Strains Of Murine Leukemia Virus Research Articles

Nearest-neighbor interactions of the major RNA tumor virus glycoprotein on murine cell surfaces.

Formaldehyde-fixed Staphylococcus aureus and monospecific antiserum to gp70, the major envelope glycoprotein of murine leukemia virus, were used to immunoadsorb gp70 from Nonidet P40 extracts prepared from surface-radioiodinated murine cells. The labeled gp70 molecules in these cells were linked to a protein of approximately 15,000 daltons via native disulfide bonding. Prior treatment of cells with the reversible, bifunctional, crosslinking reagent dimethyl-3,3'-dithiobispropionimidate, followed by immunoadsorption and two-dimensional diagonal electrophoresis, revealed apparent homodimers and homotrimers of the 85,000-dalton complex. Identical treatment of purified type C RNA tumor virus from murine cells also revealed homodimeric and homotrimeric species, demonstrating similar self-associating tendencies of this glycoprotein in both intact virus and the plasma membrane of nonproducing murine cells. One cross-linked product consistently detected on the surfaces of murine cells was not present after crosslinking of a representative strain of murine leukemia virus.

Cells nonproductively transformed by Abelson murine leukemia virus express a high molecular weight polyprotein containing structural and nonstructural components.

Cell clones nonproductively transformed by the replication-defective Abelson strain of murine leukemia virus (AbLV) were analyzed for type C viral antigen expression by competition immunoassay. AbLV-transformed mink non-producer lines were found to express a 110,000- to 130,000-molecular weight polyprotein containing murine leukemia virus gag proteins p15 and p12 covalently linked to nonstructural AbLV-coded component(s) of around 80,000-100,000 molecular weight. This polyprotein lacked detectable antigenic cross-reactivity with other virion-coded gag gene proteins such as p30, p10, the viral reverse transcriptase (RNA-dependent DNA polymerase), or the major viral envelope glycoprotein, gp70. By analogy to earlier data on feline and avian sarcoma viruses, these results suggest that a portion of this polyprotein might represent the AbLV src gene product and that in translation it is initially linked in precursor form to gag structural proteins. Superinfection of mink cells nonproductively transformed by AbLV--with either a wild mouse amphotropic type C virus isolate, 4070-A, or with the endogenous cat virus, RD114--led to production of pseudotype virus containing high concentrations of the AbLV-coded precursor polyprotein.

Type-C retrovirus maturation and assembly: Post-translational cleavage of the gag-gene coded precursor polypeptide occurs at the cell membrane

Temperature-sensitive (ts) replication mutants of the Rauscher (R-) strain of murine leukemia virus (MuLV) defective in post-translational processing of the 65,000 molecular weight gag-gene coded precursor (Pr65) have been used to study the initial stages in type-C virus assembly. In is mutant infected cells grown at the nonpermissive but not the permissive temperature, dense material was found below the cell membrane by electron microscopy. Within 30 sec following shiftdown of is mutant infected cells to the permissive temperature (31°) a decrease in the amount of this dense material was observed, coincident with the initiation of virus assembly. Virus assembly and the release of mature virions were completed within 2–4 min. Immunofluorescent studies using monospecific antibodies to several highly purified viral structural proteins revealed a high concentration of these proteins below the cell membrane which also diminished concurrently with virus release. From the evidence presented we propose that the submembranous dense material observed by electron microscopy represents a pool of the polypeptide precursor Pr65, which is cleaved and then incorporated into the developing virus within seconds following shiftdown of the cells to the permissive temperature.

