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.     

ERV-L elements: a family of endogenous retrovirus-like elements active throughout the evolution of mammals

We have previously identified in the human genome a family of 200 endogenous retrovirus-like elements, the HERV-L elements, disclosing similarities with the foamy retroviruses and which might be the evolutionary intermediate between classical intracellular retrotransposons and infectious retroviruses. Southern blot analysis of a large series of mammalian genomic DNAs shows that HERV-L-related elements-so-called ERV-L-are present among all placental mammals, suggesting that ERV-L elements were already present at least 70 million years ago. Most species exhibit a low copy number of ERV-L elements (from 10 to 30), while simians (not prosimians) and mice (not rats) have been subjected to bursts resulting in increases in the number of copies up to 200. The burst of copy number in primates can be dated to shortly after the prosimian and simian branchpoint, 45 to 65 million years ago, whereas murine species have been subjected to two much more recent bursts (less than 10 million years ago), occurring after the Mus/Rattus split. We have amplified and sequenced 360-bp ERV-L internal fragments of the highly conserved pol gene from a series of 22 mammalian species. These sequences exhibit high percentages of identity (57 to 99%) with the murine fully coding MuERV-L element. Phylogenetic analyses allowed the establishment of a plausible evolutionary scheme for ERV-L elements, which accounts for the high level of sequence conservation and the widespread dispersion among mammals.     

Discrete regions of sequence homology between cloned rodent VL30 genetic elements and AKV-related MuLV provirus genomes

Southern blot analyses using reduced stringency hybridization conditions have been employed to search for sequence homologies between rodent VL30 genes and murine leukemia virus (MuLV) proviruses. These constitute two classes of transposon-like elements previously believed to be genetically unrelated. Our results demonstrate that cloned representatives of both ecotropic and xenotropic-like proviruses share discrete regions of sequence homology with VL30 genes of both rat and mouse origin. These regions of homology exist in both 3' and 5' halves of the MuLV genome but do not include extensive portions of the long terminal repeat (LTR) or a 0.4 Kbp segment of the env gene specific for recently acquired ecotropic-type MuLV proviruses. DNA sequencing, however, revealed that the short inverted terminal repeat sequence of MuLV proviral LTRs is almost perfectly conserved at the terminus of an integrated mouse VL30 gene. These results suggest that recombination events with rodent VL30-type sequences occurred during early MuLV evolution. The strong conservation of the inverted terminal repeat sequence may reflect a common integration mechanism for VL30 elements and MuLV proviruses.     

Evidence for the Asiatic origin of endogenous AKR-type murine leukemia proviruses.

A restriction endonuclease cleavage map of the genome of AKV, the endogenous, ecotropic leukemia virus of AKR mice, has been derived. By using this map and analyzing DNA from congenic mice, we have defined four DNA fragments diagnostic for AKV proviruses. Analysis of DNAs from 10 strains of American laboratory mice revealed that all strains carrying inducible, ecotropic murine leukemia viruses yielded DNAs which contained the four DNA fragments diagnostic for AKV. Virus-negative strains lacked these fragments in their DNA. Screening DNA from 23 additional mice revealed that, among these mice, only mice from Asia gave rise to the DNA fragments diagnostic of an AKV provirus. We conclude that all of the endogenous ecotropic murine leukemia proviruses in American laboratory mice are closely related since they share a common set of restriction endonuclease cleavage sites. These proviruses appear to derive from the East Asian ancestors of these mouse strains. Analysis of DNA from six selected mice with an additional restriction endonuclease showed that greater than 97% of the nucleotide sequences in each provirus are contigous and that these endogenous proviruses are indistinguishable from proviruses introduced by exogenous infection.     

