INTRODUCTION

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Throughout evolution, the genomes of higher eukaryotes have been colonized by retroviruses and retrovirus-like sequences (2). The best-studied elements are the murine leukemia virus (MLV)-related viruses of mice. Inbred strains of mice contain multiple copies of endogenous MLVs integrated into the genome as proviruses, where they behave as any other Mendelian gene (11). Four classes of endogenous MLV-related proviruses have been isolated, and they are classified according to the structures of their env genes (34). Infectious ecotropic viruses can infect murine cells only, xenotropic viruses can infect nonmurine but not murine cells, whereas the polytropic and modified polytropic classes can infect cells of either type (2). The host mechanisms controlling endogenous retroviral gene expression, however, remain unclear, although they are likely to be complex and involve interactions between host cell factors and long terminal repeats (LTRs).

The 129 mouse is unusual among the common inbred strains in that it does not produce infectious virions from endogenous MLV sequences. This mouse strain has therefore been used to model antigen accumulation resulting from the expression of endogenous retroviruses without the confounding effects due to transcription of novel proviruses resulting from in vivo virus replication. The G_IX antigen was the first demonstration of a retroviral gene product in 129 mice. A cytotoxicity assay was established, in which antiserum prepared in inbred rats against a syngeneic leukemia induced by wild-type MLV lysed the thymocytes of some strains of mice such as 129 (designated G_IX+) and not others (G_IX) (12, 31). It was later discovered that G_IX was in fact a type-specific antigenic determinant on gp70, the major glycoprotein component of the MLV envelope (24, 37, 38). Genetic data obtained from crosses between the prototype G_IX+ strain 129 and C57BL/6 (G_IX) mice revealed that two unlinked chromosomal genes, Gv1 and Gv2, are required for the expression of the G_IX antigen on normal lymphoid cells. The Gv2 locus controls the G_IX phenotype in a dominant fashion, while Gv1 is semidominant, with heterozygote mice (Gv1^+/) demonstrating a 50% reduction in G_IX expression compared to homozygotes (Gv1^+/+) (31). Attempts have been made to genetically map Gv1, yet these studies have yielded contradictory results (30). Gv2 has been mapped to chromosome 7, although the chromosome position is based on the results of only one cross in which Gv1 was also segregating (32). By serial backcrossing to C57BL/6, an inbred congenic (129 G_IX) strain was established that only differed from its partner (129 G_IX+) at the Gv1 locus (29). Unfortunately, this strain is no longer available.

Studies of the congenic strain revealed that the G_IX phenotype was not simply due to a complete absence of viral antigens, since low levels of both gp70 and the major core protein p30 were detected (17). It was also shown that gp70 molecules found at different tissue sites of 129 G_IX+ mice, absent or reduced in the congenic strain, have distinct tryptic peptide maps (8), suggesting that multiple proviral genes are being affected by Gv1. To examine the extent of this transcriptional control, Moloney MLV DNA was used to probe RNA purified from various tissues from the congenic strain and 129 G_IX+ mice. It was concluded that a negative allele at the Gv1 locus correlated with reduced steady-state levels of retroviral RNA and that the two strains differ in the expression of the same complement of retroviral structural genes (18). Northern blotting experiments also showed that the expression of endogenous MLV structural genes appeared to be regulated in a tissue-specific manner (18). Nuclease protection with an LTR-specific probe confirmed that certain MLV transcripts were under-represented in G_IX tissues and that at least two LTR sequences were under control of Gv1 based on the pattern of protected products. It was therefore proposed that Gv1 encodes a trans-activating factor that coordinately regulates the expression of multiple proviral loci (19).

Despite this work, the mechanism by which Gv1 controls proviral expression and the role of Gv2 remain unclear. To investigate these questions and with the ultimate goal of positionally cloning Gv1, we have carried out a genetic analysis of Gv1. A reliable nuclease protection assay for the gene has been established, allowing a clearer definition of the targets for Gv1 action, an assessment of the relative roles of Gv1 and Gv2 in controlling proviral transcription, and the genetic mapping of Gv1.