Structure of the genome of moloney murine leukemia virus: a terminally redundant sequence

The genome of the Moloney strain of murine leukemia virus (Mo-MuLV) has been analyzed by digestion with ribonuclease T1 and separation of the digestion products by two-dimensional gel electrophoresis. Thirty large oligonucleotides isolated from such a fingerprint have been characterized. One of these oligonucleotides (number 21) was found to be present in twice the molar yield of the rest. The 30 oligonucleotides were mapped on the genome by determining their yields in various size classes of 3′ terminal fragments of Mo-MuLV RNA. The physical map obtained in this way suggested that oligonucleotide 21 was present very near the 3′ end of the geome as well as in another location near or at the 5′ end. The genome structure suggested by these results was confirmed by analyzing oligonucleotides in Mo-MuLV RNA complementary to strong stop DNA, which is shown to be a copy of the 5′ terminal 134 nucleotides of the Mo-MuLV genome. Some of the oligonucleotides in the RNA protected from RNAase digestion by hybridization to this DNA, including oligonucleotide 21, were present near both the 3′ and 5′ ends. Comparison of these with the nucleotide sequence of strong stop DNA shows that there is a terminal redundancy of 49–60 nucleotides in the Mo-MuLV genome RNA.

Effect of interferon on exogenous murine leukemia virus infection

When interferon (IF)-treated NIH/3T3 cells were exogenously infected with the Moloney strain of murine leukemia virus (M-MLV), no viral progeny release was detected as long as IF remained in the medium. This was evidently an inhibition of the virus replication and not a consequence of interfering with the establishment of the infection since, when IF was removed either before infection or later after infection, virus release gradually approached the rate of untreated control after a temporary delay. Furthermore, a direct examination revealed that IF treatment had no effect on the formation of infectious centers. IF treatment led to a reduced viral RNA synthesis. A similar inhibition of viral RNA synthesis was observed when the potent protein synthesis inhibitor cycloheximide (CH) was added early after infection. However, when added at a late stage, the drug had no effect on viral RNA synthesis. It appears, therefore, that IF-induced inhibition of viral RNA synthesis is not a feedback consequence of an inhibition of a later step but, rather, an inhibition of some early step. This conclusion was substantiated by the finding that when IF was eliminated from pretreated cultures up to 10 hr before infection, progeny release was still affected.

Oncogenic and immunogenic potential of cloned HIX virus in mice and cats.

Many naturally occurring and laboratory derived strains of murine leukemia viruses (MuLV) have been isolated. These have initially been ecotropic in host range in that they grew in mouse cells and produced lymphoid neoplasms in mice or rats (Gross, 1951, Friend, 1957, Moloney, 1960). Other classes of murine retroviruses have also been isolated from mouse cells which share a great deal of nucleic acid homology and antigenic determinants with MuLV; these have been labeled according to host range into "xenotropic" if they were restricted from growth in mouse cells or "amphotropic" if they had a host range encompassing both mouse cells and cells of other species (Levy, 1973, Aaronson and Stephenson, 1973; Benveniste et al., 1974; Hartley et al., 1976). Xenotropic viruses are not known to be oncogenic. Recently several unusual variants of MuLV type viruses with amphotropic properties have been isolated which appear to be hybrids (recombinants) of MuLV's and murine xenotropic virus (MuX) (Fischinger et al., 1975, Hartley et al., 1977). One of the isolates, MCF, was isolated from AKR lymphomas and was implicated as possibly the oncogenic virus causative of the AKR mouse lymphoma (Hartley et al., 1977). Our isolate, named HIX (hybrid of IC-strain of Moloney MuLV and MuX), has been tested now for almost two years for oncogenicity in several species because of its amphotropic host range. It was of interest to determine whether HIX virus, cloned by three cycles of limiting dilution isolation, was able to cause neoplasms in various species. The present report shows the induction of lymphomas by HIX virus in mice as well as the reisolation of only HIX virus in pure culture from these tumors. Despite growth of HIX to high titers in cat embryo cells, HIX virus inoculated newborn kittens have not shown any signs of disease.

