Two species of type C viral core polyprotein on AKR mouse leukemia cells.

Two species of glycosylated type C viral core polyprotein were identified on the surface of AKR spontaneous leukemia cells. One of these cell surface polyproteins was shown by immunoprecipitation to have antigenic determinants of murine leukemia virus p30, p15, p12, and p10; the other had murine leukemia virus p30, p15, and p12, but not p10, determinants. Both species were also expressed on thymocytes from 6-month-old, preleukemic AKR mice.     

Detection of AKR MuLV-specific RNA in AKR mouse cells by in situ hybridization.

Conditions for the detection of complex RNA sequences by in situ hybridization have been investigated by using a single-stranded 3H-cDNA probe complementary to the AKR MuLV genome and in vitro cultured AKR mouse cells which spontaneously produce AKR MuLV. It is shown that fixation with glutaraldehyde at low concentration allows cellular RNA to be sufficiently well retained during the annealing process and that stringent conditions in situ can be maintained by means of formamide. Some conditions which promote atypical and non-specific binding of the probe have been identified.     

Low-multiplicity infection of Moloney murine leukemia virus in mouse cells: effect on number of viral DNA copies and virus production in producer cells.

Mouse cells infected with Moloney murine leukemia virus (M-MuLV) were prepared by two methods, and the number of M-MuLV-specific DNA copies in the infected cells was measured. The number of M-MuLV-specific DNA copies detected varied from one to eight per infected cell in different cell lines. Cells in which multiple rounds of viral infection occurred during establishment had on the average more viral DNA copies than cells in which infection at low multiplicity was performed, followed by cloning of the cells. However, even in cells derived by the low multiplicity of infection method, most cell lines carried more than one copy of M-MuLV-specific DNA. Virus production per cell was also measured, and no strict correlation was observed between the number of M-MuLV DNA copies present and the amount of virus produced.     

Chromosomal assignment of two endogenous ecotropic murine leukemia virus proviruses of the AKR/J mouse strain.

The AKR/J mouse strain is genetically fixed for three different ecotropic murine leukemia virus genomes, designated Akv-1, Akv-3, and Akv-4 (Emv-11, Emv-13, and Emv-14). With recombinant inbred strains and crosses with linkage-testing stocks, Akv-3 and Akv-4 were placed on the mouse chromosome map. Akv-3, which encodes a replication-defective provirus, maps near the agouti coat color locus, a, on chromosome 2. Akv-4, which is replication competent, maps near the neurological mutant gene locus trembler, Tr, on chromosome 11. Akv-1 and Akv-2 (Emv-12), an ecotropic provirus carried by AKR/N but not AKR/J, have previously been mapped to chromosome 7 and 16, respectively. Thus, the four Akv proviruses mapped to date are on four different chromosomes. Akv-3 is the second ecotropic murine leukemia virus provirus to be mapped near the agouti locus. The results are discussed in relation to possible nonrandomness of viral integration.     

Dissociation of interferon effects on murine leukemia virus and encephalomyocarditis virus replication in mouse cells.

Two subclones of Swiss mouse cells infected with Moloney murine leukemia virus (M-MuLV) were tested for their response to interferon (IFN). Whereas M-MuLV production in the two subclones was inhibited to the same extent, one of the subclones was significantly more sensitive to IFN when the antiviral effect was measured by replication of encephalomyocarditis (EMC) virus. The same subclone was also more sensitive to the anticellular activities of IFN. Additionally, NIH 3T3 cells infected with M-MuLV were completely resistant to IFN actions when EMC virus replication or the anticellular activities were tested. However, under the same conditions, M-MuLV production was completely inhibited by IFN. These results indicate that IFN may affect cell growth functions and EMC replication through mechanisms different from those by which MuLV production is inhibited.     

Effect of recombinant human interleukin-6 on nitrite production of mouse myeloid leukemia cells

The effect of recombinant human interleukin-6 (rhIL-6) on induction of nitrite (NO(-2)) production was investigated in a mouse myeloid leukemia cell line (M1) and a subclone (Mm1). NO(-2) was induced by rhIL-6 (greater than 50 U/ml) in these cell lines. Pretreatment with rhIL-6 (100 U/ml) for 1 day synergistically enhanced the production of NO(-2) in Mm1 by LPS. Furthermore, pretreatment with IL-6 (100 U/ml) shortened the lag time of induction. These results indicate that IL-6 is involved in the regulation of the NO(-2) production in macrophage-like cells.     

