Diverse long terminal repeats are associated with murine retroviruslike (VL30) elements.

The VL30 family is a retroviruslike gene family with no apparent nucleic acid homology to any known retrovirus. Over 100 copies of VL30 DNA elements are dispersed throughout the mouse genome. Sequence analysis of the VL30 long terminal repeat (LTR) units showed that, whereas the LTR units of a given VL30 DNA element were almost identical, the LTR units associated with distinct members of the family were very different from one another. Comparison of the LTR sequences possessed by two particular VL30 DNA elements revealed a pattern of extensively homologous DNA segments adjacent to only distantly related DNA sequences. With the aid of sub-LTR probes, it was shown that a certain LTR is composed of both U5 sequences that are abundantly present in all species of the genus Mus and a U3 region detected only in Mus musculus. In addition, we isolated a VL30 DNA element in which the LTR units were replaced by the LTR units of an apparently novel retroviruslike family. These findings suggest that recombinations have played a role in generating the diverse population of VL30-associated LTRs.     

''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.     

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.     

Retrotransposon-like VL30 elements are efficiently induced in anoxic rat fibroblasts

VL30 elements are a multigene family within the class of retroviruses and retrotransposons. We have characterized the response of normal rat fibroblasts to anoxia, in which endogenous VL30 element expression is strongly induced. Optimal induction up to 500-fold occurs under complete anoxia, although a lesser response is seen under atmospheres up to 2% oxygen. Phorbol esters and diacylglycerol, which induce mouse VL30 RNA approximately eightfold, show no effect on the rat VL30 system. The hypoxic conditions optimal for rat VL30 induction represent a mild cellular stress, with no reduction in cell viability during the induction period. Although the precise physiological role of this fibroblast response to temporary anoxia is unknown, it may occur during wound healing. The induction of VL30 by anoxia provides a unique model system wherein a member of the mammalian retrovirus/retrotransposon family is highly responsive to a common physiological signal.     

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.     

Individual mouse VL30 elements transferred to rat cells by viral pseudotypes retain their responsiveness to activators of protein kinase C.

By exploiting the retroviral characteristics of mouse VL30 elements, we transferred individual copies of VL30 to Rat-1 cells by infection. Analysis of clonal isolates containing single VL30 elements integrated into the Rat-1 genome indicates that responsiveness to activators of protein kinase C is an inherent property of at least some of the VL30 sequences.     

Primer binding sites corresponding to several tRNA species are present in DNAs of different members of the same retrovirus-like gene family (VL30).

We analyzed the putative tRNA primer binding site (PBS) present in several cloned copies of the murine retrovirus-like VL30 family. In the five VL30 DNA clones analyzed, we identified PBS sequences corresponding to three different tRNA species: tRNAPro, tRNAGly, and tRNAGln. The latter two PBS sequences have not been previously encountered in other retroviral or retrovirus-like systems. A unique situation was observed in which PBS sequences complementary to two different tRNA species were flanked by otherwise identical VL30 sequences. In addition, we demonstrated the use of PBS-specific synthetic oligonucleotides for the identification of the tRNA primer and their potential utility in the direct cloning of PBS-containing DNA elements.     

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.     

A novel retroviruslike family in mouse DNA.

In the course of structural analysis of VL30 DNA elements, a recombinant retroviruslike element was encountered that contained non-VL30 long terminal repeats (LTRs) flanking internal VL30 sequences. With the aid of this novel LTR sequence probe, we cloned several DNA elements that were apparently members of a new retroviruslike family. A particular DNA element representative of this family (designated GLN) was characterized. It was approximately 8 kilobase pairs long and contained LTRs that are 430 base pairs long. It possessed an unusual primer-binding site sequence that corresponds to tRNAGln and a polypurine tract primer that is adjacent to the 3' LTR. The nucleotide sequences of the LTRs and their adjacent regions (which together housed all cis-acting retroviral functions) were different from those of known retroviruses and retroviruslike families. The comparison of three different GLN LTR sequences revealed a marked heterogeneity of U3 sequences relative to the homogeneity of R and U5 sequences. We estimate that approximately 20 to 50 copies of GLN elements are dispersed in all species of mice. GLN-related LTRs, however, are present in a much higher copy number (1,000 to 1,500 per genome). Nucleotide sequences that are more distantly related to GLN DNA are present in multiple copies in DNAs of other rodents but not in nonrodent genomes.     

Expression of viral and virus-like elements in DNA repair-deficient/immunodeficient "wasted" mice

Wasted mice bear an autosomal recessive mutation (wst) that causes neurologic abnormalities, faulty DNA repair in lymphocytes, and immunodeficiency at mucosal sites. Recent work has suggested possible viral involvement in these manifestations by demonstrating abnormal viral gp70 expression that segregates with the wasted mutation. In the experiments reported here, we examined tissue-specific expression of AKR viral (AKV) sequences and virus-like 30S (VL30) elements in wasted and control mice. Our studies showed that AKV and VL30 RNA expression were two- to fivefold higher in spleen, brain, and thymus of mice bearing the wst allele than in those of control mice. (These tissues have all been shown to display functional or developmental abnormalities in wst/wst mice.) Expression of viral mRNA in Peyer's patches and mesenteric lymph nodes was similar in wasted and control animals. The majority of the VL30-hybridizing RNA in all tissues could be attributed to long-terminal-repeat rather than to main-body sequences. These differences in expression between wasted and control mice could not be attributed to differences in VL30 or AKV gene copy number. Our results demonstrate an association between altered expression of viral and virus-like sequences and the development of tissue-restricted abnormalities in wst/wst mice.