Structure and expression of mouse VL30 genes.

DNA sequencing and blot hybridization analyses have been used to study the structure of a mouse VL30 gene and the molecular nature of VL30-related RNA which is induced upon the stimulation of cultured AKR mouse embryo cells with defined peptide growth factors. An integrated mouse VL30 gene was found to contain identical 601-base-pair long terminal repeats (LTRs) which were themselves terminated in short inverted repeats. The entire VL30 gene was flanked by a 4-base-pair direct repeat of cellular DNA. Thus, VL30 genes are structurally analogous to integrated forms of retrovirus proviruses and certain other classes of mobile genetic elements. The LTR sequence was found to contain putative promoter and polyadenylation signals and generally exhibited little sequence homology to murine leukemia virus proviral LTRs. Certain short regions of sequence conservation, however, were evident, including the inverted terminal repeat, LTR-adjacent regions corresponding to origins of murine leukemia virus proviral DNA synthesis, and a 36-base-pair direct repeat bearing homology to the 72-base-pair direct repeat (enhancer sequence) of the murine leukemia virus-related Moloney sarcoma virus. Upon mitogenic stimulation of quiescent cells with epidermal growth factor and insulin, a major 5.5-kilobase VL30-specific RNA complementary to both LTR and non-LTR sequences was rapidly induced. We conclude that a complete VL30 gene(s) is highly regulated by peptide growth factor binding to specific membrane receptors in these cells.     

Nucleotide sequence analysis of the long terminal repeat of murine virus-like DNA (VL30) and its adjacent sequences: resemblance to retrovirus proviruses

VL30 DNA represents a retrovirus-like multigene family of mice whose genetic origin is unknown. We have now determined the primary nucleotide sequences and the adjacent sequences of the long terminal direct repeats (LTRs) possessed by a randomly selected VL30 unit. The LTR of the VL30 unit comprised 435 nucleotide base pairs and had an inverted repeat of five bases at its 5' and 3' termini. At the joints with flanking mouse DNA was the VL30 sequence (5')TG . . . CA(3') and a tetranucleotide direct repeat of flanking sequences. At the inner boundary of the 5' LTR was an 18-base sequence that is complementary to tRNApro, and at the inner boundary of the 3' LTR was a purine-rich tract ending with AATG. These results suggested that VL30 DNA used the same integration strategy that is exercised by retrovirus proviruses and transposable elements and that the VL30 LTR is synthesized in a similar way that the LTR of retroviruses is synthesized. The data thus reinforce the retrovirus-like nature of VL30 genetic information.     

Apparent recombinants between virus-liKE (VL30) and murine leukemia virus-related sequences in mouse DNA.

VL30 elements are a dispersed multigene family that is ubiquitous in all murine cells. Despite not sharing nucleic acid sequence homology with natural retroviruses (exogenous or endogenous), VL30 elements are distinguished by several retrovirus-like features. By screening a mouse embryonic library, we have cloned DNA units that contain VL30 sequences linked to MuLV-related sequences. Using blot hybridization with the aid of specific subgenomic probes and heteroduplex analyses, we have established that the DNA element is composed of two VL30 long terminal repeat (LTR) units, a limited subset of VL30 information adjacent to both 5' and 3' LTRs, and an enclosure of MuLV-related information that shares homology primarily with MuLV gag and pol determinants (but lacks MuLV-related LTRs). This sequence arrangement is reciprocal in nature to the recombinations between MuLV and rat VL30 that generated the genomes of the Harvey and Kirsten strains of mouse sarcoma virus and most likely is the consequence of recombination between VL30 and MuLV-related elements and the subsequent deposition of the putative recombinant DNA in the mouse genome.     

Nucleotide sequence analysis establishes the role of endogenous murine leukemia virus DNA segments in formation of recombinant mink cell focus-forming murine leukemia viruses.