	MATERIALS AND METHODS

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Mice. F₁ and backcross mice were bred from stocks of 129/SvEv, BALB/cJ, and C57BL/6J animals maintained in the Biological Services division of the National Institute for Medical Research. The 129 mice are Gv1⁺ Gv2⁺; the C57BL/6 mice are Gv1 Gv2, and the BALB/c mice are Gv1 Gv2⁺ (30, 31).

Nucleic acid purification. Genomic DNA was prepared from fresh or snap-frozen spleen tissue as previously described (1). Total RNA was prepared from fresh or snap-frozen thymus samples as previously described (3).

Nuclease protection. Constructs containing proviral sequences from the polytropic and modified polytropic classes of MLV were subcloned from clones pMX40 (35) and pMX33 (34) for the synthesis of riboprobes. LTR sequences were amplified from these clones by using the primers PLO4 (5'-CGCAATTAACCCTCACTAAAGGG-3') and PLO5 (5'-GCGAATTCATTGGCAGACAC-3'). The PCR products were digested with PstI and EcoRI and subcloned into the pGEM 3Zf(+) vector. A 424-bp polytropic-specific sequence was amplified from the env region of pMX40 by using the primers PLO33 (5'-GGACTAAGACTGTACCGATC-3') and PLO34 (5'-CTTCGGACAGGGTCAGCTTG-3'). The amplified products were cloned into the pCRII-TOPO vector (Invitrogen) as described by the manufacturer. Riboprobes were synthesized by in vitro transcription in a reaction containing 250 ng of linearized template; 1× Transcription Optimized Buffer (Promega); 0.5 mM ATP, CTP, and GTP; 25 to 125 µM UTP; 10 mM dithiothreitol; 50 µCi of [-³²P]UTP (specific activity, 800 Ci/mmol); 10 U of rRNasin RNase inhibitor (Promega); and 20 U of T7 RNA polymerase (Promega). Riboprobes were gel purified on denaturing polyacrylamide gels, and nuclease protection was carried out according to the RPA II method (Ambion). The -actin control plasmid supplied was also used for the synthesis of riboprobes. Supplementation of the in vitro transcription reaction with up to 500 µM UTP allowed riboprobes with lower specific activities to be made for reactions in combination with the env probe to avoid interference of the -actin-protected fragment with smaller bands of interest. Typically, 25 µg total RNA was coprecipitated with 1 × 10⁵ to 3 × 10⁵ cpm of labelled MLV probe plus the -actin riboprobe as an internal control. Hybridization and RNaseA-T1 digestion was carried out as described by the manufacturer. Protected products were resolved on 5% Sequagel denaturing polyacrylamide gels (National Diagnostics), dried onto filter paper, and analyzed by autoradiography or by using a phosphorimager.

Mapping by microsatellite amplification from pooled DNA samples. Genome scanning was carried out by using the DNA pooling strategy described by Taylor et al. (36). DNA from individual backcross mice was prepared as described above, and each sample was diluted to 25 ng/µl. Pools were made by mixing DNA from 50 animals typed by nuclease protection. PCR amplification of simple sequence repeats was carried out essentially as previously described (36), although the reaction volume was reduced to 15 µl and the optimal MgCl₂ concentrations were determined empirically where necessary. Eighty-five microsatellite primer pairs, which span 97% of the genome, were purchased from Research Genetics, Inc. PCR products were run on 3% NuSeive (Flowgen) 1% agarose gels stained with ethidium bromide. For those products that could not be resolved on conventional gels, 10 pmol of one primer was end labelled with [-³²P]ATP by using T4 polynucleotide kinase (New England Biolabs), and 0.1 pmol was added per reaction to the standard PCR mix. PCR products were resolved on 5% denaturing polyacrylamide gels.