Endogenous nonecotropic proviruses mapped with oligonucleotide probes from the long terminal repeat region

We characterized endogenous proviruses in C57BL/6J, DBA/2J, and C3H/HeJ mouse strains with oligonucleotide probes derived from long terminal repeat (LTR) sequences of three classes of nonecotropic murine leukemia virus. The segregation of proviral-host DNA junction fragments was followed in BXH and BXD recombinant inbred (RI) strain sets, and most fragments mapped readily to defined chromosomal regions. Most of the LTR fragments appear to correspond to proviruses mapped previously with oligonucleotide env region probes of the same viral class. At least 22 elements represent new proviral loci, no more than half of which may be solo LTRs, and an additional six may correspond to proviruses identified previously with less specific hybridization probes. Together with proviruses identified previously with env probes, the LTR probe-reactive elements represent the majority of endogenous murine leukemia proviruses in the mouse genome.     

Most of the murine leukemia virus sequences in the DNA of NIH/swiss mice consist of two closely related proviruses, each repeated several times

The structure of the endogenous murine leukemia virus (MuLV) sequences of NIH/Swiss mice was analyzed by restriction endonuclease digestion, gel electrophoresis, and hybridization to an MuLV nucleic acid probe. Digestion of mouse DNA with certain restriction endonucleases revealed two classes of fragments. A large number of fragments (about 30) were present at a relatively low concentration, indicating that each derived from a sequence present once in the mouse genome. A smaller number of fragments (one to five) were present at a much higher concentration and must have resulted from sequences present multiple times in the mouse genome. These results indicated that the endogenous MuLV sequences represent a family of dispersed repetitive sequences. Hybridization of these same digested mouse DNAs to nucleic acid probes representing different portions of the MuLV genome allowed construction of a map of the sites where restriction endonucleases cleave the endogenous MuLV sequences. Several independent recombinant DNA clones of endogenous MuLV sequences have been isolated from C3H mice (Roblin et al., J. Virol. 43:113-126, 1982). Analysis of these sequences shows that they have the structure of MuLV proviruses. The sites at which restriction endonucleases cleave within these proviruses appeared to be similar or identical to the sites at which these nucleases cleaved within the MuLV sequences of NIH/Swiss mice. This identity was confirmed by parallel electrophoresis. We conclude that the apparently complex pattern of endogenous MuLV sequences of NIH/Swiss mice consists largely of only two kinds of provirus, each repeated multiple times at dispersed sites in the mouse genome.     

Analysis of mutant Moloney murine leukemia viruses containing linker insertion mutations in the 3'' region of pol.

Twelve linker insertion mutations have been constructed in the 3' part of the pol gene of Moloney murine leukemia virus. This region of the Moloney murine leukemia virus genome encodes IN or p46pol, which is required for integration of the retroviral DNA into the host cell chromosome. Viral proteins synthesized by these mutants were used to pseudotype a neo-containing retroviral vector. Ten of twelve linker insertion mutant pseudotypes were unable to generate stable proviruses in infected mouse cells, as measured by the formation of G418-resistant colonies. Two mutants mapping at the 3' terminus of the IN-encoding region were competent for the formation of stable vector proviruses (hundreds of G418-resistant colonies per mutant pseudotype-infected plate). Representative linker insertion mutants were also tested for the ability to synthesize viral unintegrated DNA in newly infected cells. All assayed mutants were capable of synthesizing all normal forms of viral unintegrated DNA. The structure of integrated vector proviruses generated by defective and nondefective linker insertion mutants was also analyzed. All replication-competent mutants generated normal proviruses, while the few obtainable proviruses generated by replication-defective mutants were sometimes aberrant in structure. These results argue strongly (and confirm previous data) that the IN-encoding region of pol does not play a significant role in DNA synthesis, but is absolutely required for the formation of normal proviral DNA.     

