Spontaneous germ line virus infection and retroviral insertional mutagenesis in eighteen transgenic Srev lines of mice.

SWR/J-RF/J hybrid mice spontaneously acquire new germ line ecotropic proviruses at high frequency. In the studies described here, we used these hybrids to produce 18 transgenic mouse lines, each carrying a single newly acquired Srev locus (SWR/J-RF/J ecotropic proviral locus). All of the newly acquired proviruses identified in mosaic founder SWR/J-RF/J mice that could be transmitted through the germ line were also present in somatic tissues, demonstrating that viral integration occurred before the germ line was set aside from the somatic lineages. Quantitative analysis of proviral DNA copy numbers in somatic and germinal tissues of mosaic founder parents combined with structural analysis of Srev loci indicated that these proviruses are acquired after multiple rounds of somatic viral reinfection and that most of these viral integration events occurred after DNA replication in the zygote and before DNA replication in the four-cell embryo. The frequency of provirus acquisition in Srev lines that expressed the infectious ecotropic virus was similar to that in SWR.RF mice carrying Emv-16 and Emv-17, suggesting that the chromosomal integration site of the parental locus is not an important determinant for high-frequency provirus acquisition. The frequency of recessive lethal mutations induced by spontaneous viral integration was 5%, which was similar to that induced by preimplantation embryo infection. This approach represents a simple and viable strategy for inducing and studying mutations that affect mammalian development.     

Genetic background affects integration frequency of ecotropic proviral sequences into the mouse germ line.

Germ line acquisition of ecotropic proviruses occurs at a high frequency in the progeny of SWR/J-RF/J hybrid mice carrying two genetically linked RF/J ecotropic proviral loci, Emv-16 and Emv-17 (N. A. Jenkins and N. G. Copeland, Cell 43:811-819, 1985). To determine if genetic background affects proviral integration frequency, I analyzed a series of crosses in which the two RF/J proviral loci were transferred onto different provirus-negative background strains. Unlike SWR/J-RF/J hybrid progeny, few CBA/CaJ-RF/J hybrid mice were identified that carried new germ line proviral loci. These results indicate that genetic factors other than the linked RF/J proviral loci contribute to the increased frequency of germ line provirus integration seen in the SWR/J-RF/J hybrids. The frequency of proviral acquisition appeared to increase when females carrying Emv-16, Emv-17, and at least one new proviral locus were further backcrossed, suggesting that integration frequency can be increased by genetic manipulation. The breeding data are consistent with the hypothesis that virus from the mother infects the egg or the early embryo. Analysis of the transmission frequency and cosegregation patterns of new proviral loci indicated that viral integration occurs after the first round of DNA replication and before the germ line is set aside during embryogenesis, with a majority of viral integrations occurring at the two-cell stage of development, and independent viral integrations can occur in the same or in different cells of the embryo.     

Integration of New Endogenous Mouse Mammary Tumor Virus Proviral DNA at Common Sites in the DNA of Mammary Tumors of C3Hf Mice and Hypomethylation of the Endogenous Mouse Mammary Tumor Virus Proviral DNA in C3Hf Mammary Tumors and Spleens

To understand the molecular mechanisms by which the endogenous murine mammary tumor virus (MuMTV) proviruses are expressed and produce late-occurring mammary tumors in C3Hf mice, we analyzed, by the use of restriction enzymes and the Southern transfer procedure, genomic DNA from normal organs of mammary tumor-bearing and tumor-free mice and from 12 late-occurring C3Hf mammary tumors. We found, by using the restriction enzymes EcoRI and HindIII, that in addition to the preexisting endogenous MuMTV proviruses, new MuMTV-specific proviral DNA was integrated into new sites in the host genome in all 12 of the tumors that we examined. PstI digests of C3Hf tumor DNA revealed that the new proviral DNA found in C3Hf tumors was of endogenous origin. Moreover, the respective sizes of at least one of the new DNA fragments generated by EcoRI or HindIII digestion were the same in at least 50% of the C3Hf tumors analyzed, suggesting that the integration site of this new proviral DNA could be at the same location in the host genome of these tumors. Our results may imply that mammary tumorigenesis in C3Hf mice results from activation of cellular oncogenes by an MuMTV proviral DNA promoter. Specific hypomethylation of MuMTV proviral DNA was detected in the mammary tumors and spleens of C3Hf tumor-bearing mice. Our results indicated that most, if not all, of the hypomethylated MuMTV proviral DNA sequences were derived from the endogenous MuMTV provirus located at the MTV-1 locus, a locus responsible for the production of MuMTV antigens and increased incidence of mammary carcinoma in C3Hf mice. In spleens of non-tumor-bearing mice of ages 3, 6, 9, and 12 months, there was progressive hypomethylation of proviral DNA with increasing age, suggesting a possible correlation between demethylation of MuMTV proviral DNA in the spleens of C3Hf mice and the expression of endogenous MuMTV.     

