Transformation-defective mutant of avian myeloblastosis virus that is temperature sensitive for production of transforming protein p45v-myb.

Abstract

We have characterized a mutant of avian myeloblastosis virus (strain GA907/7) that shows a reduced capacity to transform myelomonocytic cells at the nonpermissive temperature. Myelobiasts transformed by this mutant suffer a substantial decrease in the amount of the transforming protein p45v-myb when shifted from the permissive to the nonpermissive temperature. We presume that the 5-to 10-fold decrease in the amount of p45vmyb causes the loss of the transformed phenotype. The decrease is due to a reduction in the level of v-myb mRNA. Mutant GA907/7 thus provides genetic evidence that p45vmYb is the transforming protein of avian myeloblastosis virus and apparently represents an unusual defect in the production or stability of mRNA.Oncogenic transformation of myelomonocytic cells by avian myeloblastosis virus (AMV) is thought to be mediated by the expression of a viral oncogene termed v-myb (2, 5). The nucleotide sequence of v-myb represents a partial copy of a cellular gene, c-myb, and has been transduced into the viral genome from a chicken host (3,4,6,11,12). The viral gene encodes a 45,000-Mr protein, p45v-m b (1, 7), which in turn is a truncated version of the 75,000-Mr protein (p75cn`yb) encoded by chicken c-myb (7). Both proteins are located in the cell nucleus (8), but the means by which p45v-,n,b elicits oncogenic transformation and the function of its cellular counterpart, p75c-l,'b, remain obscure.Analysis of transformation-defective, temperaturesensitive mutants of oncogenic retroviruses has yielded valuable information about possible mechanisms of cell transformation. We have recently described a temperaturesensitive mutant of AMV (strain GA907/7, designated tsAMV hereafter), which shows a markedly reduced capacity to transform myeloid cells at the nonpermissive temperature (10). The results presented in this communication extend the characterization of this mutant to a biochemical analysis of viral mRNA and p45vni1b in cells transformed by tsAMV.The experiments to be described were all performed with a myeloblast cell line (GA950/2) that was derived by transforming chicken yolk sac macrophages with tsAMV (10). Cells of this line were cultivated in BT88 medium (9) supplemented with 10% tryptose phosphate broth, 5% calf serum, 5% chicken serum, and 1.6 ,ug of folic acid per ml. At the permissive temperature of 35.5°C, these cells proliferate and exhibit a blastlike morphology; when shifted to the nonpermissive temperature of 41°C, they cease to proliferate and attain a macrophagelike morphology within 2 to 5 days. * We first analyzed the level of p45v-,n,b at the permissive and nonpermissive temperatures by labeling with [35S]methionine. Myeloblasts transformed by tsAMV were incubated for 30 min in growth medium lacking methionine at either 35.5°C or 41°C (Fig. 1A). [35S]methionine (>600 Ci/mmol; Amersham Corp.) was then added to a final concentration of 500 ,uCi/ml, and the incubation was continued for 1 h at both temperatures. Cell lysates were prepared as described previously (7). Proteins of inter...

A temperature-sensitive phenotype of avian myeloblastosis virus: determinants that influence the production of viral mRNAs

“…By contrast, the surplus of subgenomic mRNA for wtAMV remained similar to that found in transient assays ( Fig. 2B; Table 2) and also resembled the results obtained previously with transformed lines of myeloid cells (10,18). We concluded that transfection under conditions that permit establishment and spread of infection can reproduce the biochemical phenotype of tsAMV in CEF.…”

“…The preceding results contrasted sharply with our previous findings for myeloid cells transformed by tsAMV, which contained genomic RNA in substantial excess of the smaller mRNA at either the permissive or the restrictive temperature (18). We speculated that this difference might be due to the use of a transient assay in the present experiments and therefore designed an experimental strategy that addressed this possibility.…”

“…Transformation of myeloid cells by tsAMV is temperature sensitive (19). Previous work in our laboratories revealed that in transformed cells, production of subgenomic mRNA and myb protein by the mutant virus is less than that by wild-type virus and that the reduction is exacerbated by the restrictive temperature (18). Therefore, we attributed the conditional phenotype to a dosage effect; i.e., production of the v-myb gene product may fall below a critical threshold at the restrictive temperature.…”

“…Previous work has identified a mutant strain of AMV (tsAMV) that elicits temperature-sensitive transformation of myeloid cells in culture (19). The lesion in tsAMV appears to reduce the amounts of subgenomic mRNA and v-myb protein that accumulate in infected cells (18). The effect is readily apparent at the permissive temperature of 35°C but is greater at the restrictive temperature of 41°C.…”

“…The effect is readily apparent at the permissive temperature of 35°C but is greater at the restrictive temperature of 41°C. These findings prompted the suggestion that the mutation in tsAMV might cause a conditional limitation on the splicing of viral RNA and that this limitation in turn would reduce production of the v-myb gene product below a critical threshold required for transformation (18).…”