Lawrence R. Boone

Specific sequence deletions in two classes of murine leukemia virus-related proviruses in the mouse genome

Characteristic long terminal repeats (LTR) of approximately 700 and 750 bp were found, respectively, in the two classes (polytropic and modified polytropic) of murine leukemia virus (MuLV)-related nonecotropic nonxenotropic proviral sequences in eight individual molecular clones of RFM/Un mouse chromosomal DNA fragments. Three proviral clones, two polytropic and one modified polytropic, contained sequence deletions in the viral structural genes. Nucleotide sequence analysis revealed that 7-bp direct repeats occur at both ends of deleted sequences in intact structures and one of the repeats remains in genomes with the deletion. Specifically, the deleted sequences were a 1487-bp gag-pol sequence with ACTGCCC repeat, a 113-bp mid-pol sequence with CAGGCAA repeat, and a 1811-bp env sequence with GGTCCAG repeat. The same specific sequence deletions were found in both classes of MuLV-related proviral structures. Examination of chromosomal DNA from eight inbred laboratory mouse strains and six wild mouse species showed that a minor population of proviruses with these specific deletions were present in Mus musculus and Mus spretus, all of which contain prominent 700-bp LTR polytropic proviral structures. The 750-bp LTR modified polytropic proviral structures were phylogenetically more restricted, being equally predominant in Mus musculus domesticus mice, but minor to undetectable in Mus spretus subspecies, and absent in other wild mouse populations.

Characterization of two solitary long terminal repeats of murine leukemia virus type that are conserved in the chromosome of laboratory inbred mouse strains

Twenty molecular clones containing sequences homologous to the long terminal repeats (LTRs) of the endogenous ecotropic murine leukemia virus (MuLV) of the RFM/Un mouse were isolated from a library of RFM/Un mouse spleen DNA in phage lambda. Three of these LTRs were not associated with any viral structural genes. Nucleotide sequence analysis demonstrated that they were solitary LTRs which were flanked by 4-bp directly repeated cellular sequences and which lacked primer binding sites. Two of the three subclones were found to be identical except for their orientations in the vector pBR322. Unique-sequence regions on either side of the two nonidentical elements were used to characterize their integration sites in genomic DNA. The solitary LTRs and their flanking regions were found to be conserved in a number of inbred mouse strains, including three strains known not to harbor endogenous ecotropic MuLV-type proviruses. Comparison of cleavage by the methylation-sensitive restriction enzyme SmaI and methylation-insensitive KpnI at the characteristic LTR SmaI/KpnI site suggested that at least one of these solitary LTRs is methylated to a lesser extent than are most endogenous proviral LTRs. These particular solitary LTRs, like endogenous proviral sequences, appear to be stably transmitted genetic elements.

Endogenous Retrovirus and Radiation-Induced Leukemia in the RFM Mouse

Publisher Summary Radiogenic myeloid leukemia in RFM/Un mice is a potentially valuable model system to study specific chromosomal rearrangements of retrovirus genes. One of the key aspects of this system is that infection by an endogenous virus is strongly restricted; therefore, an extracellular spread of the virus is not likely to play a role in the formation of additional integrated viral genomes, as is the case in other well-studied murine retrovirus models. The role RFV plays in radiogenic myeloid leukemia is uncertain. As with other endogenous ecotropic retroviruses, the virus does not transform cells in culture and does not appear to accelerate leukemogenesis in vivo . A downstream promotion model for leukemogeneis by avian leukosis virus does provide an attractive hypothesis for the involvement of RFV in the myeloid leukemia. Studies indicate that there is a single locus for the inducible ecotropic provirus that resides on chromosome 5. The myeloid leukemic cell line clearly has additional copies of the ecotropic provirus.

Analysis of Fv-1 restriction in two murine embryonal carcinoma cell lines and a series of differentiated derivatives