The sequence of 363 nucleotides near the 3' end of the pol gene and 564 nucleotides from the 5' terminus of the env gene in an endogenous murine leukemia viral (MuLV) DNA segment, cloned from AKR/J mouse DNA and designated as A-12, was obtained. For comparison, the nucleotide sequence in an analogous portion of AKR mink cell focus-forming (MCF) 247 MuLV provirus was also determined. Sequence features unique to MCF247 MuLV DNA in the 3' pol and 5' env regions were identified by comparison with nucleotide sequences in analogous regions of NFS -Th-1 xenotropic and AKR ecotropic MuLV proviruses. These included (i) an insertion of 12 base pairs encoding four amino acids located 60 base pairs from the 3' terminus of the pol gene and immediately preceding the env gene, (ii) the deletion of 12 base pairs (encoding four amino acids) and the insertion of 3 base pairs (encoding one amino acid) in the 5' portion of the env gene, and (iii) single base substitutions resulting in 2 MCF247 -specific amino acids in the 3' pol and 23 in the 5' env regions. Nucleotide sequence comparison involving the 3' pol and 5' env regions of AKR MCF247 , NFS xenotropic, and AKR ecotropic MuLV proviruses with the cloned endogenous MuLV DNA indicated that MCF247 proviral DNA sequences were conserved in the cloned endogenous MuLV proviral segment. In fact, total nucleotide sequence identity existed between the endogenous MuLV DNA and the MCF247 MuLV provirus in the 3' portion of the pol gene. In the 5' env region, only 4 of 564 nucleotides were different, resulting in three amino acid changes between AKR MCF247 MuLV DNA and the endogenous MuLV DNA present in clone A-12. In addition, nucleotide sequence comparison indicated that Moloney-and Friend-MCF MuLVs were also highly related in the 3' pol and 5' env regions to the cloned endogenous MuLV DNA. These results establish the role of endogenous MuLV DNA segments in generation of recombinant MCF viruses.     

Cloning of endogenous murine leukemia virus-related sequences from chromosomal DNA of BALB/c and AKR/J mice: identification of an env progenitor of AKR-247 mink cell focus-forming proviral DNA

Recombinant phages containing murine leukemia virus (MuLV)-reactive DNA sequences were isolated after screening of a BALB/c mouse embryo DNA library and from shotgun cloning of EcoRI-restricted AKR/J mouse liver DNA. Twelve different clones were isolated which contained incomplete MuLV proviral DNA sequences extending various distances from either the 5' or 3' long terminal repeat (LTR) into the viral genome. Restriction maps indicated that the endogenous MuLV DNAs were related to xenotropic MuLVs, but they shared several unique restriction sites among themselves which were not present in known MuLV proviral DNAs. Analyses of internal restriction fragments of the endogenous LTRs suggested the existence of at least two size classes, both of which were larger than the LTRs of known ecotropic, xenotropic, or mink cell focus-forming (MCF) MuLV proviruses. Five of the six cloned endogenous MuLV proviral DNAs which contained envelope (env) DNA sequences annealed to a xenotropic MuLV env-specific DNA probe; in addition, four of these five also hybridized to an ecotropic MuLV-specific env DNA probe. Cloned MCF 247 proviral DNA also contained such dual-reactive env sequences. One of the dual-reactive cloned endogenous MuLV DNAs contained an env region that was indistinguishable by AluI and HpaII digestion from the analogous segment in MCF 247 proviral DNA and may therefore represent a progenitor for the env gene of this recombinant MuLV. In addition, the endogenous MuLV DNAs were highly related by AluI cleavage to the Moloney MuLV provirus in the gag and pol regions.     

Nucleotide sequences of feline sarcoma virus long terminal repeats and 5' leaders show extensive homology to those of other mammalian retroviruses.

The nucleotide sequences of the Gardner-Arnstein feline sarcoma virus (FeSV) long terminal repeat and the adjacent leader sequences 5' to the viral gag gene were determined. These were compared with homologous portions of Synder-Theilen FeSV and with previously published sequences for Moloney murine sarcoma virus and simian sarcoma virus proviral DNA. More than 75% of the residues in the FeSV R and U5 regions were homologous to sequences within the same regions of the other viral long terminal repeats. Unexpectedly, alignment of the FeSV sequences with those of the Moloney murine sarcoma and simian sarcoma viruses showed similar extents of homology within U3. The homologous U3 regions included the inverted repeats, a single set of putative enhancer sequences, corresponding to a "72-base-pair" repeat, and sequences, including the CAT and TATA boxes, characteristic of eucaryotic promotors. The 5' leader sequences of both FeSV strains included a binding site for prolyl tRNA and a putative splice donor sequence. In addition, the FeSV leader contained a long open reading frame which was adjacent to and in phase with the ATG codon at the 5' end of the FeSV gag gene. The open reading frame could code for a signal peptide of about 7.4 kilodaltons. Our results support the concept that the virogenic portions of both FeSV and simian sarcoma virus were ancestrally derived from viruses of rodent origin, with conservation of regulatory sequences as well as the viral structural genes.     

