N-tropic Murine Leukemia Virus Research Articles

Restriction endonuclease mapping of unintegrated viral DNA of B- and N-tropic BALB/c murine leukemia virus.

Unintegrated linear and closed circular DNAs of B- and N-tropic endogenous BALB/c murine leukemia virus (MuLV) were extracted from newly infected mouse cells and cleaved with EcoRI, XhoI, PvuI, HindIII, SalI, XbaI, KpnI, SmaI, and PstI restriction endonucleases. The DNA fragments were separated by electrophoresis and analyzed by the Southern blot hybridization procedure. EcoRI did not cleave the two genomes. A physical map of 15 cleavage sites on B- and N-tropic genomes was constructed with the other restriction endonucleases. Identical cleavage sites of B- and N-tropic MuLV DNAs were found with all these enzymes. However, the N-tropic linear genome was found to lack about 75 base pairs at each end of the molecule. PstI, KpnI, and SmaI recognize a cleavage site at both ends of the linear molecules. And sequences derived from the 5' end of the RNA genome were found in the third left end of the linear DNA and at its extreme right-end terminus, suggesting the presence of redundant sequences. Two species of closed circular viral DNA were observed. The larger species has the same size as the linear molecule and appears to be a circularized form of linear DNA. The smaller species contains sequences common to both the linear and the larger circular viral DNA but seems to be deleted from sequences present at either one or both ends of the linear DNA. Therefore, the general structure of the linear and circular DNA species of these B- and N-tropic endogenous BALB/c MuLV appears analogous to the structure found with other retroviruses.

In vitro construction of a B-tropic virus by recombination: B-tropism is a cryptic phenotype of xenotropic murine retroviruses.

A B-tropic virus was isolated in vitro from the progeny of mouse cells doubly infected with N-tropic and xenotropic murine leukemia viruses. Biological and structural evidence is presented suggesting that the phenotypically silent structural marker for B-tropism, expressed by the xenotropic virus p30, was transferred to an N-ecotropic virus via recombination, thus resulting in the expression of a B-ecotropic murine leukemia virus.

Molecular cloning of infectious integrated murine leukemia virus DNA from infected mouse cells.

The lack of an endonuclease EcoRI site in the AKR murine leukemia virus (MuLV) DNA genome was utilized to molecularly clone, in Charon 4A lambda DNA, integrated infectious AKR MuLV DNA isolated from productively infected mouse cells. Three lambda-mouse recombinants (clones 614, 621, and 623) were selected by virtue of their reactivity with AKR MuLV [32P]cDNA. Clones 614 and 623 contained the complete AKR MuLV DNA flanked by nonviral cell sequences of which no more than 100 base pairs beyond the viral DNA appear to be shared. DNAs from both clones 614 and 623 were highly infectious for mouse cells and yielded N-tropic ecotropic MuLV; the specific infectivity of the DNA and the titer of the derived virus was more than 10-fold higher with 623. Clone 621 contained only some viral DNA and was not infectious under similar conditions.

A murine teratocarcinoma stem cell line carries suppressed oncogenic virus genomes.

Murine teratocarcinoma stem cells are nonpermissive for productive infection by a variety of DNA (polyoma and SV40 virus) and RNA (murine leukemia and sarcoma virus) tumor viruses whereas differentiated murine cells derived from the stem cells are permissive for productive (or abortive in the case of SV40) infection by these same viruses. The block to productive infection by these oncogenic viruses is at a postpenetration step in the replication cycle of these viruses but the precise level of the block has not been established for any of these viruses. In this report we describe teratocarcinoma-derived stem and differentiated cell lines which should be especially useful in determining the level of the block to replication of ecotropic murine leukemia virus in murine teratocarcinoma stem cells. The stem cell line, OTT6050AF1 BrdU, which is completely nonpermissive to productive infection by Moloney murine leukemia virus and consists of 97% pluripotent stem cells, contains DNA copies of an RNA tumor virus which is indistinguishable from the N-tropic murine leukemia virus of AKR mice. The stem cells are negative for expression of viral reverse transcriptase, p30 and gp69/71 and no virus is found by XC plaque assay or other biological tests. Differentiated cells established from the same teratocarcinoma tumor are 100% positive for viral gp69/71, p30, and produce large amounts of reverse transcriptase activity and N-tropic virus as detected by biological assay. The virus isolated from the differentiated cells is closely related, if not identical to AKR N-tropic virus by nucleic acid hybridization studies and is thus not an endogenous virus of the 129 strain of mice. The teratocarcinoma tumor from which the cell lines were established had been carried in 129 mice and perhaps at some time in the mouse passage history the tumors were infected (nonproductively) with the N-tropic virus. Regardless of the origin of this viral DNA, the OTT6050A derived stem and differentiated cell lines should be extremely useful in defining in stem cells the step at which ecotropic murine leukemia virus replication is blocked.

