Molecular biological characterization of a highly leukaemogenic virus isolated from the mouse. IV. Viral proteins.

The proteins of a highly leukaemogenic murine virus (DMBA-LV) endogenous to the CFW/D mouse have been characterized. This virus does not contain ecotropic, xenotropic or polytropic type C retroviruses capable of replicating in established tissue culture cell lines. Analysis of the viral proteins indicated that proteins characteristic of both type C and type B retroviruses were present. All the proteins characteristic of a competent, mature type B retrovirus, such as the murine mammary tumour virus, were present in the virions and were expressed at high levels in a virus-producing thymic lymphoma cell line. Type C internal structural proteins were detected in the virus and cells producing virus. Type C envelope protein was detected at very low levels in both the virions and the virus-producing tumour cell lines. Chymotryptic polypeptide profiles of this gp70 indicated similarity with the envelope glycoproteins characteristic of xenotropic and recombinant type C retroviruses.

Hepatic metallothionein production and resistance to heavy metals by rainbow trout (Salmo gairdneri)--II. Held in a series of contaminated lakes.

Rainbow trout held in net pens for 4 weeks in three locations contaminated by heavy metals developed elevated levels of hepatic metallothionein but no acclimation to a combination of zinc, copper and cadmium. Toxicity of the mixture of zinc, copper and cadmium in a ratio of 400:20:1 increased with temperature in the range of 6-21 degrees C. Hepatic metallothionein concentrations after 4 weeks of exposure in situ was correlated with the degree of contamination as measured by zinc concentrations (P less than 0.01).

Chronic exposure of coho salmon to sublethal concentrations of copper--III. Kinetics of metabolism of metallothionein.

Juvenile coho salmon (Oncorhynchus kisutch) 3-8 g body weight were exposed to sublethal concentrations of copper acetate in water of hardness 280 mg/l (as CaCO3) using flow-through conditions. Concentrations of copper were approximately 1, 50, 100 or 150 micrograms Cu/l. Uptake of copper into blood, gills, kidney and liver was measured using 67Cu and into hepatic metallothionein. Chromatography on Sephadex G-75 was used to separate the protein from the cytosol. Copper was determined using atomic absorption spectrophotometry. The ratio of rates of uptake of copper and synthesis of metallothionein was consistent with published values for the copper content of copper-thionein, i.e. 7 to 1. After 4 weeks of continuous exposure the concentrations of copper and hepatic metallothionein levelled off at concentrations that were a function of the copper concentration in the water. After the plateau or steady-state had been reached, the rate of loss of metallothionein was measured by determining the change in specific-activity of isotope with time after incorporation of 35S-L-cysteine or 3H-L-lysine into the protein. The half-life for loss of metallothionein was a function of the exposure to copper, being about 13 days for control fish and 30 days for fish exposed to the greatest concentration of copper. Salmon that had been exposed to copper for 8 weeks neither gained hepatic metallothionein in a further 4 week exposure to copper, nor lost it when transferred for 4 weeks to clean water. Tolerance to copper exhibited by fish that continued to be exposed to it was partially lost after 4 weeks depuration.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic metallothionein production and resistance to heavy metals by rainbow trout (Salmo gairdneri)--I. Exposed to an artificial mixture of zinc, copper and cadmium.

Rainbow trout developed elevated hepatic metallothionein concentrations after 4 weeks in a solution containing zinc, copper and cadmium in a fixed ratio of 400:20:1. Resistance to a combination of these metals increased in proportion to the concentration to which they were exposed for 4 weeks. The concentration of copper but not zinc or cadmium in the low molecular weight proteins separated by gel filtration was related to the concentrations of metallothionein present. The combined toxicity of the metals in the mixture was additive.

Molecular biological characterization of a highly leukaemogenic virus isolated from the mouse. III. Identity with mouse mammary tumour virus.

