Genomic organization of the mouse Tla locus: study of an endogenous retrovirus-like locus reveals polymorphisms related to different Tla haplotypes

A retrovirus element (TLev1) is located within the Thymus leukemia antigen (T7a) locus of the C57BL/10 mouse major histocompatibility complex. Low-copy probes have been isolated from sequences flanking the TLev1 integration site to examine the distribution of TLev1 among inbred mouse strains having genotypically determined variations in TL-antigen expression. It was found that the low-copy probes cross-hybridize to regions within the Tla locus in a genotype-specific manner. Although a strong association was found between TL mouse strains and TLev1, the presence or absence of the TLev1 locus did not exclusively correlate with expression or nonexpression of TL antigens. Analysis of different Mus subspecies indicates that TLev1 integrated into a common ancestor of the species Mus musculus. It is suggested that the loss of the TLev1 locus from certain mouse genomes reflects evolutionary rearrangements in the TL region; the resulting diversity may relate to the differential expression of TL antigens among mouse strains. The probes described here provide a useful tool for examining the genomic expansions and contractions which have occurred during the evolution of the Tla locus     

Somatically acquired recombinant murine leukemia proviruses in thymic leukemias of AKR/J mice.

We have probed the structure and arrangement of murine leukemia virus genomes in eight spontaneous AKR thymic leukemias by Southern hybridization with one ecotropic pol and four ecotropic env probes. These probes revealed many (in 2 cases over 15) somatically acquired proviruses that had undergone complex patterns of recombination. The large majority were not deleted and were structurally analogous to the oncogenic mink cell focus-inducing murine leukemia viruses isolated from AKR tumors in that the amino-terminal p15E-coding region derived from ecotropic AKR murine leukemia virus sequences, whereas certain gp70-coding sequences were nonecotropic. Nevertheless, we observed a few proviruses which did not appear to be gp70 recombinants; however, these proviruses were in general clearly recombinant within the p15E-coding sequences. Although the proviral recombination patterns were quite variable, in general the large majority of recombinant proviruses within each tumor appeared structurally identical, indicating that they originate from a common parent. Each tumor contained a unique pattern of provirus integrations; densitometer tracings of the Southern hybridizations indicated that many of the integrated proviruses were present at one copy per cell, suggesting that the tumors derive from a single cell which contained multiple integrated copies of a unique recombinant virus structurally similar to the mink cell focus-inducing viruses.     

Nucleotide sequence of the env-specific segment of NFS-Th-1 xenotropic murine leukemia virus.

The sequence of 863 contiguous nucleotides encompassing portions of the pol and env genes of NFS-Th-1 xenotropic proviral DNA was determined. This region of the xenotropic murine leukemia virus genome contains and env-specific segment that hybridizes exclusively to xenotropic and mink cell focus-forming but not to ecotropic proviral DNAs (C. E. Buckler et al., J. Virol. 41:228-236, 1982). The unique xenotropic env segment contained several characteristic deletions and insertions relative to the analogous region in AKR and Moloney ecotropic murine leukemia viruses. Portions of an endogenous env segment cloned from a BALB/c mouse embryo gene library that had a restriction map and hybridization properties typical of xenotropic viruses (A. S. Khan et al., J. Virol. 44:625-636, 1982) were also sequenced. The sequence of the endogenous env gene was very similar to the comparable region of the NFS-Th-1 xenotropic virus containing the characteristic deletions and insertions previously observed and could represent a segment of an endogenous xenotropic provirus.     

Nucleotide sequence of AKV murine leukemia virus.

AKV is an endogenous, ecotropic murine leukemia virus that serves as one of the parents of the recombinant; oncogenic mink cell focus-forming viruses that arise in preleukemic AKR mice. I report the 8,374-nucleotide-long sequence of AKV, as determined from the infectious molecular clone AKR-623. The 5'-leader sequence of AKV extends to nucleotide 639, after which lies a long open reading frame encoding the gag and pol gene products. The reading frame is interrupted by a single amber codon separating the gag and pol genes. The pol gene overlaps the env gene within the 3' region of the AKV genome. The nucleotide sequence of the 5' region of AKV reveals the following features. (i) The 5'-leader sequence lacks any AUG codon to initiate translation of gPr80gag, suggesting that gPr80gag is not required for the replication of AKV. (ii) A short portion of the leader region diverges in sequence from the closely related Moloney murine leukemia virus and appears to be related to a sequence highly repeated in eucaryotic genomes. (iii) As in Moloney murine leukemia virus, there is a potential RNA secondary structure flanking the amber codon that separates the gag and pol genes. This structure might function as a regulatory protein binding site that controls the relative levels of synthesis of the gag and pol precursors. The nucleotide sequence of the 3' region of AKV is compared with sequences reported previously from both infectious and noninfectious molecular clones of AKV.     