Retroviruses as probes for mammalian development: allocation of cells to the somatic and germ cell lineages

Preimplantation mouse embryos were infected with a recombinant retrovirus, which serves as a genetic marker for the progeny of an infected blastomere. Quantitative analysis of proviruses carried in mosaic animals indicated that the molarity of individual proviral bands was equal in almost all tissues. The cells that give rise to the embryo proper must therefore intermingle extensively before final tissue allocation to ensure equal contribution of founder cells to all somatic tissues. The distribution of molarities of individual proviruses suggested that somatic lineages are derived from at most eight founder cells. About half of the proviruses were present in the germ line and the somatic tissues of mosaic animals, while the remaining proviruses were found either in the germ line or in the somatic tissues, but not in both. Our results suggest that at least three cells form the germ line and are set aside prior to somatic tissue allocation.     

The endogenous ecotropic murine retroviruses Emv-16 and Emv-17 are both capable of producing new proviral insertions in the mouse genome.

New germ line proviral insertions are acquired at a high frequency by the progeny of SWR/J-RF/J hybrid female mice that carry the endogenous ecotropic murine leukemia proviruses Emv-16 and Emv-17. The tight linkage of these RF/J strain proviral loci has prevented genetic segregation of the retroviral genomes. Hence, it is not known whether both of these proviruses are capable of giving rise to new proviral insertions. We have molecularly cloned Emv-16 and Emv-17 and have characterized them in vitro and in vivo. Restriction enzyme analysis of the recombinant clones revealed that the proviral genomes are very similar to each other and closely resemble the wild-type AKR virus. A comparison of the flanking cellular DNA suggests that the Emv-16 and Emv-17 loci did not arise by simple duplication of a viral insertion site within the RF/J genome but most likely are independent integration events. Both proviruses produce infectious virus when transfected into NIH 3T3 cells, indicating that they are nondefective retroviruses. Exogenous infection of SWR/J mice with either Emv-16 or Emv-17 leads to viremia in the host animals, and in both cases, progeny of viremic females acquire new proviral insertions. The ability of these retroviruses to generate novel retroviral integration sites in the mouse genome provides a simple method for inducing insertional mutations in mice.     

Multiple integrations of human foamy virus in persistently infected human erythroleukemia cells

Foamy viruses are complex retroviruses whose replication strategy resembles that of conventional retroviruses. However, foamy virus replication also resembles that of hepadnaviruses in many respects. Because hepadnaviruses replicate in an integrase-independent manner, we were interested in investigating the characteristics of human foamy virus (HFV) integration. We have shown that HFV requires a functional integrase protein for infectivity. Our analyses have revealed that in single-cell clones derived from HFV-infected erythroleukemia-derived cells (H92), there were up to 20 proviral copies per host cell genome as determined by Southern blot and fluorescent in situ hybridization analysis. Use of specific probes has also shown that a majority of the proviruses contain the complete tas gene, which encodes the viral transactivator, and are not derived from Deltatas cDNAs, which have been shown to arise rapidly in infected cells. To demonstrate that the multiple proviral sequences are due to integration instead of recombination, we have sequenced the junctions between the proviral sequences and the host genome and found that the proviruses have authentic long terminal repeat ends and that each integration is at a different chromosomal site. A virus lacking the Gag nuclear localization signal accumulates fewer proviruses, suggesting that nuclear translocation is important for high proviral load. Since persistently infected H92 clones are not resistant to superinfection, the relative importance of an intracellular versus extracellular mechanism in proviral acquisition has yet to be determined.     