Molecular properties of a gag- pol- env+ murine leukemia virus from cultured AKR lymphoma cells.

We have described the isolation of a replication-defective murine leukemia virus from a culture of AKR lymphoma cells [Rein et al., Nature (London) 282:753-754, 1979]. To facilitate the characterization of this murine leukemia virus, we transmitted it to mink cells and analyzed its genome by restriction mapping of the mink cellular DNA. This genome resembled the Akv genome quite closely, but it had an additional KpnI cleavage site at 1.3 kilobase pairs from the 5' end of the provirus and a small (approximately 50-base-pair) deletion between 1.8 and 3.0 kilobase pairs from the 5' end. When we tested these mink cells by immune precipitation or by competition radioimmunoassay, we found that they synthesized gPr82env, but contained no detectable gag or pol proteins. It seems likely that the KpnI cleavage site at 1.3 kilobase pairs reflects an abnormal sequence at or near the beginning of the gag gene, which prevents gag or pol translation by introducing a frameshift or termination codon into this region.     

Rapid metabolism of moloney leukemia virus precursor polypeptides in virus infected Swiss mouse embryo cells.

Viral protein synthesis in Moloney murine leukemia virus infected high passage mouse embryo cells was studied utilizing monospecific antisera to the viral core protein p30 and envelope protein gp71. Pulse-chase analysis of [³⁵S]methionine-labeled polypeptides in combination with the demonstration of the presence of either gp71 or p30-specific antigenic determinants in them indicated a 84,000-dalton polypeptide as the precursor of viral glycoproteins and four metabolically unstable polypeptides of approximate molecular weights 88,000, 72,000, 62,000, and 39,000 as the precursors of viral core protein, p30. The p30-containing 88,000 and 72,000-dalton polypeptides were distinctly seen in this system under normal growth conditions. Further, the processing of p30 precursors was very rapid and was complete during a 40 min chase while only partial processing of glycoprotein precursor was observed during the same period.     

Oncogenicity of AKR endogenous leukemia viruses.

Four biologically distinct groups of endogenous murine leukemia virus (MuLV) have been isolated from AKR mice. These viruses included (i) ecotopic XC+ MuLV that occur in high titer in normal tissues and serum of AKR mice throughout their life span, (ii) ecotropic XC- MuLV that are produced in high titers by leukemia cells, (iii) xenotropic MuLV that are readily demonstrable only in aged mice, and (iv) polytropic MuLV thatarise in the thymuses of aged mice as a consequence of genetic recombination between ecotropic and xenotropic MuLV. Virus of each of these biological classes were assayed in AKR mice for their ability to accelerate the occurrence of spontaneous leukemia. Certain isolates of ecotropic XC- MuLV and polytropic MuLV were found to have high oncogenic activity. These viruses induced 100% leukemias within 90 days of inoculation. In contrast, ecotropic XC+ MuLV that were obtained from AKR embryo fibroblasts and xenotropic MuLV that were obtained from the lymphoid tissues of aged AKR mice did not demonstrate oncogenic activity. These findings demonstrate fundamental differences between XC- and XC+ ecotropic MuLV that are found in leukemic and normal tissues, respectively. Furthermore, these findings point to the role of ecotropic XC- and polytropic MuLV in the spontaneous leukemogenesis of AKR mice.     

Interaction of whole-body hyperthermia and irradiation in the treatment of AKR mouse leukemia

Whole-body hyperthermia (WBH) to 41-42 degrees C combined with fractionated total-body irradiation (TBI) was studied in mice with transplanted AKR leukemia. Mice treated with both TBI and WBH survived longer than mice treated with either modality alone. From other groups of similarly treated mice the spleens were removed, weighed, and assayed for their content of leukemic colony-forming units (CFU) by injecting single-cell suspensions into normal syngeneic recipients. Using this methodology it was determined that the thermal enhancement ratio for WBH combined with TBI was 1.6, and that enhanced killing of leukemia cells occurred irrespective of the sequence of WBH and TBI. Data are presented which relate variables, such as duration of WBH or heating time to target temperature, to the response of neoplastic disease. The implications of these preclinical findings to clinical trials are discussed.     