Comparison of the restriction endonuclease maps of unintegrated proviral DNAs from four xenotropic murine leukemia viruses.

From purified linear and superhelical DNAs, the restriction endonuclease maps of four xenotropic murine leukemia virus DNAs from NFS, NZB, BALB/c, and AKR mice were determined with ten restriction endonucleases. Each xenotropic proviral DNA was found to be a unique restriction endonuclease map, with differences in the gag, pol, env, and terminal repeated sequence regions. However, type-specific SacI and EcoRI sites in the env region were identical in all four xenotropic murine leukemia virus DNAs and were not found in ecotropic murine leukemia virus DNA. Comparison of the xenotropic murine leukemia virus DNA maps with maps of ecotropic murine leukemia virus DNA showed that the pol and terminal repeated sequence regions were highly conserved. Other similarities in ecotropic and some xenotropic viral DNAs suggest common origins.     

Origin of pathogenic determinants of recombinant murine leukemia viruses: analysis of Bxv-1-related xenotropic viruses from CWD mice.

The acquisition of U3 region sequences derived from the endogenous xenotropic provirus Bxv-1 appears to be an important step in the generation of leukemogenic recombinant viruses in AKR, HRS, C58, and some CWD mice. We report here that each of three CWD lymphomas produced infectious xenotropic murine leukemia virus related to Bxv-1. In Southern blot experiments, these proviruses hybridized to probes that were specific for the xenotropic envelope and Bxv-1 U3 region sequences. Nucleotide sequence analysis of a cloned CWD xenotropic provirus, CWM-S-5X, revealed that the envelope gene was closely related to but distinct from those of other known xenotropic viruses. In addition, the U3 region of CWM-S-5X contained a viral enhancer sequence that was identical to that found in MCF 247, a recombinant AKR virus that is thought to contain the Bxv-1 enhancer. Finally, restriction enzyme sites in the CWM-S-5X provirus were analogous to those reported within Bxv-1. These results establish that the virus progeny of Bxv-1 have the potential to donate pathogenic enhancer sequences to recombinant polytropic murine leukemia viruses. Interestingly, the three CWD polytropic viruses that were isolated from the same tumor cells that produced the Bxv-1-like viruses had not incorporated Bxv-1 sequences into the U3 region.     

Cas-Br-E murine leukemia virus: sequencing of the paralytogenic region of its genome and derivation of specific probes to study its origin and the structure of its recombinant genomes in leukemic tissues.

The ecotropic Cas-Br-E murine leukemia virus (MuLV) and its molecularly cloned derivative pBR-NE-8 MuLV are capable of inducing hind-limb paralysis and leukemia after inoculation into susceptible mice. T1 oligonucleotide fingerprinting, molecular hybridization, and restriction enzyme analysis previously showed that the env gene of Cas-Br-E MuLV diverged the most from that of other ecotropic MuLVs. To analyze proviruses in leukemic tissues, we derived DNA probes specific to Cas-Br-E sequences: two from the env region and one from the U3 long terminal repeat. With these probes, we found that this virus induced clonal (or oligoclonal) tumors and we documented the presence of typical mink cell focus-forming-type proviruses in leukemic tissues and the possible presence of other recombinant MuLV proviruses. Since the region harboring the determinant of paralysis was mapped within the pol-env region of the virus (L. DesGroseillers, M. Barrette, and P. Jolicoeur, J. Virol. 52:356-363, 1984), we performed the complete nucleotide sequence of this region covering the 3' end of the genome. We compared the deduced amino acid sequences of the pol carboxy-terminal domain and of the env gene products with those of other nonparalytogenic, ecotropic, and mink cell focus-forming MuLVs. This amino acid comparison revealed that this part of the pol gene product and the p15E diverged very little from homologous proteins of other MuLVs. However, the Cas-Br-E gp70 sequence was found to be quite divergent from that of other MuLVs, and the amino acid changes were distributed all along the protein. Therefore, gp70 remains the best candidate for harboring the determinant of paralysis.     

Structure, distribution, and expression of an ancient murine endogenous retroviruslike DNA family.

An endogenous retroviruslike DNA, B-26, was cloned from a BALB/c mouse embryo gene library by using a generalized murine leukemia virus DNA probe. Southern blot hybridization and nucleotide sequence analyses indicated that B-26 DNA might be a novel member of the GLN DNA family (A. Itin and E. Keshet, J. Virol. 59:301-307, 1986) which contains murine leukemia virus-related pol and env sequences. Northern analysis indicated that B-26-related RNAs of 8.4 and 3.0 kilobases were transcribed in thymus, spleen, brain, and liver tissues of 6-week-old BALB/c mice.     

