Moloney murine leukemia virus-induced tumors: recombinant proviruses in active chromatin regions.

The DNase I sensitivity of chromosomal DNA regions carrying integrated proviral genomes of Moloney (M-MuLV) and AKR Murine Leukemia Virus (AKR-MuLV), and the cellular homologue of the mos-gene (c-mos) of Moloney Sarcoma Virus (MSV) were studied in tumor tissues of leukemic mice. The genetically transmitted sequences of M-MuLV, AKR-MuLV, and the c-mos gene are all in DNase I resistant chromatin conformations in M-MuLV-induced tumors. Each M-MuLV-induced tumor contained at least one somatically acquired integrated recombinant MuLV genome that displayed two main characteristic features of active chromatin: a) a configuration hypersensitive to DNase I, and b) extensive hypomethylation. DNase I hypersensitive sites were mapped at the junction of cellular sequences and the 5'-viral large terminal repeat (LTR). Expression of a recombinant MuLV seems therefore to be a necessary feature to maintain the transformed state.     

Integration of spleen focus-forming virus proviruses in Friend tumor cells.

Using the Southern blot procedure, we studied the presumed spleen focus-forming virus (SFFV) provirus integration sites in the genome of the premalignant and the malignant cells isolated during the course of Friend erythroleukemia. Two restriction endonucleases, PstI and BamHI, discriminated the presumed integrated SFFV proviruses from the endogenous xenotropic-mink cell focus-forming viral sequences. No SFFV integration sites were detectable in the premalignant cells, suggesting a random integration of SFFV proviruses in the genome of these cells. In contrast, SFFV proviruses were detected at a single or very few sites in the genome of all malignant cells we analyzed. These results indicate that the event leading to the malignant transformation in acute Friend leukemia is clonal. In two of the six animals examined, tumors cells isolated from the spleens and the livers of individual mice showed identical SFFV integration patterns. This last result suggests that in some cases different tumors in a same leukemic animal could be derived from a unique clonal event.     

Genetic identification of endogenous polytropic proviruses by using recombinant inbred mice.

Forty-seven endogenous polytropic murine viruses (Pmv) were identified by examination of proviral-cellular DNA junction fragment segregation in recombinant inbred (RI) mice. Most Pmv loci were found in more than one of the seven RI progenitor strains analyzed, but only four were present in all strains. Chromosomal assignments for 41 Pmv loci were determined by comparing their RI strain distribution patterns with those of known genetic markers. Pmv loci were found dispersed throughout the genome, with chromosomes 1, 3, 4, 5, 7, 11, 12, 15, and 16 each carrying three or more proviruses. Linkage analysis in the AKXD RI set suggested that the gene encoding mink cell focus-forming virus resistance (Rcmfr) of DBA/2J mice is probably not a Pmv provirus. It was also deduced that no single, AKR/J-specific Pmv provirus is required as an env gene donor for thymomagenic mink cell focus-forming viruses. In addition, a Pmv provirus was very closely associated with the albino mutation on chromosome 7.     

Genome structure of Abelson murine leukemia virus variants: proviruses in fibroblasts and lymphoid cells.

We have prepared full-length DNA clones of the Abelson murine leukemia virus (A-MuLV) genome. A specific probe homologous to the central portion of the A-MuLV genome was prepared by nick translation of a subcloned restriction fraction from the cloned DNA. The probe was used to examine the genome structure of several A-MuLV variants. The conclusions are: (i) three viruses coding for Abelson-specific proteins of molecular weight 120,000, 100,000, and 90,000 had genomes indistinguishable in size, suggesting that the shorter proteins are the result of early translational termination; (ii) compared with the genome encoding the 120,000-dalton (120K) protein, a genome coding for a 160K protein was 0.8 kilobase larger in the A-MuLV-specific region; and (iii) a genome coding for a 92K protein had a 700-base pair deletion internal to the coding region. This mutant was transformation defective: its 92K protein lacked the protein kinase activity normally associated with the A-MuLV protein, and cells containing the virus were not morphologically transformed. In addition, we determined the number of A-MuLV proviruses in each of several transformed fibroblast and lymphoid cells prepared by infection in vitro. These experiments show that a single copy of the A-MuLV provirus is sufficient to transform both types of cells and that nonproducer cells generally have only one integrated provirus.     

Somatically acquired recombinant murine leukemia proviruses in thymic leukemias of AKR/J mice.

We have probed the structure and arrangement of murine leukemia virus genomes in eight spontaneous AKR thymic leukemias by Southern hybridization with one ecotropic pol and four ecotropic env probes. These probes revealed many (in 2 cases over 15) somatically acquired proviruses that had undergone complex patterns of recombination. The large majority were not deleted and were structurally analogous to the oncogenic mink cell focus-inducing murine leukemia viruses isolated from AKR tumors in that the amino-terminal p15E-coding region derived from ecotropic AKR murine leukemia virus sequences, whereas certain gp70-coding sequences were nonecotropic. Nevertheless, we observed a few proviruses which did not appear to be gp70 recombinants; however, these proviruses were in general clearly recombinant within the p15E-coding sequences. Although the proviral recombination patterns were quite variable, in general the large majority of recombinant proviruses within each tumor appeared structurally identical, indicating that they originate from a common parent. Each tumor contained a unique pattern of provirus integrations; densitometer tracings of the Southern hybridizations indicated that many of the integrated proviruses were present at one copy per cell, suggesting that the tumors derive from a single cell which contained multiple integrated copies of a unique recombinant virus structurally similar to the mink cell focus-inducing viruses.     

