The hr-t Gene of Polyoma Virus

The hr-t gene of polyoma virus encodes both the small and middle T (tumor) antigens and exerts pleiotropic effects on cells. By mutating the 3' splice site for middle T mRNA, we have constructed a virus mutant, Py8O8A, which fails to express middle T but encodes normal small and large T proteins. The mutant failed to induce morphological transformation or growth in soft agar, but did stimulate postconfluent growth of normal cells. Cells infected by Py8O8A became fully agglutinable by lectins while retaining normal actin cable architecture and normal levels of extracellular fibronectin. These properties of Py8O8A demonstrated the separability of structural changes at the cell surface from those in the cytoskeleton and extracellular matrix, parameters which have heretofore been linked in the action of the hr-t and other viral oncogenes.

“…Enzymes and the synthetic octadecamer 5 (78) were used to grow viral stocks. The hr-t mutant NG59 and the marker rescued 59RA wild type have been described previously (24).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

A polyoma mutant that encodes small T antigen but not middle T antigen demonstrates uncoupling of cell surface and cytoskeletal changes associated with cell transformation.

Liang¹,

Carmichael²,

Benjamin³

1984

“…Expression of the large T antigen can confer serum independence of growth and lead to the establishment of primary rodent cells (6,7,23). The hr-t gene, encoding both the small and middle T antigens, is required for the expression of essentially all phenotypic changes that accompany transformation (2). No ts mutants in the hr-t gene have been isolated, although a cold-sensitive mutation affecting the middle T antigen (mT) was recently described (36).…”

mentioning

confidence: 99%

Regulation of cellular phenotype and expression of polyomavirus middle T antigen in rat fibroblasts.

Raptis¹,

Lamfrom²,

Benjamin³

1985

118

Polyoma middle T antigen (mT) was expressed in rat F-111 cells under control of the dexamethasoneregulatable mouse mammary tumor virus promoter. Graded phenotypic responses to levels of mT induction by the hormone were seen, with morphological transformation, focus formation, and anchorage-independent growth requiring increasing levels of mT expression. The ability of different clones to form tumors reflected their maximum level of induction of mT-associated kinase and their ability to grow in soft agar. Expression of transformation parameters and tumorigenicity correlates with the level of mT phosphorylated by pp6O-src in immune complexes and not with the total amount of mT determined by metabolic labeling. We suggest that cellular factors regulate mT activity by forming a kinase-active fraction of mT molecules that controls the transformed state.

“…Cellular transformation by polyoma virus requires the hr-t gene (3,5,56), and polyomatransformed cells express the products of this viral gene (6,14). Cells containing an integrated hr-t mutant genome remain phenotypically normal (37).…”

mentioning

confidence: 99%

“…The hr-t gene encodes two proteins: the 56K middle T and 22K small T antigens (5). The relative roles of these two proteins in various aspects of transformation are not yet clear.…”

mentioning

confidence: 99%

Polyoma virus middle T antigen: relationship to cell membranes and apparent lack of ATP-binding activity.

Schaffhausen¹,

Dorai²,

Arakere³

et al. 1982

Middle T antigen of polyoma virus is associated principally with the plasma rmembrane. Comparison of the trvpsin sensitivity of middle T in intact cells and "inside out" membrane preparations showed that middle T is oriented towards the inside of the cell. This was confirmed by labeling of middle T in permeabilized cells, but not in intact cells, using [y-32P]ATP. Middle T molecules active in the in vitro kinase reaction could be differentiated from the bulk (metabolically labeled) middle T based on resistance to trypsin treatment. The active fraction also behaved differently from the bulk when cell frameworks were prepared with Triton-containing buffers; whereas the bulk middle T was evenly distributed in the soluble and cell framework fractions, the kinase-active forms were largely associated with the framework. Middle T molecules labeled in vivo with 32P04were found largely in the framework fraction, like the molecules that show kinase activity in vitro. Experiments with ATP affinity reagents 8-azido-ATP and 2,3-dialdehyde ATP have failed to label the middle T antigen. However, 2,3-dialdehyde ATP could be used to inhibit the kinase reaction. This raises the question of whether middle T antigen possesses intrinsic kinase activity or, rather, associates with a cellular tyrosine kinase.