REVEAL-PCR. Repeat element-viral element amplified locus PCR (REVEAL-PCR) was carried out essentially as previously described (16). First, 10 pmol of an intracisternal A-type particle (IAP)-specific oligonucleotide (JS139 or JS140) was end labelled with [-³²P]ATP by using T4 polynucleotide kinase and combined in a PCR reaction with one of three B1 or B2 short interspersed element (SINE) primers (JS134, JS135, or JS136). Labelled PCR products were resolved on 5% denaturing polyacrylamide gels which were dried onto filter paper and exposed to X-ray film overnight.

Radiation hybrid mapping. PCR analysis of the T31 mouse radiation hybrid panel (Research Genetics) was carried out by using the REVEAL-1-specific primers JS139 (5'-GTTTACCACTTAGAACACAG-3') and PLO16 (5'-GGAAATAGTAAGTCACACC-3'). First, 20 ng of all 100 hybrid clone DNA samples were amplified in 15 µl of PCR reactions containing 1× PCR Buffer II (Perkin-Elmer), 2.5 mM MgCl₂, 0.2 mM concentrations of each deoxynucleoside triphosphate (dNTP), 2 pmol of primers, and 0.5 U of AmpliTaq DNA polymerase (Perkin-Elmer). PCR conditions were identical to the REVEAL-PCR protocol, and all reactions were carried out in triplicate. Based on the analysis of products on 2% agarose gels, the hybrid clones were scored according to the presence or absence of a mouse-specific band. The screening results were processed by the software Map Manger QT (20) at The Jackson Laboratory.

Microsatellite mapping. Markers were chosen based on polymorphism between the 129 and the C57BL/6 or BALB/c strains where data were available. The sequence of primers for microsatellites in the region of interest were obtained from The Jackson Laboratory Mouse Genome Informatics World Wide Web site (23). DNA samples (50 ng) were amplified in 15-µl PCR reactions containing 1× PCR buffer I (Perkin-Elmer), 0.2 mM concentrations of each dNTP, 2 pmol each primer, and 0.2 U of AmpliTaq DNA polymerase. The initial denaturation step was 2 min at 94°C, followed by 45 cycles of denaturation for 20 s at 94°C, annealing for 30 s at 55°C, and extension for 30 s at 72°C before a final 10-min extension at 72°C. PCR products were resolved on conventional or polyacrylamide gels as described above. Linkage and quantitative trait locus (QTL) data were analyzed by using the Map Manager QT program (20).

	RESULTS

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Characterization of Gv1 controlled transcription. To allow studies of the inheritance of Gv1, we set out to devise a reliable nuclease protection assay for quantifying proviral expression. U3 probes were designed to allow the independent analysis of both the polytropic and modified polytropic classes of endogenous MLV that are present in multiple copies in all inbred strains of mice. Previous studies (34) have shown that the LTRs of modified polytropic proviruses contain two inserts, of 5 and 42 nucleotides, compared to polytropic proviruses in the U3 region (Fig. 1A). Probes to this region will therefore yield clearly distinguishable full-length protected fragments in nuclease protection experiments. Total thymus RNA from 129, C57BL/6, BALB/c, and F₁ (129 × C57BL/6 and 129 × BALB/c) mice were hybridized to the polytropic and modified polytropic LTR probes by using -actin as an internal control. Quantitative studies with the polytropic probe revealed that the full-length protected product of 272 nucleotides was clearly reduced in the Gv1 strains compared to 129 (Fig. 1B, compare lane 4 to lanes 5 and 6). Expression of this LTR sequence was consistently lower in the C57BL/6 strain than in the BALB/c strain, and this was reflected in the F₁ mice, which showed an intermediate level of proviral expression compared to the 129 (Gv1⁺) animals. These levels of expression are consistent with the semidominant nature of Gv1 inheritance. The full-length protected fragment, with the modified polytropic probe, did not differ in intensity between the Gv1⁺ and Gv1 strains (Fig. 1B, lanes 9 to 13), implying that the Gv1 locus does not affect the expression of the modified polytropic class of endogenous MLV.