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.     

Polymorphism and complexity of the human DC and murine I-A alpha chain genes

A cDNA clone encoding the human B cell alloantigen DC alpha chain (pDCH1) has been used to analyse the structure of the human and murine major histocompatibility complexes by the DNA filter hybridization technique. The pDCH1 probe hybridizes to a single DNA sequence present on chromosome 17 in the mouse genome. A restriction enzyme polymorphism enables us to map this sequence to the I-A subregion. Extensive restriction enzyme polymorphism detected in HLA-DR homozygous typing cells is reminiscent of the DR/MT linkage disequilibrium groups, suggesting that the pDCH1 probe could be useful for haplotype typing in the human population. The HLA-DR region appears more complex than the I region since a second DC-like hybridizing sequence is detected in the human genome in these experiments.     

Harvey sarcoma virus genome contains no extensive sequences unrelated to those of other retroviruses except ras.

The Harvey murine sarcoma virus genome contains two rat-derived sets of genetic information recombined with the Moloney mouse leukemia virus. The rat sequences represent a ras oncogene and a rat VL30 element. The VL30 sequences have several discrete regions of similarity with retroviral sequences which were detected by searching a protein database for similarities with predicted polypeptide sequences from the VL30 regions. On the 5' side, the most similar sequences were those of feline sarcoma viruses; on the 3' side, murine leukemia viruses were the most similar. Some of the regions of similarity could also be detected directly by searching a nucleic acid sequence database with the viral DNA sequences. The most extensive region of similarity was that which corresponded to the endonuclease in the pol gene of a murine leukemia virus. The majority of the rat-derived sequences present in the Harvey sarcoma virus genome can now be attributed exclusively to ras or retrovirus- or retrotransposon-related sequences.     

Molecular properties of a gag- pol- env+ murine leukemia virus from cultured AKR lymphoma cells.

We have described the isolation of a replication-defective murine leukemia virus from a culture of AKR lymphoma cells [Rein et al., Nature (London) 282:753-754, 1979]. To facilitate the characterization of this murine leukemia virus, we transmitted it to mink cells and analyzed its genome by restriction mapping of the mink cellular DNA. This genome resembled the Akv genome quite closely, but it had an additional KpnI cleavage site at 1.3 kilobase pairs from the 5' end of the provirus and a small (approximately 50-base-pair) deletion between 1.8 and 3.0 kilobase pairs from the 5' end. When we tested these mink cells by immune precipitation or by competition radioimmunoassay, we found that they synthesized gPr82env, but contained no detectable gag or pol proteins. It seems likely that the KpnI cleavage site at 1.3 kilobase pairs reflects an abnormal sequence at or near the beginning of the gag gene, which prevents gag or pol translation by introducing a frameshift or termination codon into this region.     

Lack of AKR ecotropic provirus amplification in AKR leukemic thymuses.

A DNA fragment from the 3' region of a molecularly cloned AKR ecotropic provirus was identified to be specific for the AKR ecotropic murine leukemia virus (MuLV). This selected DNA fragment was used to analyze the integrated MuLV proviruses in normal and leukemic tissue DNAs of AKR mice. In comparison with a DNA fragment from the 5' region of the cloned AKR genome or one representing the entire genome, this selected probe hybridized to only a few MuLV proviruses. By comparing transformed and nontransformed tissue DNAs, it appeared that no amplification of proviral sequences related to the AKR ecotropic MuLV had occurred in thymomas of AKR mice during the development of leukemia in these animals. Analysis of the AKR ecotropic MuLV proviruses revealed a significant degree of polymorphism for these sequences among individuals in the AKR/J strain of mouse.     

The Chromosomal Organization of the Human Endogenous Retrovirus-like Sequence HERV-H: Clustering of the HERV-H Sequences in a 300-kb Region Close to the GRPR Locus on the X Chromosome

Within the haploid genome there are approximately 1,000 copiesof the human endogenous retroviruslike sequence, HERV-H. Althoughthese sequences are scattered throughout the entire genome,in situ hybridization experiments revealed that there are discreteclusters positioned on chromosomes 1p and 7q. In this study,we have located three HERV-H sequences which were unexpectedlyclustered within a 300-kilobase region close to the GRPR locuson the X chromosome. In previous studies, no clusteringof thissequence has been reported at this locus. Our finding demonstratesthat, like other repetitive sequences, clustering of HERV-Hoccurs in the human genome, although these sequences may notalways be detected by in situ hybridization methods.     