Immunocytochemical detection of a murine leukemia virus-related nuclear antigen in mouse oocytes and early embryos

The expression of murine leukemia virus (MuLV)-related antigens in oocytes and early embryos of Swiss mice was studied by the unlabeled antibody peroxidase-antiperoxidase method using an antiserum to the major core protein, p30, of AKR MuLV. This procedure resulted in an intense staining, at antibody dilutions up to 1:500, of the germinal vesicles of oocytes and the interphase nuclei of early embryos. Nuclear staining was restricted to a specific period of oocyte growth and embryo development. It developed gradually in oocytes having reached one third to one half the full-grown size and persisted until meiotic maturation. In early embryos, nuclear staining was present from the one-cell stage to the morula, but disappeared during transition from morula to blastocyst and was not seen in expanded blastocysts. Nuclear staining was abolished by absorption of the anti-p30 serum with detergent-disrupted virions of Gross(A), AKR, Kirsten and Moloney MuLV, and Kirsten MSV(MuLV), but not with the Friend and Rauscher strains of MuLV, the xenotropic BALB:virus 2, a mouse tropic and an amphotropic clone of wild mouse MuLV, or with FeLV and RSV. On the basis of these results, it is suggested that the nuclear antigen (termed germinal vesicle antigen) reacting with the anti-p30 serum is the product of a cellular gene having a normal function in early embryonic development, and that sequences related to this gene are incorporated into the genome of AKR-type MuLV.

Identification of mouse chromosomes required for murine leukemia virus replication

Abstract The replication patterns of five ecotropic and two amphotropic strains of murine leukemia virus (MuLV) were studied by infecting 41 Chinese hamster x mounse hybrid primary clones segregating mouse (Mus musculus) chromosomes. Ecotropic and amphotropic strains replicated in mouse and some hybrid cells, but not in hamster cells, indicating that replication of exogenous virus requires dominantly expressed mouse cellular genes. The patterns of replication of the five ecotropic strains in hybrid clones were similar; the patterns of replication of the two amphotropic strains were also similar. When compared to each other, however, the replication patterns of ecotropic and amphotropic viruses were dissimilar, indicating that these two classes of MuLV require different mouse chromosomes for replication. Chromosome and isozyme analyses assigned a gene, Rec-1 (replication of ecotropic virus), to mouse chromosome 5 that is necessary and may be sufficient for ecotropic virus replication. Because of preferential retention of mouse chromosomes 15 and 17 in the hybrid clones, however, the possibility that these chromosomes carry genes that are necessary but not sufficient for ecotropic virus replication cannot be excluded. Similarly, the data indicate that mouse chromosome 8 (or possibly 19) carried a gene we have designated Ram-1 (replication of amphotropic virus) which is necessary and may be sufficient for amphotropic virus replication. Because chromosomes 8 and 19 tended to segregate together and two of the three clones excluding 19 have chromosome reaggrangements, we cannot exclude 19 as being independent of amphotropic virus replication. In addition, because of preferential retention, chromosomes 7, 12, 15, 16 and 17 cannot be excluded as being necessary but not sufficient. Hybrid cell genetic studies confirm the assignment of the Fv-1 locus to chromosome 4 previously made by sexual genetics. In addition, our results demonstrate that hybrid cells which have segregated mouse chromosome 4 but have retained 5 become permissive for replication of both N and B tropic strains of MuLV.

Isolation of transforming murine leukemia viruses from mice with a high incidence of spontaneous lymphoma.

Murine leukemia viruses capable of malignant transformation of mink tissue culture cells have been isolated from an AKR thymoma cell line and from a spontaneous reticulum cell sarcoma in an NIH Swiss mouse partially congenic for the AKR ecotropic virus-inducing locus Akv-2. In contrast to the recently described mink cell focus-inducing strains of murine leukemia virus, at least one of the two transforming strains is replication defective. Nonproducer mink cells carrying the genome of the transforming virus of AKR origin have been isolated, and pseudotype transforming viruses generated.

Envelope glycoproteins of Rauscher murine leukemia virus: isolation and chemical characterization

The envelope glycoproteins (designated gp70 and gp45) of the Rauscher strain of murine leukemia virus were solubilized by osmotic shock and freeze-thawing in chaotropic solutions. The viral glycoproteins were then purified by phosphocellulose chromatography and gel permeation chromatography on Bio-Gel A-1.5m. Yields by this procedure were 6.2% for gp70 and 1.3% for gp45 on a protein input basis. The apparent molecular weights were respectively 67 500 and 47 500 with a polypeptide chain molecular weight of approximately 45 000 for both glycoproteins. Amino acid analysis showed a high degree of similarity for both components, with some differences subject to further evaluation. The total carbohydrate content was approximately 32% for gp70 and 6-9% for gp45. In keeping with the amino acid compositional similarity suggesting relationships, alanine was found to ba the amino-terminal amino acid of both glycoproteins, and cross-reactivity was demonstrated by immunologic tests. The data suggest that the chief difference between gp70 and gp45 lies in the carbohydrate content.