Induction of B-Tropic and N-Tropic Murine Leukemia Virus From B10.BR/SgLi Mouse Embryo Cell Lines by 5-lodo-2′-deoxyuridine<xref ref-type="fn" rid="fn2">2</xref>

In contrast to the original B10.BR/SgSn congenic mouse strain, adult mice of the B10.BR/SgLi subline showed a high level of expression of B-tropic ecotropic murine leukemia virus (MuLV). Both B-tropic and N-tropic ecotropic MuLV could be included in cultures of virus-free cell lines derived from embryos of B10.BR/SgLi mice. Both various were also inducible from each of several clonal cell lines and from cultures of F1 embryos of matings of B10.BR/SgLi males with females of strains NFS/N and A/J, which are negative for B-tropic virus. Thus the information for B-tropic MuLV as well as that for N-tropic MuLV was transmitted as a genetic element in the B10.BR/SgLi subline.

Isolation and characterization of a mouse cell line containing a defective Moloney murine leukemia virus genome

A culture of mouse cells containing a 1,000-nucleotide deletion mutant of Moloney murine leukemia virus has been isolated. The deletion did not affect the size or function of the 21S mRNA that encodes the env gene products. Both the deleted RNA and the 21S mRNA were recovered in polyribosomes. Cells containing the deleted virus made no detectable Pr180gag-pol. Pr65gag synthesis with also absent, but a 45,000-molecular-weight gag gene product was found that might be encoded by the deleted genome. Biosynthesis of Pr80env proceeded normally in these cells; the intracellular precursor was cleaved and migrated to the cell surface as gp70. The cells could not be superinfected by homologous Moloney murine leukemia virus presumably because of surface restriction due to the gp70. Although the cells express the Moloney murine leukemia virus gp70 on their surface, they will not make pseudotypes after infection with vesicular stomatitis virus implying that Pr65gag may play a critical role in pseudotype formation. Induction of endogenous virus expression in the cells carrying the deletion mutant generated an N-tropic murine leukemia virus that can fuse XC cells. This may represent a recombinant between the deletion mutant and an endogenous virus.

Fv-1 restriction of xenotropic and amphotropic murine leukemia virus genomes phenotypically mixed with ecotropic virus

Xenotropic murine leukemia virus (MuLV) genomes rendered capable of infecting mouse cells by phenotypic mixing with an ecotropic MuLV exhibited the Fv-1 restriction pattern of that ecotropic virus. Like Fv-1-restricted ecotropic viruses, xenotropic genomes phenotypically mixed with N-tropic ecotropic MuLV showed two-hit dose-response kinetics when titrated on restrictive Fv-1 b cells and the kinetics were converted to one-hit by the addition of N-tropic ecotropic virus. When SC-1 cells chronically infected with N-tropic amphotropic MuLV were superinfected with B-tropic ecotropic virus, each genome tended to maintain its homologous tropism, but with intermediate N:B infectivity ratios, and these ratios changed between the acute and chronic phases of the infection. In clonal lines chronically producing both viruses, the relative N- or B-tropism of the two genomes tended to vary in the same direction, and this correlated with which genome was produced in greater abundance. It is concluded that there is some recognition of amphotropic and ecotropic genomes by their homologous Fv-1 tropism determinants, and that the tropism of the virions in the mixed harvest is determined by this recognition factor and by the relative abundance of the two types of determinant.

Abrogation of Fv-1b restriction with murine leukemia viruses inactivated by heat or by gamma irradiation.

Fv-1b restriction in BALB/3T3 cells is temporarily abrogated following infection with N-tropic murine leukemia virus. The mechanism of this phenomenon was investigated by comparing the inactivation rates for viral infectivity and for the ability of the same virus to abrogate Fv-1 restriction. Inactivation of the abrogating ability of N-tropic murine leukemia virus following graduated doses of gamma radiation proceeded at half the rate of that for viral infectivity. This result indicates that viral RNA must function in abrogating Fv-1b restriction but that only a portion of the viral genome is required. The inactivation kinetics of N-tropic murine leukemia virus were also determined following incubation of virus at 43 degrees C. Abrogating ability of N-tropic murine leukemia virus was found to be about six times as stable under these conditions as was viral infectivity. Interestingly, virion-associated reverse transcriptase activity was inactivated at the same rate as was viral infectivity, indicating that this enzyme may not need to function during abrogation. Virus heated at 43 degrees C was used to study the kinetics of the abrogation phenomenon itself. Abrogation was shown to be transient, requiring 6 to 9 h after virus infection to become maximally effective and beginning to disappear after about 18 h. The data reported here confirm the idea that abrogation of Fv-1 restriction can be separated experimentally from virus replication, and they raise the possibility that a separate biochemical pathway exists for incoming viral RNA in Fv-1 restrictive cells.

