Mutagenesis of a functional chimeric gene in yeast identifies mutations in the simian virus 40 large T antigen J domain.
Simian virus 40 large T antigen contains an amino terminal J domain that catalyzes T antigen-mediated viral DNA replication and cellular transformation. To dissect the role of the J domain in these processes, we exploited the genetic tools available only in the yeast Saccharomyces cerevisiae to isolate 14 loss-of-function point mutations in the T antigen J domain. This screen also identified mutations that, when engineered into simian virus 40, resulted in T antigen mutants that were defective for the ability to support viral growth, to transform mammalian cells in culture, to dissociate the p130-E2F4 transcription factor complex, and to stimulate ATP hydrolysis by hsc70, a hallmark of J domain-containing molecular chaperones. These data correlate the chaperone activity of the T antigen J domain with its roles in viral infection and cellular transformation and support a model by which the viral J domain recruits the cytoplasmic hsc70 molecular chaperone in the host to rearrange multiprotein complexes implicated in replication and transformation. More generally, this study presents the use of a yeast screen to identify loss-of-function mutations in a mammalian virus and can serve as a widely applicable method to uncover domain functions of mammalian proteins for which there are yeast homologues with selectable mutant phenotypes.
Fewell
Sheara W
SW
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Pipas
James M
JM
Brodsky
Jeffrey L
JL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000957:Antigens, Viral, Tumor / Q000737:chemistry*; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics* / Q000276:immunology; D013696:Temperature; D013997:Time Factors
D000957:Antigens, Viral, Tumor; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D000255:Adenosine Triphosphate