lupus-prone mice

The predominant transcription of Mpmv11 in livers of B6.Sgp3 mice cannot be attributed to its U3 regulatory sequence as it is identical to that of Mpmv1 and Mpmv13 (D2) (Fig. S1 and S2). We explored the possibility that the highly predominant expression of Mpmv11 in B6.Sgp3 mice was promoted by enhancer elements of host genes flanking this provirus. However, this possibility appears unlikely because of the lack of upregulated expression in livers of B6.Sgp3 mice of two neighboring genes, Arf6 and Mettl21d, located ~170 and ~22 Kb upstream and downstream of Mpmv11 (mean of 5 mice ± SEM: Arf6 mRNA; B6.Sgp3: 0.9 ± 0.1, B6: 1.0 ± 0.2; Mettl21d mRNA; B6.Sgp3: 1.4 ± 0.2, B6: 1.0 ± 0.2).

The Sgp3 allele of lupus-prone BXSB mice is also responsible for the predominant and abundant expression of full-length mPT env transcripts in livers, as demonstrated by the analysis of B10.Sgp3 congenic mice [20]. Notably, the analysis of B10 mice bearing the Sgp3 locus derived from BXSB mice confirmed the selectively enhanced expression of Mpmv11 in their livers (mean of 3 mice ± SEM: 60.3 ± 6.5%; Fig. 2B), as in the case of B6 mice bearing the NZB-derived Sgp3 locus (Fig. 2A). However, to our surprise, livers of SB/Le mice failed to display the predominant expression of full-length mPT env RNAs (Fig. 2C), despite the fact that the BXSB-Sgp3 allele is inherited from SB/Le mice (BXSB is a recombinant strain derived from a cross of B6 and SB/Le mice). Thus, the lack of predominant expression of full-length mPT env transcripts in livers of SB/Le mice could be due to the absence of the Sgp3-responsive Mpmv11 provirus in the SB/Le genome. This was indeed confirmed by genomic PCR analysis using primers for the U3 region coupled with primers for Mpmv11 5’-and 3’-flanking sequences (Fig. 2D).

3.5. Differential effect of Sgp3 on the transcription of mPT proviruses in thymi and livers of B6 mice

Gv1 (Gross virus antigen 1) which is known to control the transcription of endogenous retroviral sequences in different tissues, including the thymus and liver [32], directly overlaps with the Sgp3 locus [1618, 33]. However, it has been shown that the Gv1 locus regulated the transcription of PT proviruses, but not mPT proviruses in thymus [33], while Sgp3 controlled the transcription of both PT and mPT proviruses in livers [15]. Therefore, we compared the abundance of PT, mPT and xenotropic gp70 RNAs in thymi and livers between B6 and B6.Sgp3 mice. B6.Sgp3 mice displayed 6.1- and 9.6-fold increased levels of PT (P < 0.005) and xenotropic (P < 0.001) gp70 RNAs, respectively, but only a minimal (1.6-fold) increase of mPT (P < 0.01) gp70 RNA in thymi, as compared with B6 mice (Fig. 3A). In contrast, Sgp3 promoted high levels of transcription of all three retroviral sequences in livers: 4.3-, 12.9- and 7.9-fold increases in PT (P < 0.005), mPT (P < 0.0001) and xenotropic (P < 0.001) gp70 RNAs, respectively. Despite the minimal enhancing effect of Sgp3 on the expression of mPT viruses in thymi, clonal analysis of mPT transcripts revealed a heterogeneous expression profile of mPT proviruses in thymi of B6.Sgp3 mice, though the frequency of Mpmv6 remained the highest (mean of 4 mice ± SEM: 48.9 ± 6.3%), followed by that of Mpmv11 (19.1 ± 3.5%; Fig. 3B).