Methylation state and DNase I sensitivity of chromatin containing Moloney murine leukemia virus DNA in exogenously infected mouse cells

The nature of Moloney murine leukemia virus (M-MuLV)-specific proviral DNA in exogenously infected mouse cells was studied. M-MuLV clone A9 cells, NIH-3T3 fibroblasts productively infected with M-MuLV, were used. These cells contain 10 to 15 copies of M-MuLV proviral DNA. The state of methylation of M-MuLV proviral DNA was examined by cleaving A9 cell DNA with restriction endonucleases which have the dinucleotide CpG in their cleavage sequences. Analysis with such enzymes, which recognized nine different sites in M-MuLV DNA, indicated that most if not all of the M-MuLV proviruses in A9 cells were completely unmethylated. An individual proviral integration was examined, using as probe adjacent single-copy cellular sequences. These sequences were obtained from a lambda phage recombinant clone containing an M-MuLV provirus from the A9 cells. This individual integration also showed no detectable methylation. In contrast, endogenous MuLV-related sequences present in NIH-3T3 cells before infection were largely methylated. The configuration chromatin containing M-MuLV proviruses was also investigated by digesting A9 nuclei with DNase I, followed by restriction analysis of the remaining DNA. Endogenous MuLV-related DNA was in chromatin relatively resistant to DNase I digestion, whereas the majority of M-MuLV-specific proviruses were in domains of intermediate DNase I sensitivity. Two proviral copies hypersensitive to DNase I digestion were identified. Analogy to the DNase I sensitivity of expressed and nonexpressed globin genes suggested that the proviral copies containing DNase I-hypersensitive sites were transcribed.     

HIV-1 Virion Production from Single Inducible Proviruses following T-Cell Activation Ex Vivo

Quantifying induced virion production from single proviruses is important for assessing the effects of HIV-1 latency reversal agents. Limiting dilution ex vivo cultures of resting CD4⁺ T cells from 14 HIV-positive volunteers revealed that virion production after T-cell activation from individual proviruses varies by 10,000-fold to 100,000-fold. High-producing proviruses were associated with increases in cell-associated HIV-1 DNA levels, suggesting that reactivated proviruses proliferate. Single-cell analyses are needed to investigate differences in proviral expansion and virus production following latency reversal.     

A Linkage Map of Endogenous Murine Leukemia Proviruses

Thirty endogenous proviruses belonging to the modified polytropic (Mpmv) class of murine leukemia virus (MLV) were identified by proviral-cellular DNA junction fragment segregation in several sets of recombinant inbred mice. Twenty-six Mpmv loci were mapped to chromosomal regions by matching proviral strain distribution patterns to those of previously assigned genes. Like other endogenous nonecotropic MLVs, Mpmv loci were present on several chromosomes in all strains examined. We pooled recombinant inbred strain linkage data from 110 MLV loci and selected marker genes in order to construct a chromosomal linkage map. Every mouse chromosome was found to harbor at least one proviral insertion, and several regions contained multiple integrations. However, the overall distribution of the 110 mapped proviruses did not deviate significantly from a random distribution. Because of their polymorphism in inbred strains of mice, and the ability to score as many as 57 proviruses per strain using only three hybridization probes, the nonecotropic MLVs mapped in common strains of mice offer a significant advantage over older methods (e.g., biochemical or individual restriction fragment polymorphisms) as genetic markers. These endogenous insertion elements should also be useful for assessing strain purity, and for studying the relatedness of common and not-so-common inbred strains.     

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.     

Adeno-associated virus general transduction vectors: analysis of proviral structures.