Characterization of Purely Ecotropic and Amphotropic Naturally Occurring Wild Mouse Leukemia Viruses

Two new strains of murine leukemia virus, one (strain 4996) purely ecotropic and the other (strain 1313) purely amphotropic, were isolated from spontaneous lymphomas in aged wild mice (Mus musculus). The 4996 virus is the first wild mouse field isolate which consists solely of ecotropic virus without the concomitant presence of amphotropic virus. The 1313 isolate is distinct in host range from seven other previously described wild mouse amphotropic isolates and is also the only murine leukemia virus shown to replicate in chicken cells.

Amphotropic host range of naturally occuring wild mouse leukemia viruses

Seven murine leukemia virus field isolates (uncloned) from wild mice (Musmusculus) of four widely separated areas in southern California show an unusually wide in vitro host range. They replicate well in human, feline, canine, guinea pig, rabbit, rat, and mouse cells, whereas bovine, hamster, and avian cells are resistant. Since this host range includes that of both mouse tropic (ecotropic) and xenotropic murine leukemia viruses, they are designated as "amphotropic". No purely xenotropic virus component is detectable in these field isolates. They may represent the "wild" or ancestral viruses from which the ecotropic and xenotrophic murine leukemia virus strains of laboratory mice have been derived.

Naturally occurring murine leukemia viruses in wild mice: characterization of a new "amphotropic" class.

A new class of murine leukemia viruses, isolated from wild Mus musculus trapped in California, is described. These viruses, designated "amphotropic," replicate in mouse, rabbit, mink, human, guinea pig, and rat cells, but not in hamster, quail, or duck cells. They show N-tropism for mouse cells, and do not trigger the XC cell response. They are distinct by interference and virus neutralization testing from the previously recognized mouse-tropic and xenotropic MuLV classes. Mouse-tropic viruses occuring along with three of the four amphotropic isolates were found to be distinguishable by virus neutralization from other mouse-tropic murine leukemia virus strains of laboratory mouse origin.

Characterization of the type and group specificities of the immune response in mice to murine leukemia viruses

Normal sera from a variety of strains of inbred mice have precipitating antibodies to murine type C viruses that are detected by radioimmune precipitation assays. The results demonostrate that this humoral immune response is primarily directed against the AKR strain of murine leukemia virus (MuLV) proteins gp71, gp43, and p15(E). These sera also react with Friend- or Rauscher-MuLV in radioimmune precipitation assays. This reaction is not due to a separate immune response, but rather is primarily a consequence of the cross-reactivity of antibodies to the AKR strain of MuLV p15(E) with the p15(E) of these viruses. These data, using autogenous immune sera, emphasize the serological differences of the virion glycoproteins and the serological similarity of the p15(E) virion component of the viruses. Furthermore, based on the serological reactivities to the glycoproteins, the results suggest that the AKR strain of MuLV is endogenous to and expressed in mice, but that the Friend-Moloney-Rauscher virus group is not.

Murine leukemia virus mutants with temperature-sensitive defects in precursor polypeptide cleavage

Among temperature-sensitive conditional lethal mutants of the Rauscher strain of murine leukemia virus (R-MuLV), several were previously shown to express viral antigens in the absence of complete virus release at the nonpermissive temperature. In the present study, analysis of the size distribution of viral polypeptides of molecular weight 30,000 and 12,000 daltons in extracts of cells infected with two temperature-sensitive mutants at the nonpermissive temperature revealed a marked accumulation of both antigens in a size region corresponding to 60,000–70,000 daltons. The precursor was partially purified and shown to contain the polypeptides of molecular weight 15,000 daltons, as well as 30,000 and 12,000 daltons, in relative concentrations similar to those found in purified virions.

