Translation of bovine leukemia virus virion RNAs in heterologous protein-synthesizing systems

Abstract

Bovine leukemia virus 60 to 70S RNA was heat denatured, the polyadenylic acid-containing species were separated by velocity sedimentation, and several size classes were translated in a micrococcal nuclease-treated cell-free system from rabbit reticulocytes. The major RNA species sedimented at 38S and migrated as a single component of molecular weight 2.95 x 10' when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The predominant polypeptides of the in vitro translation of bovine leukemia virus 38S RNA were products with molecular weights of 70,000 and 45,000; minor components with molecular weights of 145,000 and 18,000 were also observed. Two lines of evidence indicate that the 70,000and 45,000-molecular weight polypeptides represent translation products of the gag gene of the bovine leukemia virus genome (Pr709"t' and Pr45,"('). First, they are specifically precipitated by a monospecific antiserum to the major internal protein, p24, and second, they are synthesized and correctly processed into virion proteins p24, p15, and plO in Xenopus laevis oocytes microinjected with bovine leukemia virus 38S RNA. The 145,000-molecular weight polypeptide was immunoprecipitated by the anti-p24 serum and not by an antiserum to the major envelope glycoprotein, gp6O. It contained all the tryptic peptides of Pr7g"t'(l and additional peptides unique to it, and thus represents an elongation product of Pr7e"'g' in an adjacent gene, presumably the pol gene. The 18,000-molecular weight product was antigenically unrelated to p24 and gp6O and shared no peptides in common with Pr701'', Pr451"', or the 145,000-molecular weight polypeptide. It was maximally synthesized on a polyadenylic acid-containing virion 16 to 18S RNA, and we present evidence that this RNA is a 3' endderived subgenomic fragment of the bovine leukemia virus genome rather than a contaminating cellular RNA.

Bovine leukemia virus protease: purification, chemical analysis, and in vitro processing of gag precursor polyproteins

“…Not only the precise localization and reading frame assignment as described in this paper, but also the results of our biochemical studies with in vitro synthesized proteins are also compatible with this hypothesis. As reported previously by Ghysdale et al (7) and as shown in Fig. 4, the 66K and 44K proteins were gag-related precursors.…”

Activation of protein kinase C or cAMP-dependent protein kinase increases phosphorylation of the c-erbA-encoded thyroid hormone receptor and of the v-erbA-encoded protein.

“…At the end of the labelling period, cells were rinsed in ice-cold phosphate-buffered saline, and lysed in 50 mM Tris-HCI (pH 7.4), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, 10 mM sodium fluoride, 40 mM p-nitrophenylphosphate, 1% aprotinin (Sigma), 0.1 mg/ml PMSF and 1 pg/ml leupeptin (Sigma). The lysates were centrifuged at 100 000 g for 60 min and immunoprecipitations were carried out as described previously (Ghysdael et al, 1979), except that 10 mM sodium fluoride was included in all buffers. Immunoprecipitates were analysed on 10% polyacrylamide slab gels (acrylamide:bisacrylamide 30:0.4) in the presence of 0.1% SDS.…”

Induction of Transformed Phenotypes in Sheep Fibroblasts by Culture Fluids from Cells Persistently Infected with Bovine Leukemia Virus

“…FLK is the best cell line as a source of BLV and is widely distributed throughout the world (24). Purified BLV materials from FLK cells were negative for other retroviruses (3)(4)(5)(6). Cells were grown in Eagle's minimum essential medium containing 10% heat-inactivated BLV-free fetal calf serum (FCS, GIBCO).…”

“…Fetal lamb kidney (FLK) clones persistently infected with BLV have been widely used as a source of BLV (1- 6,19). The virological properties of BL V from FLK cells have been well characterized (2)(3)(4)(5)(6).…”