Temperature-Sensitive Mutants of Fujinami Sarcoma Virus: Tumorigenicity and Reversible Phosphorylation of the Transforming p140 Protein
Authors: Lee Wen-Hwa , Bister Klaus , Moscovici Carlo , Duesberg Peter H. 
Published: January 2020

Several clones of Fujinami sarcoma virus (FSV) isolated from a laboratory stock or from mutagenized virus were temperature sensitive (
ts
) in transformation of cells in culture. When shifted from the permissive (37°C) to the nonpermissive (41.5°C) temperature, the cellular phenotype reverted to normal within 2 h, but it required about 48 h at 37°C to revert back to the transformed morphology. A temperature-resistant (
tr
) FSV clone was isolated from a tumor of an animal. All
ts
mutants were tumorigenic in animals but induced tumors only after latent periods of 12 to 25 days, compared to 5 to 6 days with
tr
virus. The
ts
lesions of the FSV mutants affected 90% of the phosphorylation of the nonstructural,
gag
-related 140,000-kilodalton phosphoprotein coded by FSV (p140), but did not affect virus replication or the synthesis of p140. Upon shifting from the permissive to the nonpermissive temperature, p140 was 90% dephosphorylated with an approximate
32
P half-life of 20 min. When shifted back to the permissive temperature, the preexisting p140 was rephosphorylated in the absence of protein synthesis within a 90-min test period. Likewise, most of the phosphate of fully phosphorylated p140 was exchanged at the permissive temperature within 30 to 90 min even when protein synthesis was inhibited. However, the protein structure of p140 had a half-life of 5 h at both temperatures. These results prove p140 to be a substrate of reversible phosphorylation. Superinfection and transformation of
ts
FSV-infected cells maintained at the nonpermissive temperature with acute leukemia virus MC29 failed to phosphorylate p140. It would follow that in vivo phosphorylation of
ts
p140 is controlled by an FSV-specific mechanism and is a prerequisite, not a consequence, of transformation. p140 of
ts
FSV recovered from cells maintained at 41.5°C with anti-
gag
serum was over 10 times less phosphorylated by associated kinase than the same protein recovered from cells at 37°C if assayed in vitro at 20°C. This kinase activity associated with or dissociated from p140 with a half-life of less than 30 min during temperature shifts of
ts
FSV-infected cells. However, p140 recovered from
ts
FSV-infected cells maintained at 37°C was phosphorylated by associated kinase in vitro not only at 20°C but also, and essentially at the same level, at 41.5°C. This suggests that the kinase associated with the immunocomplex of p140 of
ts
FSV is not temperature sensitive. p140 translated in vitro from
ts
and
tr
FSV RNA lacked kinase activity. We conclude that a fully phosphorylated p140 is necessary for the maintenance of transformation by FSV. This is consistent with the notion that other highly oncogenic viruses also code for nonstructural phosphoproteins with probable transforming function. A model which postulates that p140 is a substrate of reversible phosphorylation and that the lesion of the
ts
FSV clones described herein affects association of p140 with a cellular kinase rather than a hypothetical intrinsic kinase activity of the protein is most compatible with our data.