Thermolabile reverse transcriptase of a mammalian leukemia virus mutant temperature sensitive in its replication and sarcoma virus helper functions.

Three temperature-sensitive mutants of the Rauscher strain of murine leukemia virus are defective in early post-penetration functions required both for leukemia virus infection and for initiation of transformation of cells by their pseudotypes of murine sarcoma virus. In the present study, the reverse transcriptase of one of these mutants (ts 29) is shown to be thermolabile compared with the enzymes of the wild-type virus and several other temperature-sensitive mutants. These findings provide evidence that the reverse transcriptase is required both for leukemia virus infection and for initition of transformation by the replication-defective murine sarcoma virus genome.

Assay in the mouse for delayed-type hypersensitivity to murine leukemia virus.

Delayed-type hypersensitivity (DTH), assayed by footpad swelling, was induced in 6- to 8-week-old BALB/cCr mice immunized with formalin-inactivated, sucrose-banded murine type-C viruses. The DTH response was inducible with as little as 11.25 mug sensitizing antigen, was greatest after sc sensitization as compared to im and ip sensitization, and was optimally elicited with a 7-day challenge. A statistical evaluation of the DTH assay revealed that the test was consistently reproducible and limited only by biologic variability of the mouse and the standardization of the antigen preparation. The DTH response was specific for type-C virus subtypes because it could distinguish the Rauscher strain of murine leukemia virus from AKR leukemia virus when the challenge antigen was extracted with Tween 80-ether. Immunized mice that gave DTH responses were resistant to challenge with exogenous, live murine leukemia viruses.

Temperature-sensitive mutants of murine leukemia virus. V. Impaired leukemogenic activity in vivo.

The in vivo biologic activities of a clonal isolate of the Rauscher strain of murine leukemia virus and several conditional lethal mutants with impaired replicative functions at the non-permissive temperature were investigated. The parental virus induced a rapidly developing lymphoid leukemia in a high percentage of NIH Swiss mice inoculated as newborns. Mice inoculated in parallel with temperature-sensitive mutant viruses showed no evidence of disease. The implications of these findings with regard to the requirements for type-C virus replication to permit oncogenic transformation of lymphoid cells in vivo are discussed.

Distribution of murine type B and type C viral nucleic acid sequences in template active and template inactive chromatin

Template active chromatin and template inactive chromatin have been fractionated from mouse cells infected with the Moloney strain of murine leukemia virus. In vivo the cells produce abundant RNA homologous to Moloney leukemia virus, but do not produce either globin mRNA or RNA homologous to type B mouse mammary tumor virus. The DNA extracted from the template active chromatin or template inactive chromatin contained equal amounts of sequences homologous to Moloney type C virus, to type B virus, or to globin mRNA. The results are discussed with regard to the in vivo structure of chromatin and the difficulties in fractionating chromatin in vitro.

Nucleic acid homology of murine type-C viral genes.

The nucleic acid sequence homology between various murine, endogenous type-C viruses (three host range classes of BALB/c virus, the AT-124 virus, and the CCL 52 virus) and two laboratory strains of murine leukemia virus (Rauscher and Kirsten) was determined by DNA:RNA hybridization. The viral sequences exhibit varying degrees of partial homology. DNA:DNA hybridizations were performed between [(3)H]DNA probes prepared from N- and X-tropic BALB/c endogenous viruses and cellular DNAs from BALB/c, NIH Swiss, and AKR inbred mouse strains as well as from California feral mice and the Asian mouse subspecies Mus musculus molossinus and M. musculus castaneus. All of these strains of mice are shown to possess multiple (six to seven per haploid genome), partially related copies of type-C virogenes in their DNAs. Thermal melting profiles of the DNA:RNA and DNA:DNA hybrids suggest that the partial homology of the viral nucleic acid sequences is the result of base alterations throughout the viral genomes, rather than the loss of discrete segments of viral sequences.