Heteroduplex analysis of the sequence relations between the RNAs of mink cell focus-inducing and murine leukemia viruses.

The sequence relationships betwen AKR ecotropic virus and an AKR-derived "mink cell focus-inducing" (MCF) isolate (AKR MCF 247), between Moloney murine leukemia virus (M-MLV) and an M-MLV MCF isolate (M-MLV83), and between AKR and M-MLV were studied by electron microscopic heteroduplex analysis. The MCF-specific sequences were found to map from 1.95 kilobases (kb) to 2.75 kb (+/- 0.15 kb) from the 3' end of the RNAs for both MCF isolates. The major sequence nonhomology regions between AKR and M-MLV lie between 0.9 and 3.5 kb from the 3' end. However, the AKR and M-MLV sequences immediately adjacent to the 1.95- and 2.75-kb junctions with MCF-specific sequences are relatively similar in AKR and M-MLV. Our results suggest that the env gene of MLVs maps from 1 kb to 3 kb from the 3' end of the genomic RNA and that the carboxyl end of the glycoprotein of each MCF strain is similar (or identical) to that of its ecotropic parent.     

Localization of the leukemogenic determinants of SL3-3, an ecotropic, XC-positive murine leukemia virus of AKR mouse origin.

SL3-3 is a potent leukemogenic retrovirus that closely resembles the non-leukemogenic virus Akv. Both viruses were isolated from AKR mice, have ecotropic host ranges, and form plaques in the XC assay. They differ at only 1 to 2% of the nucleotides in the viral genomes but differ markedly in virulence properties. SL3-3 induces leukemia in a high percentage of inoculated AKR, C3H, CBA, and NFS mice, whereas Akv does not induce disease in any of these strains. To determine which region of the genome accounts for the leukemogenic potential of SL3-3, we constructed recombinant genomes between molecular clones of SL3-3 and Akv. Recombinant, viral DNA genomes were cloned and then were transfected onto NIH 3T3 fibroblasts to generate infectious virus. The recombinant viruses were tested for leukemogenicity in AKR/J, CBA/J, and C3Hf/Bi mice. We localized the primary leukemogenic determinant to a 3.8-kilobase fragment of the SL3-3 genome containing the viral long terminal repeat, 5' untranslated sequences, gag gene, and 5', 30% of the pol gene. Reciprocal recombinants containing the equivalent region from Akv, linked to the env gene and the remainder of the pol gene from SL3-3, did not induce leukemia. We conclude that the primary virulence determinant of SL3-3 lies outside the region of the genome that encodes the envelope proteins gp70 and p15E.     

Genes of the H-2 complex regulate the antibody response to murine leukemia virus

The influence of the major histocompatibility complex of the mouse (H-2 complex) on the antibody response against murine leukemia virus (MuLV) was investigated after 3 different ways of virus presentation (milk transmission of virus, spontaneous virus activation, and immunization with inactivated virus). The antibody response against MuLV was measured in the sera of H-2 congenic C57BL V+ sublines (V+ denotes positive for milk-transmitted MuLV), (B10.AV+ X C57BL)F1 mice, (C57BL X AKR)F1 mice and of C57BL animals after immunization with inactivated AKR virus. The pattern of immune responsiveness of the different C57BL strains to MuLV was independent of virus replication and was similar for the 3 ways of virus presentation. Serum antibody levels were controlled by 2 genes within the H-2 complex. The first gene (Ir-MuLV-1) was located in the I-A region and was complemented by a second gene (Ir-MuLV-2), which was situated in the regions to the right of the I-B region. Presence of 2 responder alleles (Ir-MuLV-1+,2+) led to an optimal antibody response (H-2b haplotype). Animals that only expressed a responder allele in the I-A region (Ir-MuLV-1+,2-) were intermediate responders. Animals lacking a responder allele in the I-A region (Ir-MuLV-1-,2+ or Ir-MuLV-1-,2-) were low responders.     

YAC-assisted cloning of a putative G-protein mapping to the MHC class I region.

We report the successful use of whole yeast artificial chromosomes (YACs) as probes for direct positional cloning of novel expressed sequences in a given genomic fragment. The class I region of the human major histocompatibility complex, in particular the chromosomal fragment spanning the HLA-E locus, was investigated. The screening of a cDNA library with a 210-kb-long YAC clone led to the identification of a new gene, positionally conserved in the major histocompatibility complex of the mouse genome and encoding a putative GTP binding protein. Although its precise function remains unknown, the interspecies conservation of both sequence and map position suggests a regulatory or functional link with the histocompatibility cluster.