A substitution in a nonconserved region of polyomavirus large T antigen which causes a thermosensitive mutation

The temperature-sensitive defect of the tsP155 mutant of polyomavirus (Py) maps in the large T antigen (LT) coding sequence of a viral DNA diverging markedly from that of extensively characterized wild-types (WTs) such as A2 and CSP. We have sequenced about 600 base pairs (bp) "early" DNA encompassing the mutated site in tsP155, as well as the corresponding DNA segment from a revertant virus (RtsP155). As expected, tsP155 was found to be more closely related to CSP than to A2. Out of 3 single bp differences between tsP155 and CSP, 2 were common to tsP155 and RtsP155. The only substitution exclusive to tsP155 was a G----C transversion at bp 2658 which canceled the HaeIII site at bp 2657. Heteroduplexes inclusive of tsP155 DNA and of a 312-bp-long fragment of RtsP155 DNA yielded recombinant viruses growing under restrictive conditions whose DNAs had all regained the HaeIII site at bp 2657. These findings clearly identify the ts mutation with the tranversion at bp 2658, which is expected to change Ala 701 for a Pro in LT. We discuss this substitution in relation to the phenotype of tsP155.