View larger version (49K): [in this window] [in a new window]   FIG. 1.   Quantitative nuclease protection demonstrates differential expression of the polytropic class of endogenous MLV LTR sequences in Gv1 strains of mice. (A) The MLV LTR region cloned for the synthesis of riboprobes. Shaded areas indicate two regions, of 5 and 42 bp, of the modified polytropic sequence absent in the polytropic clone. (B) Quantitative nuclease protection of thymus total RNA with an LTR probe and a -actin internal control probe. Lanes 4 to 8, protected products with polytropic probe; lanes 9 to 13, modified polytropic probe. The strains of mice analyzed plus the relative intensities of the full-length protected products, corrected for -actin are as follows: lane 4, 129, 0.118; lane 5, C57BL/6, 0.006; lane 6, BALB/c 0.032; lane 7, 129 × C57BL/6, 0.030; lane 8, 129 × BALB/c, 0.064; lane 9, 129, 0.108; lane 10, C57BL/6, 0.128; lane 11, BALB/c, 0.112; lane 12, 129 × C57BL/6, 0.109; and lane 13, 129 × BALB/c, 0.121. The undigested -actin, modified polytropic, and polytropic probes (lanes 1, 2, and 3, respectively) are longer than the full-length protected products due to retention of plasmid sequence from the in vitro transcription reaction (regions shaded black in panel A).

View larger version (58K): [in this window] [in a new window]   FIG. 2.   Quantitative nuclease protection demonstrates differential expression of the polytropic class of endogenous MLV env sequences in Gv1 strains of mice. (A) The region of MLV env cloned for the synthesis of riboprobes. The shaded area indicates a 27-bp region absent in the modified polytropic clone. Solid black regions indicate plasmid sequence retained in the probes from the in vitro transcription reaction. (B) Quantitative nuclease protection of thymus total RNA with a polytropic env probe and a -actin internal control probe. The strains of mice analyzed and the relative intensities of the full-length protected products, corrected for -actin are as follows: lane 1, 129, 0.214; lane 2, C57BL/6, 0.013; lane 3, BALB/c, 0.032; lane 4, 129 × C57BL/6, 0.096; and lane 5, 129 × BALB/c, 0.123. The relative intensities of the 212- and 185-nucleotide modified polytropic-specific fragments are as follows (respectively): lane 1, 0.086 and 0.110; lane 2, 0.080 and 0.114; lane 3, 0.075 and 0.108; lane 4, 0.091 and 0.122; and lane 5, 0.093 and 0.127. No -actin probe was added to the 129 × C57BL/6 RNA sample in lane 6.

Genetic mapping of Gv1. C57BL/6 and BALB/c backcrosses were set up to follow the segregation of Gv1. Nuclease protection data was collected from 230 animals from the backcross populations by using the polytropic LTR riboprobe (Table 1). In each set of reactions, the corrected intensity of the full-length protected product was compared to a reaction by using RNA from the appropriate F₁ mouse. If the band intensity was over 75% of the F₁ value, the mouse was classified as heterozygous at the Gv1 locus, whereas Gv1 animals were those that demonstrated intensity values of <55%. An example from the C57BL/6 backcross is shown in Fig. 3. The nuclease protection data from both backcross populations show a bimodal distribution (Fig. 4). Of the 230 animals typed for Gv1 by using the assay, 111 (48%) carry the 129 allele, which is a finding consistent with the segregation of a single gene. These results also indicate that Gv2 does not play a role in regulating proviral expression since essentially equivalent distributions were seen whether (129 × C57BL/6) or not (129 × BALB/c) the gene was segregating in the cross.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Nuclease protection of C57BL/6 backcross mice indicates Gv1 genotype. Thymus total RNA from the mouse strains indicated was analyzed by quantitative nuclease protection by using the polytropic LTR riboprobe and a -actin internal control probe. Relative intensity of the 272-nucleotide fragments corrected for -actin as follows: lane 1, 0.225; lane 2, 0.065; lane 3, 0.009; lane 4, 0.076; lane 6, 0.016; lane 6, 0.005; lane 7, 0.056; lane 8, 0.009; lane 9, 0.066; lane 10, 0.018; and lane 11, 0.015. Mice were classified as Gv1+/ (lanes 4, 7, and 9) by similarity (>75%) of the band intensity to RNA from the F1 animal (lane 2) or as Gv1/ (lanes 5, 6, 7, 10, and 11) if the value obtained was under 55% of the F1 protected product intensity.