Recombinant BALB and Harvey sarcoma viruses with normal proto-ras-coding regions transform embryo cells in culture and cause tumors in mice.

The ras genes of BALB and Harvey sarcoma viruses contain point mutations in codon 12 or codons 12 and 59, relative to proto-ras from normal animal and human cells. By in vitro recombination between cloned rat proto-ras and cloned BALB and Harvey sarcoma proviruses, we constructed recombinant proviruses with normal proto-ras-coding regions. These recombinant proviruses transformed mouse 3T3 cells upon transfection. However, when the transforming efficiencies of proviral DNAs were compared after transfection with helper provirus, recombinant proviruses were 2 to 30 times less efficient than the corresponding wild-type proviruses. Recombinant sarcoma viruses isolated from cells transformed by cloned proviral DNA contained the expected normal ras-coding region. They transformed rat embryo cells and induced erythroblastosis and sarcomas in newborn mice as efficiently as wild-type viruses did. We conclude that conversion of normal proto-ras genes to viral ras genes depends on truncation of normal proto-ras regulatory elements and substitution by retroviral (long terminal repeat) promoters and that the transforming function of long terminal repeat-ras genes is enhanced by point mutations.     

Anoxia-inducible rat VL30 elements and their relationship to ras-containing sarcoma viruses.

VL30 elements are associated with cancer by their overexpression in rodent malignancies, their induction in a fibroblast response to anoxia which shares features with the malignant phenotype, and their presence recombined into Harvey murine sarcoma virus (HaSV) and Kirsten murine sarcoma virus. These sarcoma viruses contain ras oncogenes flanked on both sides by retrotransposon VL30 element sequences, in turn flanked by mouse leukemia virus sequences. Three very basic questions have existed about the VL30 element sequences found in sarcoma viruses: (i) how did they become recombined, (ii) what are their exact boundaries, and (iii) why are they there? To help decipher the nature of VL30 elements in sarcoma viruses, we examined VL30 clones isolated from an anoxic fibroblast cDNA library and independently by polymerase chain reaction cloning from rat cell DNA. Sequence comparisons with HaSV revealed that HaSV was formed by the substitution of 0.7 kb of VL30 sequences by 0.9 kb of c-Ha-ras sequences, with this event possibly facilitated by the presence of an identical Alu-like repeat found upstream of the 5' recombination point in both the VL30 element and c-Ha-ras. Recombination occurred 42 bases beyond the Alu-like sequences in VL30 and 1596 bases beyond them in c-Ha-ras, at position 926 of HaSV. The 3' ras-VL30 recombination event in HaSV occurred within a seven-base region of shared sequence identity, between HaSV bases 1825 and 1825 and 1831. Recombination between Moloney leukemia virus (MoLV) and VL30 appears to have occurred at a point corresponding to base 218 or 219 of MoLV and was near a TAR-like VL30 sequence; such recombination at the 3' end was between positions 7445 and 7456 of MoLV (HaSV positions 4694 to 4703). Kirsten murine sarcoma virus was found to be closely analogous to HaSV, and limited similar features were also seen with Rasheed sarcoma virus.     

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.     

Long terminal repeat-like elements flank a human immunoglobulin epsilon pseudogene that lacks introns