The integrated genome of murine leukemia virus

The Southern gel filter transfer technique has been used to characterize the integrated genome of Moloney murine leukemia virus (M-MuLV) and the genomes of the endogenous viruses of the mouse. Study of 10 clones of rat cell independently infected by M-MuLV indicates a minimum of 15 integration sites into which the M-MuLV provirus can be inserted. No common integration site is observed among these clones. Clones productively infected by M-MuLV acquire multiple proviruses, whereas infected cells unable to produce virus contain only one M-MuLV provirus. Once established, the integrated genomes are stable for at least two years after initial infection.The use of M-MuLV probe allows detection of a spectrum of Eco RI-cleaved mouse DNA fragments containing endogenous MuLV genomes. DNAs of different inbred laboratory mouse strains yield similar patterns of provirus with each strain showing minor characteristic differences. In some instances, mouse cells infected by M-MuLV reveal additional proviruses beyond those seen in the uninfected cell. DNAs from three different M-MuLV-induced thymomas indicate, as in rat cells, multiple possible integration sites.     

Two subfamilies of murine retrotransposon ETn sequences

Early transposon (ETn) elements are 5.7-kb retrotransposons found in the murine genome. We have sequenced large portions of two ETn elements that have apparently transposed within the DNA of a murine myeloma cell line, P3.26Bu4. One of the transposed ETn elements has 5' and 3' long terminal repeats (LTRs) that are exact duplicates of each other and has a 6-bp target site duplication. These results suggest that this element, which inserted into an immunoglobulin gamma 1 switch region, moved by a retrotransposition process. Our nucleotide sequences confirm that individual ETn elements are very similar to one another and lack open reading frames. However, the ETn sequences reported here and those previously described differ significantly near their 5' LTRs, including 200 bp of weak similarity and 240 bp of complete disparity. Southern hybridization analysis suggests that both subfamilies of ETn sequences are represented many times in the mouse genome. The possibility that the disparate sequences have a role in transposition by ETn elements is discussed.     

Murine endogenous retrovirus MuERV-L is the progenitor of the "orphan" epsilon viruslike particles of the early mouse embryo

Viruslike particles which displayed a peculiar wheellike appearance that distinguished them from A-, B- or C-type particles had previously been described in the early mouse embryo. The maximum expression of these so-called epsilon particles was observed in two-cell-stage embryos, followed by their rapid decline at later stages of development and no particles detected at the zygote one-cell stage. Here, we show that these particles are in fact produced by a newly discovered murine endogenous retrovirus (ERV) belonging to the widespread family of mammalian ERV-L elements and named MuERV-L. Using antibodies that we raised against the Gag protein of these elements, Western blot analysis and in toto immunofluorescence studies of the embryos at various stages disclosed the same developmental expression profile as that observed for epsilon particles. Using expression vectors for cloned, full-length, entirely coding MuERV-L copies and cell transfection, direct identification of the epsilon particles was finally achieved by high-resolution electron microscopy.     

Characterization, mapping, and distribution of the two XMRV parental proviruses

Xenotropic murine leukemia virus-related virus (XMRV) was previously reported to be associated with human prostate cancer and chronic fatigue syndrome. Our groups recently showed that XMRV was created through recombination between two endogenous murine retroviruses, PreXMRV-1 and PreXMRV-2, during the passaging of a prostate tumor xenograft in nude mice. Here, multiple approaches that led to the identification of PreXMRV-2, as well as the distribution of both parental proviruses among different mouse species, are described. The chromosomal loci of both proviruses were determined in the mouse genome, and integration site information was used to analyze the distribution of both proviruses in 48 laboratory mouse strains and 46 wild-derived strains. The strain distributions of PreXMRV-1 and PreXMRV-2 are quite different, the former being found predominantly in Asian mice and the latter in European mice, making it unlikely that the two XMRV ancestors could have recombined independently in the wild to generate an infectious virus. XMRV was not present in any of the mouse strains tested, and among the wild-derived mouse strains analyzed, not a single mouse carried both parental proviruses. Interestingly, PreXMRV-1 and PreXMRV-2 were found together in three laboratory strains, Hsd nude, NU/NU, and C57BR/cd, consistent with previous data that the recombination event that led to the generation of XMRV could have occurred only in the laboratory. The three laboratory strains carried the Xpr1(n) receptor variant nonpermissive to XMRV and xenotropic murine leukemia virus (X-MLV) infection, suggesting that the xenografted human tumor cells were required for the resulting XMRV recombinant to infect and propagate.