A highly leukaemogenic virus isolate (DMBA-LV) endogenous to the CFW/D mouse has been found to contain two viral genomes. One was closely related to the type B milk-borne mouse mammary tumour virus (MMTV) and present in tenfold excess over a type C viral genome which was only partially related to xenotropic and polytropic isolates from the CFW/D mouse as well as to the ecotropic Moloney murine leukaemia virus isolate. The thymic lymphoma cell line that produced DMBA-LV expressed high levels of MMTV viral RNA (35S and the 24S envelope mRNA). Both the virus and the virus-producing cell line expressed multiple species of type C viral RNA. Similar species of type C viral RNA were also associated with non-infectious, non-leukaemogenic viral particles present in both normal lymphoid cells and in a MMTV-free thymic lymphoma cell line established from a second chemical carcinogen-induced tumour.

Hepatic metallothionein and resistance to copper in juvenile coho salmon.

1. Four groups of 600 juvenile coho salmon (Oncorhynchus kisutch) of 3-8g body weight were exposed to sublethal concentrations of copper acetate in water of hardness 280 mg/l (as CaCO3). 2. Flow-through conditions were maintained for a period of 10 weeks, during which the copper concentrations to which the groups of fish were exposed were approximately 1, 50, 100 and 150 micrograms Cu/l. 3. Hepatic metallothionein concentrations were measured using differential pulse polarography in filtrates of heat-denatured saline extracts of the tissues. 4. These values increased as a function of the copper concentration to maximum levels after 4 weeks of continuous exposure and thereafter remained more or less constant. 5. Flow-through 168 hr LC50 values for fish tested at intervals throughout the experiment increased to maximum values (2.5 X control values) at 1 week and during the next 3 weeks fell somewhat to stable but elevated values. 6. At 4 weeks, these were related to the metallothionein (MT) values by the regression equation 168 hr LC50 = 2.423 (+/- 0.455) X MT + 48.6 (+/- 31.1). 7. The probability that the relationship was not accidental exceeded 97%.

Chronic exposure of coho salmon to sublethal concentrations of copper.--I. Effect on growth, on accumulation and distribution of copper, and on copper tolerance.

1. Coho salmon, exposed to sublethal levels of aqueous copper (1/4 and 1/2 LC50), lost appetite and ceased growing or showed decreased rates of growth. 2. Recovery of appetite and growth rate was faster in fish exposed to 1/4 of the LC50 than in those exposed to 1/2 of the LC50. 3. Copper levels were elevated in liver gill and kidney of exposed fish with the liver tissue accumulating a much larger amount of the metal than any other tissue. 4. The concentration of liver copper became constant at about the time that growth rate recovered. 5. The exposed fish exhibited much higher resistance to elevated aqueous copper levels than did the controls. 6. The results suggest that coho salmon may become acclimated to higher levels of copper and that acclimated fish are more tolerant to copper than control animals.

Chronic exposure of coho salmon to sublethal concentrations of copper--II. Distribution of copper between high- and low-molecular-weight proteins in liver cytosol and the possible role of metallothionein in detoxification.

1. Liver cytosol samples from juvenile coho salmon exposed over a 14-week period to 70 micrograms/l (1/4 LC50) or 140 micrograms/l (1/2 LC50), were separated into high-molecular-weight and low-molecular-weight fractions by column chromatography on Sephadex G-75, and compared to similar samples obtained from control fish. 2. The levels of copper in the low-molecular-weight function of fish exposed to 70 micrograms/l were not significantly increased over control values for the first 6 weeks but then increased rapidly, while those of fish exposed to 140 micrograms/l increased substantially in the first 2-4 weeks of exposure, then levelled off. 3. In the exposed fish the levels of copper in the high-molecular-weight fraction were not significantly elevated above control levels over the first 8-10 weeks but then increased significantly. 4. The low-molecular-weight fractions of exposed fish were shown to contain increasing levels of metallothionein. The metallothionein was isolated on DEAE-cellulose and characterized by amino acid analyses.

Biological and molecular biological characterization of the virus progeny from transformed clones MuSV-124 and MuSV-349: evidence for MuLV-specific nucleotide sequences in the MoMuSV size class of RNA from MoMuSV-124.