View larger version (22K): [in this window] [in a new window]   FIG. 4.   A single locus is responsible for the proviral expression levels observed in two backcross populations. A distribution of the quantitative nuclease protection data with the polytropic LTR riboprobe from the two backcross populations indicated is shown. Data are shown as a percentage of the relative intensity of the full-length protected product compared to RNA from F1 mice. The bimodal distribution of the data suggests that a single locus is controlling the proviral expression levels observed.

View larger version (16K): [in this window] [in a new window]   FIG. 5.   Linkage of Gv1 is excluded from 74% of the mouse genome. Chromosomal locations of the 41 microsatellite markers successfully amplified and analysed by the DNA pooling method are shown. The Mit number (6) of each marker is shown, and the centromeric ends of the chromosomes are indicated by black circles. Linkage to Gv1 has been excluded from the regions represented by thickened lines, which correspond to 74% of the mouse genome.

View larger version (70K): [in this window] [in a new window]   FIG. 6.   Two IAP proviral markers are linked to Gv1. (A) Relative positions of the primer pairs used to generate two sets of REVEAL-PCR products. (B) REVEAL-PCR products amplified with JS140 and JS134, in the presence of end-labelled primer JS140, from the genomic DNA samples indicated. The arrow indicates the position of REVEAL-2, which is absent in C57BL/6 mice but was amplified from 129 mice. Of 23 backcross animals predicted to be heterozygous at the Gv1 locus, 20 show the presence of this band, indicating linkage. (C) Genomic DNA from the same 23 C57BL/6 backcross mice as in panel B was amplified by using primers JS139 and PLO-16 derived from the marker REVEAL-1. Products were run on a 2% agarose gel against the x174/HaeIII marker (M). The arrow indicates the position of the D13Nimr1 product at 229 bp. Of 23 backcross animals, 22 show the presence of the band, indicating linkage.

View larger version (36K): [in this window] [in a new window]   FIG. 7.   Gv1 maps to chromosome 13. (A) Haplotype analysis of the 116 C57BL/6 backcross progeny typed for markers flanking the REVEAL-PCR product, REVEAL-1. Each column represents the allele inherited from the (129 × C57BL/6)F1 parent. The number of offspring inheriting each allele is indicated at the bottom of each column; shaded boxes indicate the presence of a 129 allele, and the open boxes indicate the presence of a C57BL/6 allele. (B) Partial linkage map of chromosome 13. The relative position of Gv1 with respect to the linked markers indicated was determined by using the Map Manager QT program, without taking into account the double-recombinant animals. Map distances (in cM) are indicated on the left. (C) QTL analysis of all 116 backcross animals based on proviral expression levels observed.

	DISCUSSION

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Despite intensive studies, we know little about the factors controlling expression of endogenous retroviruses (2). Elucidation of the mode of action of Gv1 might be expected to shed light on these processes. The genetic mapping of Gv1 reported here represents the first step towards the eventual cloning and characterization of the gene.

Throughout this study we have assumed that the gene we have mapped to chromosome 13 does indeed correspond to the locus controlling G_IX antigen expression that was originally described. Of particular concern was the possibility that we might have been following merely the inheritance of a single provirus rather than the trans-acting Gv1. Unfortunately, the congenic G_IX strain is no longer available, so direct tests for the presence of chromosome 13 markers of C57BL/6 origin in the congenic strain are not possible. Nevertheless, our assumption of identity does not seem unreasonable since the properties of our gene appear concordant with those of Gv1.