Fig. 3
Levels of PT, mPT and xenotropic gp70 RNAs in thymi and livers of B6.Sgp3 and WT B6 mice as well as frequencies of different Mpmv transcripts in thymi of B6.Sgp3 mice

3.6. Enhanced transcription of the potentially replication-competent Mpmv4 provirus in livers of B6. Sgp3 mice after stimulation of TLR7

Injection of TLR7 or TLR9 agonists, in addition to LPS (TLR4 agonist), induced the acute phase expression of serum gp70 in B6.Sgp3 but not B6 mice [15, 17, 19, 34]. Thus, we investigated whether injection of 1V136 (TLR7 agonist) could alter the expression profile of Mpmv proviruses in B6.Sgp3 mice. B6.Sgp3 mice displayed a 2-fold upregulation of mPT gp70 RNA levels in livers 9h after injection of 1V136 (P < 0.05; Fig. 4A). Although no induced expression of additional Mpmv proviruses was observed, the expression pattern of Mpmv proviruses in B6.Sgp3 mice changed after injection of 1V136: the frequency of the potentially replication-competent Mpmv4 transcript was markedly enhanced, compared with that of control B6.Sgp3 mice (mean of 4 mice ± SEM: 45.6 ± 1.5% vs. 4.9 ± 2.3%; P < 0.0001; Fig. 2A and and4B).4B). Notably, the expression profile of Mpmv proviruses was not changed in 1V136-injected B6 mice (data not shown).

Fig. 4
Levels of mPT gp70 RNA and frequencies of different Mpmv transcripts in livers of B6.Sgp3 mice injected with 1V136

4. Discussion

Endogenous retroviruses are implicated in the pathogenesis of murine SLE and there is remarkable high-level expression of the mPT retrovirus which is promoted by the Sgp3 locus in lupus-prone mice [20]. We attempted in the present study to define the specific mPT proviruses expressed in B6 mice carrying or not the Sgp3 locus derived from lupus-prone mice. The clonal analysis of mPT transcripts revealed the transcription of only 3 of the 13 mPT proviruses present in the B6 genome, possibly as a result of co-regulated expression with the three host genes in which these mPT proviruses are integrated in the same transcription direction. In contrast, we observed that Sgp3 induced the transcription of distinct and selective subpopulations of mPT proviruses, including proviruses which are potentially replication-competent. Finally, a highly enhanced transcription of a potentially replication-competent mPT virus was detected in B6.Sgp3 mice after stimulation of TLR7. These observations further highlight the role of Sgp3 in the pathogenesis of SLE by promoting the generation of potentially infectious retroviruses which could act as a triggering factor for the development of autoimmune responses via TLR7.

Among 13 Mpmv proviruses present in the B6 genome, the expression of only Mpmv6, Mpmv10 and Mpmv13 was detected in B6 mice. Their unique integration in the same transcription direction within the introns of three host genes and the correlation of levels of these three mPT RNAs with those of the host genes between livers and thymi support the idea that these Mpmv proviruses are actively transcribed as a result of co-regulated expression with their corresponding host genes. In contrast, Mpmv proviruses integrated in the opposite transcription direction within the host genes are not expressed despite transcription of host genes in B6 mice. However, this is clearly not a general mechanism for the transcription of other endogenous retroviruses. Indeed, only two xenotropic viruses (Xmv10 and Xmv14) are expressed in livers among 14 Xmv proviruses present in B6 mice, in which Xmv10 is integrated in the opposite transcription direction within the intron of the actively transcribed host gene (4921517L17Rik) encoding a hypothetical protein, LOC70873, and Xmv14 is not integrated within the host genes (34).

It is intriguing that the effect of the Sgp3 locus is selective for subpopulations of mPT proviruses as it neither detectably induced the transcription of Mpmv1, Mpmv2, Mpmv3 and Mpmv12 nor upregulated the transcription of the three mPT viruses expressed in WT B6 mice. In addition, among the multiple mPT proviruses newly expressed in B6.Sgp3 mice, the transcription of one mPT provirus, Mpmv11, was more selectively induced by Sgp3. This was not due to its U3 regulatory sequence or an Sgp3-induced enhanced expression of its neighboring genes. Notably, we have also recently observed that Sgp3 induced the transcription of only 3 of the 14 xenotropic proviruses in B6 mice [34]. Taken together, these data suggest the presence of a unique genetic mechanism responsible for the selective expression of subpopulations of endogenous retroviruses by Sgp3.

