Preformed hexamers of SV40 T antigen are active in RNA and origin-DNA unwinding.
Preformed hexamers of simian virus 40 (SV40) large tumor antigen (T antigen) constitute the bulk of T antigen in infected cells and are stable under physiological conditions. In spite of this they could not be assigned a function in virus replication or transformation. We report that preformed hexamers represent the active T antigen RNA helicase. Monomers and smaller oligomeric forms of T antigen were inactive due to the lack of hexamer formation under RNA unwinding conditions. In contrast to the immunologically related cellular DEAD-box protein p68, the T antigen RNA helicase is found to act in a much more processive way and it does not catalyze rearrangements of structured RNAs. Thereby, it rather seems to resemble other virus-encoded RNA helicases, like vaccinia virus NPH-II. Surprisingly, in our hands preformed hexamers also strikingly bound to and unwound the SV40 replication origin, pointing to a possible role of preformed hexamers in the initiation step of viral DNA replication. Furthermore, we have detected an extra hexamer-specific, high-affinity T antigen ATP binding site with a very slow exchange rate constant, the function of which is discussed.
Uhlmann-Schiffler
Heike
H
Medizinische Biochemie und Molekularbiologie, Universität des Saarlandes, Gebäude 44/45, D-66421 Homburg, SAAR, Germany.
Seinsoth
Stephanie
S
Stahl
Hans
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D001667:Binding, Competitive; D002460:Cell Line; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D019281:Dimerization; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D020365:RNA Helicases / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D018741:Replication Origin / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics / Q000276:immunology; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D012367:RNA, Viral; D000255:Adenosine Triphosphate; D020365:RNA Helicases