G_IX was described as a thymus antigen expressed by 129 mice but not by BALB/c or C57BL/6 mice (31). Inheritance was semidominant. We have monitored the thymic expression of polytropic MLV sequences in crosses between 129 (positive) and BALB/c or C57BL/6 (negative), and the transcription levels are consistent with the inheritance of a single, semidominant gene. The protected products that appeared absent in the thymus RNA from congenic 129 mice in the experiments of Levy et al. (19) are in fact consistent with the use of a modified polytropic probe for detecting polytropic LTR sequences. We note that one polytropic provirus, Pmv41 (10), from DBA mice, has been shown to lie within the interval containing the gene we have mapped. However, this provirus is not present in 129 mice, and no other polytropic proviruses have been mapped to this region of chromosome 13 (9). Hybridization studies with the polytropic-specific oligonucleotide probe JS5 (35) have confirmed the absence of Pmv41 in 129/SvEv mice (33). Analysis of a small panel of backcross mice revealed no linkage between 129 proviruses and polytropic virus expression (33), a result consistent with the proposition that Gv1 encodes a trans-acting gene product.

The observation that polytropic but not modified polytropic sequences are responsive to Gv1 are consistent with the predicted G_IX phenotypes of these classes of MLV sequence. The appearance of the G_IX antigen is correlated with the absence of a glycosylation signal in the env gene of ecotropic MLV (7); sequence data (34) from the equivalent region of polytropic and modified polytropic MLV reveals that the former, but not the latter, class of sequence also lacks a glycosylation signal. Polytropic MLV (bases 1277 to 1285 [34]) is predicted to encode Asp-Leu-Thr, whereas modified polytropic MLV (bases 1250 to 1258) encodes Asn-Leu-Thr.

The expression of the G_IX antigen is controlled by two genes, Gv1 and Gv2, and it has been hypothesized that both genes play a role in proviral transcription. Although there is a slight difference in the distribution of the nuclease protection data from the C57BL/6 and BALB/c backcrosses, this study suggests otherwise. It has been shown here that only Gv1 has any transcriptional control activity, as a bimodal distribution was observed in both backcross populations. This suggests that Gv2 must act posttranscriptionally and may play a role in MLV surface protein expression.

One of the major functions of the LTR is to regulate the transcription of retroviral genes by providing signals recognized by cellular transcriptional control machinery. Positive and negative regulatory elements have been identified in the LTR, and many transcription factors are known to bind to the U3 region directly. The factor recognition sequences are usually short and may be repeated several times in each LTR, and the signals themselves have also been shown to have different effects in different cell types (4). Since polytropic and modified polytropic MLVs differ in their responsiveness to Gv1, U3 sequences polymorphic between the two classes may therefore be a target for such a trans-activating factor. Attempts will be made to identify possible binding sites by using a gel mobility assay; a factor present in the 129 mice but not in the C57BL/6 or BALB/c strains that binds to polytropic-specific U3 probes could be a good candidate for the gene product of Gv1.

Based on the map position of Gv1, we have attempted to identify possible candidate genes within the interval between D13Mit13 and D13Mit231 (28). A number of potential DNA binding factors, for example, a number of zinc-finger-related genes, Zfp68-rs1, Zfp71-rs1, and Zfp85-rs1 (21), as well as sequences related to the gene encoding the high-mobility-group chromatin protein, Hmg-1 (25), were identified; however, none provides a compelling candidate for Gv1. Possibly more interesting are two genes with trans effects, mdac, modifier of Dac, (15) and Rsl, regulator of sex-limited protein, Slp (14). The latter case is particularly intriguing because a potential target for Rsl is the LTR of an ancient endogenous retrovirus which imposes androgen control over the expression of Slp (27). We are currently refining the map position of Gv1 with the intention of cloning the gene by using the positional or positional candidate approach (5).