We have used antibiotic-resistant retrovirus vectors rescued by Fv-1-sensitive murine leukaemia viruses (MuLV) to examine the Fv-1 phenotype of two undifferentiated embryonal carcinoma (EC) cell lines derived from teratocarcinomas of mouse strain 129. In addition, a set of EC cell-derived differentiated cell lines was analysed. Restriction of both B-tropic and endogenous N-tropic virus is characteristic of the Nr-type restriction reported in mouse strain 129. However, results indicate that Fv-1 restriction is not expressed in the PCC4.aza1R EC cell line. In contrast, the F9 EC cell line showed a strong restriction of the B-tropic pseudotyped vector but failed to restrict endogenous N-tropic pseudotypes. The Fv-1 gene thus seems to be differentially expressed in two EC cell lines derived from the same mouse strain. Furthermore, the selective restriction of B-tropic but not endogenous N-tropic MuLV in F9 cells suggests that these activities function independently of each other. Analysis of PCC4.aza1R-derived differentiated cell lines revealed that three fibroblast cell lines derived by retinoic acid-induced differentiation were also phenotypically silent for Fv-1. However, a pre-adipocyte line established following simultaneous exposure to retinoic acid and 5-azacytidine showed strong restriction of both B-tropic and endogenous N-tropic MuLV. Although additional data suggest that there is no correlation between the differentiated pre-adipocyte phenotype and Fv-1 expression, our results nonetheless show that Nr restriction can be observed in some derivatives of PCC4.aza1R cells, presumably by activating expression of the Fv-1 gene.

Differential Expression of Fv‐1 in Fibroblasts Derived from Embryonal Carcinoma Cells

The Fv-1 locus in mice is the major genetic determinant influencing susceptibility to murine leukemia virus (MuLV) infection.’ With few exceptions, mouse cells in culture exhibit Fv-1 restriction. Typically, this is observed as a 100to 1000-fold reduction in titer relative to cells of the permissive genotype. Little is known about Fv-I gene expression is undifferentiated mouse embryonal carcinoma (EC) cell lines. Murine EC cell lines have been established from primary cell cultures of pluripotent teratocarcinomas. EC lines have the capacity to differentiate in vitro and have been proposed as models for normal cell differentiation. It has previously not been possible to demonstrate whether EC cells express Fv-1 restriction, because MuLV replication is blocked at the transcriptional level in these cells. Recently, however, several retrovirus vectors have been constructed which are expressed in EC cells. One of these, designated RSV linker1 (generously provided by Elwood Linney, Duke University), contains a neo resistance gene driven from a Rous sarcoma virus promoter/enhancer region. RSV linker-1 rescued by N-, B-, and NB-tropic MuLV was used to determine the Fv-1 phenotype of F9 and PCC4.azalR. These two clonal, undifferentiated EC lines were derived from the 129 mouse strain, which carries the Fv-I”’ allele. Our results indicate that F9 cells do express Fv-1; however, PCC4.azalR cells are relatively permissive for all viruses tested, suggesting that Fv-I is not expressed in this line (TABLE 1). We additionally determined the Fv-1 phenotype of cells derived by differentiation of PCC4.azalR cells by using the same G418-resistant colony assay, except with the MSV-DHFR-neo vector.* Retinoic acid (RA) is known to promote in vitro differentiation of PCC4.azalR into mesenchymal cell types.’ Subsequent 5-azacytidine ( 5 azaC) treatment further induces a preadipocyte phenotype in some cells, which differentiate into either brown or white fat cells when grown to confluence in the presence of insulin and dexamethasone. Three fibroblast cell lines (PCC4D2, PCC4D4, PCC4D7) and one epithelial cell line (Diff 5 ) derived by RA-induced differentiation of PCC4.azalR cells were examined for their Fv-I phenotype. The quantitative assay for G418 resistance indicated that titers of NB-tropic (Moloney MuLV), N-tropic (Gross passage A and RFM MuLV), B-tropic ( WN1802B) and amphotropic (4070A)

Variation of Long-Terminal-Repeat Size in Molecular Clones of the BALB/c Endogenous Ecotropic Murine Leukemia Virus

Publisher Summary This chapter discusses the analyses of molecular clones of an endogenous ecotropic virus of BALB/c mice, WN1802N, to identify possible changes that occur in the long terminal repeat (LTR) region. In these analyses, covalently-closed circular DNA intermediates were isolated from cell cultures 48 hours post-infection and cloned by constructing Hin dIII recombinants with Charon 9 bacteriophage. The DNA of 15 independent isolates was first analyzed by Hin dIII restriction and endonuclease digestion, agarose gel electrophoresis, and Southern blot hybridization. Seven of the clones (λWN7, λWN12, λWN26, λWN30, λWN36, and λWN41) have an insert size that corresponds approximately to the complete MuLV genome containing two LTRs. There are four different size LTRs in this population of clones. The most abundant size class is 520 bp and it is probable that this is the size of the endogenous provirus LTR. Seven of these are infectious. There are three isolates with a 600-bp LTR, one of which is infectious, one with 570 bp, and one with 670 bp. Neither of these last two are infectious, but it is yet to be determined whether this is related to the LTR.