Isolation of recombinant DNA clones carrying complete integrated proviruses of Moloney murine leukemia virus

EcoRI DNA fragments from a Moloney murine leukemia virus (M-MuLV)-infected mouse fibroblast line (M-MuLV clone A9) were cloned in lambda phage Charon 4A cloning vector to derive clones containing integrated M-MuLV proviral DNA. A 10- to 16-megadalton class of EcoRI fragments was chosen for cloning, based on (i) its ability to induce XC-positive virus upon transfection of NIH/3T3 cells, and (ii) its content of a 0.8-megadalton viral KpnI fragment diagnostic for M-MuLV. Six recombinant DNA clones were isolated which contain a complete M-MuLV provirus, as judged by (i) restriction endonuclease mapping and (ii) the fact that all of the clones gave rise to XC-positive, NB-tropic virus upon DNA infection in NIH/3T3 cells. The sizes of the inserts were 12.0 (for three clones) or 12.5 megadaltons (for three clones). Restriction mapping indicated that these six clones represent five different M-MuLV proviral integrations into different cellular DNA sites.     

Pathogenesis of feline leukemia virus T17: contrasting fates of helper, v-myc, and v-tcr proviruses in secondary tumors.

A naturally occurring feline thymic lymphosarcoma (T17) provided the unique observation of a T-cell antigen receptor beta-chain gene (v-tcr) transduced by a retrovirus. The primary tumor contained three classes of feline leukemia virus (FeLV) provirus, which have now been characterized in more detail as (i) v-tcr-containing recombinant proviruses, (ii) v-myc-containing recombinant proviruses, and (iii) apparently full-length helper FeLV proviruses. The two transductions appear to have been independent events, with distinct recombinational junctions and no sequence overlap in the host-derived inserts. The T17 tumor cell line releases large numbers of FeLV particles of low infectivity; all three genomes are encapsidated, but passage of FeLV-T17 on feline fibroblast and lymphoma cells led to selective loss of the recombinant viruses. The oncogenic potential of the T17 virus complex was, therefore, tested by infection of neonatal cats with virus harvested directly from the primary T17 tumor cell line. A single inoculation of FeLV-T17 caused persistent low-grade infection culminating in thymic lymphosarcoma and acute thymic atrophy, which was accelerated by coinfection with the weakly pathogenic FeLV subgroup A (FeLV-A)/Glasgow-1 helper. Molecularly cloned FeLV-tcr virus (T-31) rescued for replication by a weakly pathogenic FeLV-A/Glasgow-1 helper virus was similarly tested in vivo and induced thymic atrophy and thymic lymphosarcomas. Most FeLV-T17-induced tumors manifested either v-myc or an activated c-myc allele and had undergone rearrangement of endogenous T-cell antigen receptor beta-chain genes, supporting the proposition that the oncogenic effects of c-myc linked to the FeLV long terminal repeat are targeted to a specific window in T-cell differentiation. However, neither the FeLV-T17-induced tumors nor the T-31 + FeLV-A-induced tumors contained clonally represented v-tcr sequences. Only one of the FeLV-T17-induced tumors contained detectable v-tcr proviruses, at a low copy number. While v-tcr does not have a readily transmissible oncogenic function, a more restricted role is not excluded, perhaps involving antigenic peptide-major histocompatibility complex recognition by the T-cell receptor complex. Such a function could be obscured by the genetic diversity of the outbred domestic cat host.     

Continuing germ line integration of AKV proviruses during the breeding of AKR mice and derivative recombinant inbred strains.

The gel electrophoresis-hybridization technique of Southern was used to analyze genetically transmitted proviruses coding for the AKV strain of murine leukemia virus. We were able to identify the restriction endonuclease EcoRI fragments containing two previously unidentified, genetically transmitted AKV proviruses of AKR mice. Comparison of different sublines of AKR mice revealed considerable heterogeneity in their complement of germ line proviruses. This heterogeneity provides evidence that the provirus complement of AKR mice is not stable. Rather, the number of genetically transmitted proviruses increases during inbreeding. Examination of a series of sublines of the C3H strain indicated that this amplification is dependent on viremia. We estimate that, in viremic strains of mice, one new provirus becomes fixed in the germ line every 15 to 30 years.     