The Sgp3 locus has been narrowed down to an interval between 64.5 and 70.0 Mb of chromosome 13 (unpublished data), in which a cluster of 21 KRAB (Krüppel-associated box)-ZFPs (zinc finger protein) has been identified [35, 36]. ZFPs have been reported to influence the expression of endogenous retroviruses [29, 37, 38]. Notably, ZFP809 suppresses the transcription of a retroviral gene through recognition of a point mutation in the tRNA primer-binding site [29], indicating that a subtle difference, even a single nucleotide polymorphism, in the retroviral sequence could be the target of different KRAB-ZFPs. Moreover, we observed differential effects of Sgp3 on the transcription of mPT, but not PT and xenotropic viruses, between livers and thymi. These data strongly suggest that several of the Zfp genes present in the Sgp3 locus could be involved in the regulated expression of distinct classes of endogenous retroviruses and even subpopulations of the same class of retroviruses.

Our previous and present analyses raised the possibility that the Sgp3 locus could induce the generation of replication-competent xenotropic and mPT retroviruses [34]. Although the polymorphic form of the xenotropic and polytropic retrovirus receptor (XPR1) expressed in most laboratory strains of mice does not confer susceptibility to xenotropic viruses, mPT retroviruses are able to utilize XPR1 for infectious entry [39]. In addition, the remarkably enhanced expression of mPT env RNA in lupus-prone mice could lead to the generation of chimeric virions containing mPT gp70 proteins and xenotropic genomes [40]. Consequently, such infectious retroviruses could enter plasmacytoid dendritic cells and the activation of these cells through TLR7 could play a substantial role in the accelerated development of SLE through the secretion of IFNα, a cytokine prominently involved in the pathogenesis of SLE [41, 42]. Such a mechanism could account for the spontaneous formation of anti-DNA autoantibodies in Sgp3 congenic mice [17, 18] and for the development of autoimmune responses against nuclear autoantigens in mice infected with murine leukemia virus [43]. It should also be stressed that infectious retroviruses are not necessarily required for the activation of gp70-specific B cells, since these B cells could be activated through TLR7 following internalization of endogenous retroviruses recognized by the BCR. Thus, the high-level production of non-infectious endogenous retroviruses by Sgp3 could be sufficient to trigger anti-gp70 autoimmune responses in mice predisposed to SLE.

It is significant that the stimulation of TLR7 enhanced the production of serum gp70 as well as potentially replication-competent retroviruses in B6.Sgp3 mice as a result of increased secretion of IL-6 and TNF by activated monocytes/macrophages [19, 34]. In addition, the activation of TLR7 induced the development in lupus-prone mice of monocytosis with expansion of a hyperactive monocyte subset in response to IgG IC [21, 44, 45]. Thus, during the course of SLE, an excessive activation of monocytes/macrophages by IgG Fc receptors interacting with RNA-containing IgG IC and then by TLR7 may promote the production of serum gp70 and endogenous retroviruses, thereby providing an additional source for antigenic stimulation and nephritogenic IC formation. Notably, elevated levels of serum gp70 were associated with increased production of gp70 IC and accelerated development of lupus nephritis [46].

Collectively, our data indicate multiple roles of TLR7 in the pathogenesis of SLE. Isolation of replication-competent mPT viruses from lupus-prone mice and assessment of their potential pathogenic activity in mice predisposed to SLE would help define to which extent endogenous retroviruses indeed contribute to the development of SLE. Moreover, the identification of the Sgp3 gene(s) would help elucidate the molecular basis responsible for the expression of endogenous retroviruses implicated in murine SLE. This would enable us to address the relevance of their human counterparts, thereby providing a clue for the potential role of endogenous retroviruses in human SLE.

Highlights

  • Lupus-prone mice express extremely high levels of modified polytropic retroviruses.
  • Their expression is controlled by the Sgp3 locus derived from lupus-prone mice.
  • Sgp3 promotes the expression of unique subpopulations of modified polytropic viruses.
  • Replication-competent retroviruses can be highly induced 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 Guy Brighouse and Ms Montserrat Alvarez for their excellent technical assistance and Drs Dominique Belin and Thomas Moll for critically reading the manuscript.

Footnotes

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