Transfection of Fv-1 permissive and restrictive mouse cells with integrated DNA of murine leukemia viruses.

Whole-cell DNA preparations isolated from SC-1 cells chronically infected with N- or B-tropic murine leukemia viruses (MuLV) were tested for infectious activity in an Fv-1n (NIH-3T3) and two Fv-Ib (C57BL/6 and SV-A31) cell cultures. Efficiency of transfection for all DNAs was better in the NIH-3T3 cells than in C57BL/6 or SV-A31 cells; and an [N-tropic MuLV]SC-1 cell DNA preparation was slightly more infectious than a [B-tropic MuLV]SC-1 cell DNA preparation in all three cell cultures, regardless of their Fv-1 genotypes. Progeny viruses from the transfection showed N- or B-tropism corresponding to that of the parent viruses produced by the infected SC-1 cells that were used for the DNA preparation. DNA dose-response studies in NIH-3T3 cells revealed a one-hit mechanism for both the [B-tropic MuLV]SC-1 cell DNA and the [N-tropic MuLV]SC-1 cell DNA preparation. These results demonstrate that, in contrast to virion infection, transfection of N- and B-tropic MuLV with DNA preparations from chronically infected cells is not affected by the Fv-1 gene.

Genetic studies of the Fv-1 locus of mice: linkage with Gpd-1 in recombinant inbred lines

Multiple recombinant inbred lines, derived from crosses between strains permissive to N-tropic murine leukemia viruses (Fv-1n) and strains permissive to B-tropic murine leukemia viruses (Fv-1b), have been characterized as to Fv-1 genotype and other chromosome 4 markers, including the closely linked hexose-6-phosphate dehydrogenase isozyme locus (Gpd-1). Only one recombinant between Fv-1 and Gpd-1 was found among 45 lines tested. On this basis, the distance between Fv-1 and Gpd-1 is estimated to be 0.6 centimorgans. None of the lines was either resistant or susceptible to both N- and B-tropic viruses. Nineteen other inbred strains, previously untested, were characterized as either Fv-1n or Fv-1b.

Expression of endogenous murine leukemia viruses during the course of a protracted immunological disorder.

Mice of the low leukemia (BALB/cJ x A/J)F1 hybrid (CAF1) strain express B-and N-tropic infectious murine leukemia virus (MuLV) after the age of 6 mo. Initation of a protracted immunological disorder, the graft-versus-host reaction (GVHR), at 7 wk of age, accelerates the induction of both these mouse-tropic endogenous viruses, and preferentially enhances the replication of B-tropic MuLV. The earlier appearance of B-tropic MuLV in a greater proportion of mice and in higher titer is thought to be casually related to the eventual development of lymphoreticular tumors in the GVHR mice, since previous studies have shown that these same tumors can be reproduced by inoculating syngeneic recipients with serially passaged GVHR extracts containing B-tropic MuLV.

A new class of murine leukemia virus associated with development of spontaneous lymphomas.

A new type of murine leukemia virus has been detected in thymuses of leukemic and late preleukemic AKR mice, in lymphomas developing in NIH Swiss mice carrying the AKR ectopic virus-inducing loci Akv-I or Akv-2, and in the thymus of a preleukemic C58 mouse. The viruses induce focal areas of morphologic alteration in a mink lung cell line and are tentatively referred to as "mink cell focus-inducing" (MCF) strains. They have the host range of both xenotropic and N-tropic ecotropic murine leukemia viruses, are neutralized by antisera to both ecotropic and xenotropic viruses, and are interfered with by both viruses. They may represent a particular type of genetic recombinant which emerges during the preleukemic period in high-ecotropic-virus mouse strains, and they may play a significant role in the etiology of spontaneous lymphomas.

N-Tropic variants obtained after co-infection with N- and B-tropic murine leukemia viruses

Sc-1 cells co-infected with small XC plaque-forming N-tropic and large XC plaque-forming B-tropic murine leukemia viruses produced, in addition to parental types, progeny with the phenotype, large XC plaque morphology, and N-tropism. This phenotype remained stable through end point titration and plaque purification on NIH/3T3 cells and growth on BALB/3T3 cells. These N-tropic viruses (XLP-N virus) grow to unusually high titer and make very large XC plaques.

Growth of pseudotypes of vesicular stomatitis virus with N-tropic murine leukemia virus coats in cells resistant to N-tropic viruses.

Formation of pseudotypes between murine RNA tumor viruses and vesicular stomatitis virus (VSV) has been confirmed. Pseudotypes of VSV genomes coated by the surface envelope from an N-tropic tumor virus grew equally well in cells homozygous for either the Fv-1(n) or Fv-1(b) alleles. Therefore, the product of the Fv-1 locus, which restricts growth of murine RNA tumor viruses, must act on an intracellular aspect of tumor virus replication, a step after attachment and penetration.