The genetic information of MoMuSV-349 and MoMuSV-124, two clones of productively transformed TB cells, was distributed between two size classes of RNA (mol. wt. 2.9 x 10(6)) in the proportions of 5:1. Some preparations of MoMuSV-124 lacked the large RNA. The virions produced by both clones also contained all the nucleotide sequences of Moloney leukaemia virus and the ratio of MuSV:MuLV produced by the two clones differed markedly. The distribution of the sequences specific for Moloney murine leukaemia virus (MoMuLV) between the two size classes of RNA was studied using molecular hybridization to DNA probes complementary to and representative of: (i) the Moloney murine sarcoma virus (MoMuSV) RNA genome (mol. wt. 1.9 x 10(6)); (ii) those nucleotide sequences shared by MoMuSV and MoMuLV; (iii) nucleotide sequences specific for MoMuSV; (iv) nucleotide sequences specific for MoMuLV. The only detectable Moloney leukaemia virus-specific nucleotide sequences present in MoMuSV-124 virions were in the RNA of mol. wt. 1.9 x 10(6), whereas these sequences were detected in the RNA of mol. wt. 2.9 x 10(6) isolated from MoMuSV-349 virions. The biological properties of the replicating information in MoMuSV-124 suggest that, consistent with the small size of RNA, it is defective. whereas MoMuSV-349 produces virions containing an intact MoMuLV genome, competent for replication.

Sub-genomic RNA in Moloney leukemia virus grown in lymphoid-derived cell lines consists primarily of homologous viral RNA.

Moloney murine leukemia virus (MoMuLV) grown in 2 lymphoid-derived murine cell lines (JLS-V9 and TB) contained 2 size classes of RNA subunits: 2.8 +/- 0.4 x 10(6) (n = 30) and 1.6 +/- 0.1 x 10(6) (n = 15) daltons. Detectable levels of low molecular weight viral RNA (LMW vRNA) were not present in MoMuLV grown in mouse embryo fibroblasts, nor rat cells, nor was it produced by them when they were infected with MoMuLV containing both species of vRNA. DNA probes complementary to both subunits were synthesized separately using purified reverse transcriptase (R.T.) and calf thymus DNA as primer. Polyadenylated LMW vRNA was selected and, by molecular hybridization was found to be completely homologous with the large RNA subunit. This was confirmed using cDNA probes representing defined regions of the genome. The LMW vRNA therefore contains multiple subsets of the viral RNA, which could correspond to multiple deletion mutants possibly generated by an efficient RNA splicing mechanism. In addition, low levels of non-homologous virus-like RNA were detected in MoMuLV grown in TB (2%) AND NIH/3T3 cells (0.4%).

Lower limb paralysis induced in mice by a temperature-sensitive mutant of Moloney leukemia virus.

A temperature-sensitive mutant of Moloney murine leukemia virus defective in an early function and injected into newborn mice produced lower limb paralysis. Susceptible mice were inbred strains CFW/D, CBA/H, C3H/Bi/Ka, and outbred NIH Swiss stock. Inbred W/Fu rats and C57BL/Ka mice did not develop the paralysis, though the latter were infected with virus; the sera from these mice produced paralysis in susceptible CFW mice.

Genetic studies of the ploidy of Moloney murine leukemia virus.

An assay for Moloney murine leukemia virus was developed that made use of the production of morphologically altered foci in nonproducer mouse cells (15F) carrying murine sarcoma virus. Wild-type (wt) virus gave a ratio of titers at 39 degrees C/34degrees C = 1.05 +/- 0.45 (standard deviation;n = 20). A spontaneous, thermosensitive (ts) mutant of Moloney murine leukemia virus, ts3, defective in a late viral function, gave 39 degrees C/34degrees C = 0. A murine cell line (TB) was mixedly infected with ts3 and wt (multiplicities of infection, 7.8:4.3), cloned after infection, and shown to be infected by both viruses. At 34 degrees C it produced wt, ts, and particles of mixed parentage. The heterozygotes (hz) had ratios of assays 39 degrees C/34 degrees C = 0.06 to 0.84 (mean, 0.36). To eliminate possible interference by multiploid particles with determination of the proportions of the three types of particles, the virus produced by the mixedly infected, cloned cell line at 34 degrees C was distributed by velocity sedimentation in a sucrose gradient, and virus was picked from the lightest part of the gradient. The proportions of ts, wt, and hz were 0.27, 0.26, and 0.47. Those particles identified as hz segreated ts, wt, and hz in the proportions 0.24, 0.27, and 0.49, respectively. These values were not significantly different from those predicted from a diploid model of the genome.