Defectiveness of avian myelocytomatosis virus MC29: Isolation of long-term nonproducer cultures and analysis of virus-specific polypeptide synthesis

Abstract

Nonproducer (NP) clones of chicken and quail cells transformed by avian myelocytomatosis virus MC29-A were isolated in focus and agar colony assays. Several quail MC29 NP clones were developed into long-term cultures. They have been kept in continuous culture for 6 months and about 35 passages. They do not release virus particles detectable by [ 3H]uridine incorporation or reverse transcriptase assay. Rescue of transforming virus is possible at any time by superinfection of the NP cell clones with avian leukosis viruses such as Rous associated virus type 1 or ring-necked pheasant virus (RPV). [ 35S]Methionine pulse-labeled protein extracts of NP and of superinfected MC29 transformed cell cultures were analyzed by immune precipitation and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In NP cells the common precursor polypeptide for the structural proteins of avian RNA tumor viruses (pr76) is not synthesized. Instead, a major polypeptide with a molecular weight of 110,000–120,000 was observed (MC29–110K). In superinfected cells, both MC29–110K and pr76 are synthesized. The MC29–110K polypeptide was precipitated by an antiserum against whole virus (PR RSV-B) as well as by a monospecific anti-p27 serum. It was not precipitated by an anti-glycoprotein serum. Pulse-chase experiments showed that the MC29–110K polypeptide turned over at a rate comparable to that of pr76. However, none of the major structural proteins (p27, p19, p15) could be detected after the chase. Competition radioimmune assays demonstrated that protein extracts of NP MC29 cells contain inhibitory activity for precipitation of p19 and p27, but not for p15.