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Link to original content: https://pubmed.ncbi.nlm.nih.gov/19494335/
Selective up-regulation of intact, but not defective env RNAs of endogenous modified polytropic retrovirus by the Sgp3 locus of lupus-prone mice - PubMed Skip to main page content
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. 2009 Jun 15;182(12):8094-103.
doi: 10.4049/jimmunol.0900263.

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  1 , Lucie Baudino, Marie-Laure Santiago-Raber, Naoki Morito, Isabelle Dunand-Sauthier, Bernard J Morley, Leonard H Evans, Shozo Izui
Affiliations

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 et al. J Immunol. .

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.

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Figures

FIGURE 1
FIGURE 1
Expression levels of different retroviral gp70 RNAs in livers of various strains of mice. Levels of ecotropic, xenotropic, PT and mPT gp70 RNAs in livers of 2–3 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, and the mean values are indicated by the horizontal line.
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 2–3 mo-old NZB and B6 male mice was determined by RT-PCR with xenotropic, 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 2–3 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 analyzed from lupus-prone NZB, NZW, BXSB and MRL mice, non-autoimmune NFS, 129, AKR and DBA/2 mice with high serum concentrations of gp70 (>15 µg/ml) and non-autoimmune B6, B10, BALB/c, CBA and C3H/He mice with low serum concentrations of gp70 (<5 µg/ml) are shown. 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, 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). Notably, the amplification of only a single fragment of an expected size of corresponding mPT env sequences confirmed the specificity of these PCR analysis. Lane 1, NZB; lane 2, NZW; lane 3, BXSB, lane 4, MRL; lane 5, B6.
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) 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. Notably, the D1 sequence is identical to that of the NL1 mPT provirus derived from NFS mice (GenBank accession number: AY219562). The GenBank accession number for the D2 sequence is FJ182230.
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. 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 amino-acid 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 amino acids presumed to span the membrane is underlined. Numbers indicate amino-acid positions of the WT mPT env polyprotein.
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 2–3 mo-old B6 and B10 congenic male mice bearing 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. Representative results of three individual mice analyzed are shown. As a control, a mixture of three different plasmids containing WT, D1 and D2 clones obtained from B6 mice was included (Ctl). (B) Semi-quantitative 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 carried out 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, while three 5-fold dilutions of cDNAs were examined for D1/D2 mPT env RNAs and GAPDH mRNA. Representative results of three individual mice analyzed are shown. (C) The abundance of three different species of mPT env RNAs (WT, D1 and D2) in livers of 2–3 mo-old BXSB, SB/Le and B6 male mice was determined by RT-PCR with mPT858F forward and p15E-R reverse primers.
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 indicate nucleotide identity, and dots indicate 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. Notably, the U3 sequence of the WT and D1 mutant is identical to that of the MX33 and NL1 mPT proviruses, respectively.
FIGURE 7
FIGURE 7
Serum levels of gp70 IC in TLR7 sufficient or deficient B6.Nba2 female mice and control B6 (WT) female mice at 8 months of age. Each symbol represents an individual animal. Results are expressed as µg/ml for gp70 IC. The mean values are indicated by the horizontal line.
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