Granulopoeisis and leukemogenesis: lessons from congenital neutropenia

Congenital neutropenia are extremely rare diseases, defined by a permanent or cyclic decrease of blood neutrophils. Molecular basis of several congenital neutropenia has been recently determined, involving gene coding for the neutrophil elastase gene (ELA2), GFI1, WAS protein and mitochondrial HAX1 protein. These mutations, dominant (ELA2, GFI1), X-linked (WAS) and autosomal recessive (HAX1), result in instability of the contents of the granules- particularly the neutrophil elastase- or in abnormalities of the cytoskeleton, and possibly, in an increased apoptosis. ELA2 mutations resulting both in profound and permanent neutropenia, and in cyclic--pseudo sinusoidal--neutropenia lead to consider that time pattern is very close in the two apparently distinct phenotypes. This observation suggests that temporal variations of neutrophils could be represented by non linear functions. Congenital neutropenia, specifically ELA2 mutated, are also characterized by a high rate of leukemia (about 15% at 20 years of age). Leukemia risk does not appear to be related to an oncogenic effect of ELA2 mutations, but much likely to the deepness of the neutropenia, and the intensity of G-CSF therapy.     

Proteolytic processing in development and leukemogenesis

There are now numerous examples in the hematopoietic system of genes that are critical for normal hematopoietic development, but when mutated, rearranged, or overexpressed, contribute to leukemogenesis. Two papers in this issue of Cell provide a fascinating twist on this paradigm, and suggest that proteolytic processing of certain of these genes plays an important role both in development and in leukemogenesis. These findings also suggest the possibility that proteases may be therapeutic targets in leukemia.     

Murine myeloid cell lines derived by in vitro infection with recombinant c-myb retroviruses express myb from rearranged vector proviruses.

To date, cellular transformation in vitro by the myb oncogene has been described for avian haemopoietic cells only. In order to exploit the well-characterized murine haemopoietic system to study transformation by myb, we have infected fetal liver cells with retroviral vectors carrying cDNAs that encode either complete or carboxy-terminally truncated c-myb proteins. We describe four cell lines which, despite our ability to efficiently infect haemopoietic target cells, were generated at low frequency. This was due, as least in part, to the requirement for a rearrangement within the vector that allowed expression of myb sequences. Three of the lines express a truncated myb protein while the fourth apparently expresses a normal c-myb protein, and thus constitutes an exception to the general association of truncation with transformation by myb. All four cell lines resemble immature cells of the myelomonocytic lineage and are dependent on colony-stimulating factors (CSFs) for their growth in vitro. One representative line could be converted to CSF-independence by infection with either Abelson murine leukaemia virus or a recombinant granulocyte-macrophage-CSF-encoding retrovirus; unlike the parental line, the resultant sublines were highly tumorigenic when injected into syngeneic mice.     

Microviridae goes temperate: microvirus-related proviruses reside in the genomes of Bacteroidetes

The Microviridae comprises icosahedral lytic viruses with circular single-stranded DNA genomes. The family is divided into two distinct groups based on genome characteristics and virion structure. Viruses infecting enterobacteria belong to the genus Microvirus, whereas those infecting obligate parasitic bacteria, such as Chlamydia, Spiroplasma and Bdellovibrio, are classified into a subfamily, the Gokushovirinae. Recent metagenomic studies suggest that members of the Microviridae might also play an important role in marine environments. In this study we present the identification and characterization of Microviridae-related prophages integrated in the genomes of species of the Bacteroidetes, a phylum not previously known to be associated with microviruses. Searches against metagenomic databases revealed the presence of highly similar sequences in the human gut. This is the first report indicating that viruses of the Microviridae lysogenize their hosts. Absence of associated integrase-coding genes and apparent recombination with dif-like sequences suggests that Bacteroidetes-associated microviruses are likely to rely on the cellular chromosome dimer resolution machinery. Phylogenetic analysis of the putative major capsid proteins places the identified proviruses into a group separate from the previously characterized microviruses and gokushoviruses, suggesting that the genetic diversity and host range of bacteriophages in the family Microviridae is wider than currently appreciated.     

The chromatin structure of Rous sarcoma proviruses is changed by factors that act in trans in cell hybrids.

In several lines of Rous sarcoma virus (RSV)-transformed rat cells the proviruses are in a configuration typical of active eukaryotic genes. They are sensitive to pancreatic DNase I, with sites hypersensitive to nuclease near the 5' end of the genome, they are close to the nuclear 'cage' and they show a low level of cytosine methylation in CpG doublets. In contrast, in phenotypically untransformed hybrids between these cells and uninfected rat or mouse cells, RSV inactivity is associated with hypermethylation of the provirus, reduced DNase I sensitivity (in two out of three examples) and, where examined, relative remoteness from the nuclear cage. These changes in proviral configuration, which occur rarely in spontaneous reversion of transformed cells, can thus be induced at high frequency and stability in cell hybrids by trans-acting influences of the uninfected parents.     

Disulfide structure of alfimeprase: a recombinant analog of fibrolase

The disulfide structure of alfimeprase, a recombinant analog of fibrolase, was experimentally determined by a combination of peptide mapping, Edman degradation, and mass spectrometry. The three disulfide bonds were determined to be Cys-116/196, Cys-156 /180, and Cys-158/163 with the residue number system of alfimeprase.