There are at least three immunoglobulin epsilon genes (C epsilon 1, C epsilon 2, and C epsilon 3) in the human genome. The nucleotide sequences of the expressed epsilon gene (C epsilon 1) and one (C epsilon 3) of the two epsilon pseudogenes were compared. The results show that the C epsilon 3 gene lacks the three intervening sequences entirely and has a 31-base A-rich sequence 16 bases 3' to the putative poly(A) addition signal, indicating that the C epsilon 3 gene is a processed gene. The C epsilon 3 gene sequence is homologous to the five separate DNA segments of the C epsilon 1 gene; namely, a segment in the 5'-flanking region (100 bases) and four exons, which are interrupted by a spacer region or intervening sequences. Long terminal repeat (LTR)-like sequences which contain TATAAA and AATAAA sequences as well as terminal inverted repeats are present in both 5'- and 3'-flanking regions. The 5' and 3' LTR-like sequences do not, however, constitute a direct repeat, unlike transposable elements of eukaryotes and retroviruses. The 3' LTR-like sequence is repetitive in the human genome, but is not homologous to the Alu family DNA. Models for the evolutionary origin of the processed gene flanked by the LTR-like sequences are discussed. The C epsilon 3 gene has a new open frame which codes potentially for an unknown protein of 292 amino acid residues.     

Molecular characterization of the Akvr-1 restriction gene: a defective endogenous retrovirus-borne gene identical to Fv-4r.

A dominant restriction allele, Akvr-1r, from California wild mice (Mus musculus domesticus) confers resistance to exogenous ecotropic murine leukemia virus (MuLV) infection. The presence of an ecotropic MuLV envelope-related glycoprotein in uninfected virus-resistant cells suggests that viral interference is a possible mechanism for this resistance. We molecularly cloned the ecotropic MuLV envelope-related sequence from the genomic DNA of a wild mouse homozygous for the Akvr-1r locus. The cloned provirus was defective and contained a C-terminal end of the pol gene, a complete envelope gene, and a 3' long terminal repeat. The presence of this provirus was directly correlated with Akvr-1r-mediated virus resistance in cell cultures and hybrid mice. The Akvr-1r provirus restriction map and partial DNA sequence were identical to those of the Fv-4r allele, an ecotropic MuLV resistance locus from Japanese feral mice (M. musculus molossinus), which was previously shown to be allelic with the Akvr-1r gene. The 3' host flanking sequences of Fv-4r and Akvr-1r also had identical restriction maps. These findings indicate that Akvr-1r and Fv-4r are the same gene. It was probably acquired by interbreeding of these feral species in recent times. Conservation of this locus might be favored by the useful function that it performs in protection against ecotropic MuLV infection endemic in both populations of wild mice.     

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Two chimeric helper proviruses were derived from the provirus of the ecotropic Moloney murine leukemia virus by replacing the 5'long terminal repeat and adjacent proviral sequences with the mouse metallothionein I promoter. One of these chimeric proviruses was designed to express the gag-pol genes of the virus, whereas the other was designed to express only the env gene. When transfected into NIH 3T3 cells, these helper proviruses failed to generate competent virus but did express Zn2+-inducible trans-acting viral functions needed to assemble infectious vectors. One helper cell line (clone 32) supported vector assembly at levels comparable to those supported by the Psi-2 and PA317 cell lines transfected with the same vector. Defective proviruses which carry the neomycin phosphotransferase gene and which lack overlapping sequence homology with the 5' end of the chimeric helper proviruses could be transfected into the helper cell line without generation of replication-competent virus. Mass cultures of transfected helper cells produced titers of about 10(4) G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 X 10(4) CFU/ml. In contrast, defective proviruses which share homologous overlapping viral sequences with the 5' end of the chimeric helper proviruses readily generated infectious virus when transfected into the helper cell line. The deletion of multiple cis-acting functions from the helper provirus and elimination of sequence homology overlapping at the 5' ends of helper and vector proviruses both contribute to the increased genetic stability of this system.     

''Solo'' large terminal repeats (LTR) of an endogenous retrovirus-like gene family (VL30) in the mouse genome.

VL30 genetic elements constitute a murine multicopy gene family that is retrovirus-like, despite the lack of sequence homology with any known retrovirus. Over one hundred copies of VL30 units are dispersed throughout the mouse genome. We report here that the mouse genome also contains 'solo' VL30 long terminal repeats (LTRs). These are structures which contain the LTR detached from the rest of the VL30 sequences. The isolation of solo LTRs from a mouse embryonic gene library with the aid of sub-genomic VL30 probes is described. Direct DNA sequencing established that the solo LTR unit is grossly similar to a standard VL30 LTR and that the LTR is flanked by a 4-base pair duplication. The analogy to the occurrence of solitary LTR units of transposable elements is discussed.