We used two kinds of adeno-associated virus (AAV) vectors to transduce the neomycin resistance gene into human cells. The first of these (dl52-91) retains the AAV rep genes; the second (dl3-94) retains only the AAV terminal repeats and the AAV polyadenylation signal (428 base pairs). Both vectors could be packaged into AAV virions and produced proviral structures that were essentially the same. Thus, the AAV sequences that are required in cis for packaging (pac), integration (int), rescue (res), and replication (ori) of viral DNA are located within a 284-base-pair sequence that includes the terminal repeat. Most of the G418r cell lines (73%) contained proviruses which could be rescued (Res+) when the cells were superinfected with the appropriate helper viruses. Some produced high yields of viral DNA; other rescued at a 50-fold lower level. Most of the lines that were Res+ (79%) contained a tandem repeat of the AAV genome (2 to 20 copies) which was integrated randomly with respect to cellular DNA. Junctions between two consecutive AAV copies in a tandem array contained either one or two copies of the AAV terminal palindrome. Junctions between AAV and cellular sequences occurred predominantly at or within the AAV terminal repeat, but in some cases at internal AAV sequences. Two lines were seen that contained free episomal copies of AAV DNA. Res+ clones contained deleted proviruses or tandem repeats of a deleted genome. Occasionally, flanking cellular DNA was also amplified. There was no superinfection inhibition of AAV DNA integration. Our results suggest that AAV sequences are amplified by DNA replication either before or after integration and that the mechanism of replication is different from the one used during AAV lytic infections. In addition, we have described a new AAV general transduction vector, dl3-94, which provides the maximum amount of room for insertion of foreign DNA and integrates at a high frequency (80%).     

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.     

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.     

Variation in the number of copies and in the genomic organization of ecotropic murine leukemia virus proviral sequences in sublines of AKR mice.

DNAs isolated from individual mice of four AKR sublines (AKR/J, AKR/N, AKR/Cum, and AKR/Boy) were examined by hybridization of electrophoretically separated restriction enzyme fragments to a 500-base pair, 32P-labeled probe specific for env sequences of ecotropic murine leukemia virus. Variation in the number of proviral DNA copies and in their genomic organization, as reflected by the location of restriction enzyme sites in flanking cellular sequences, was observed both between and within AKR sublines. Evidence is presented for the continual acquisition of new proviruses in the four sublines studied. The ecotropic proviral DNA copies present in the four AKR sublines can be related to their genealogy; each subline contains two or three copies of proviral DNA in common with other sublines and from one to six unique ecotropic proviruses. Overall, a new copy appears about every 12 generations of inbreeding. Some of the unique proviral DNA copies contain internal alterations, as reflected by restriction enzyme maps that differ from those of prototype ecotropic proviruses.     

The dispersion of defective endogenous murine retroviral elements suggests retrotransposition-mediated amplification.

The dispersion of four replication-defective endogenous proviruses, originally detected in 129 strain mice and shown to have extensive deletions of gag, pol, and env gene regions, was investigated in 13 inbred strains and substrains of mice. Using probes to sequences flanking the integration sites in 129 mice, unique genomic Eco RI fragments were assigned to each of the four endogenous proviral elements. Analyses revealed that certain of these proviral elements are present both in strains closely related to strain 129 (i.e., strains 101 and LP/J) and in more distantly related strains (i.e., strains BALB/cJ, A/J, and C3H/HeJ). In mouse strains lacking proviral integration at a particular locus, the size of the corresponding Eco RI genomic fragment and absence of a characteristic Kpn I site indicated the lack of a residual solitary long terminal repeat. Hybridization of oligonucleotide probes that distinguish the specific deletions present within these elements identified additional analogous proviral integrations at many different sites in all strains investigated. These data indicate that the diversification of these proviral elements found in inbred strains is generated by integration of new copies, rather than excision through homologous recombination. Moreover, the results are consistent with other endogenous retroviruses providing the trans-acting proteins necessary to package the defective viral RNA.     

Transgenic mouse model of kidney disease: insertional inactivation of ubiquitously expressed gene leads to nephrotic syndrome.

Transgenic mouse strains carrying proviruses were generated by exposing mouse embryos to a recombinant retrovirus. Animals carrying a single provirus were intercrossed to derive mice homozygous for a given proviral insertion. Adult mice homozygous for the Mpv17 integration developed nephrotic syndrome and chronic renal failure. Histologically, affected kidneys showed progressive glomerular sclerosis. Similar lesions are seen in patients with progressive renal function deterioration. A probe to DNA sequences flanking the provirus detected a 1.7 kb RNA ubiquitously expressed during embryogenesis and in adults with high levels in kidney, brain, and heart. This RNA was not detected in tissues of homozygous animals, suggesting that the provirus interferes with RNA expression. Sequence analysis of the cDNA revealed that the gene encodes a 176 amino acid peptide containing hydrophobic regions, suggesting membrane association of the putative protein. The Mpv17 mutant is a potentially useful experimental system for studying mechanisms leading to renal disorders in man.