Effects of interferon on hemoglobin synthesis and leukemia virus production in Friend cells

Establishment of the antiviral state by interferon does not impair differentiation of Friend cells. Interferon actually produces an increase in dimethylsulfoxide-induced hemoglobin synthesis. However, both the constitutive production and the induction of leukemia virus in these cells are inhibited by interferon.     

Molecular cloning of AKR xenotropic murine leukemia virus unintegrated proviral DNA

Suprahelical proviral DNA of AKR xenotropic murine leukemia virus was purified from agarose gels and cloned in lambda Charon 28 DNA (BamHI sites). Nine viral DNA recombinants were identified and mapped with 12 restriction endonucleases. Three calsses of cloned viral DNA inserts were found: (1) Six inserts were apparently full-length 9.0-kb DNA with tandem long terminal repeat (LTR) elements; (2) two inserts contained DNAs with deletions in or adjacent to the LTR regions; (3) a single isolate contained an inversion of 2.3 kb around the LTR in the envelope gene.     

Methionine-mediated lethality in yeast cells at elevated temperature.

Saccharomyces cerevisiae cells grown at 30 degrees C in minimal medium containing methionine lose viability upon transfer to 45 degrees C, whereas cells grown in the absence of methionine survive. Cellular levels of two intermediates in the sulfate assimilation pathway, adenosine 5'-phosphosulfate (APS) and adenosine 5'-phosphosulfate 3'-phosphate, are increased by a posttranslational mechanism after sudden elevation of temperature in yeast cultures grown in the absence of methionine. Yeast cells unable to synthesize APS because of repression by methionine or mutation of the MET3 gene do not survive the temperature shift. Thus, methionine-mediated lethality at elevated temperature is linked to the inability to synthesize APS. The results demonstrate that APS plays an important role in thermotolerance.     

Localization of the leukemogenic determinants of SL3-3, an ecotropic, XC-positive murine leukemia virus of AKR mouse origin.

SL3-3 is a potent leukemogenic retrovirus that closely resembles the non-leukemogenic virus Akv. Both viruses were isolated from AKR mice, have ecotropic host ranges, and form plaques in the XC assay. They differ at only 1 to 2% of the nucleotides in the viral genomes but differ markedly in virulence properties. SL3-3 induces leukemia in a high percentage of inoculated AKR, C3H, CBA, and NFS mice, whereas Akv does not induce disease in any of these strains. To determine which region of the genome accounts for the leukemogenic potential of SL3-3, we constructed recombinant genomes between molecular clones of SL3-3 and Akv. Recombinant, viral DNA genomes were cloned and then were transfected onto NIH 3T3 fibroblasts to generate infectious virus. The recombinant viruses were tested for leukemogenicity in AKR/J, CBA/J, and C3Hf/Bi mice. We localized the primary leukemogenic determinant to a 3.8-kilobase fragment of the SL3-3 genome containing the viral long terminal repeat, 5' untranslated sequences, gag gene, and 5', 30% of the pol gene. Reciprocal recombinants containing the equivalent region from Akv, linked to the env gene and the remainder of the pol gene from SL3-3, did not induce leukemia. We conclude that the primary virulence determinant of SL3-3 lies outside the region of the genome that encodes the envelope proteins gp70 and p15E.     

Enhanced production of Rauscher leukemia virus after infection of high-passaged JLS-V9 cells.

JLS-V9, a mouse bone marrow cell line infected with Rauscher leukemia virus at high passage level, produced larger amounts of virus than the standard JLS-V10 cells. The enhanced virus production was attributed to the increased saturation density of JLS-V9 cells.     

Virus-specific RNA synthesis in interferon-treated mouse cells productively infected with Moloney murine leukemia virus.

Mouse cells productively infected with Moloney murine leukemia virus were treated with interferon, and intracellular virus-specific RNA was studied by hybridization with complementary DNA. The steady-state concentration of virus-specific RNA in interferon-treated cells was somewhat greater than that in untreated cells, and the rates of virus-specific RNA synthesis were approximately equal in treated and untreated cells.