HIGH-FREQUENCY OF SINGLE-BASE TRANSITIONS AND EXTREME FREQUENCY OF PRECISE MULTIPLE-BASE REVERSION MUTATIONS IN POLIOVIRUS

We employed independent clones of a temperature-sensitive mutant of ty pe 1 poliovirus, 3AB-310/4, to quantitate the frequency of specific U --> C transitions at nucleotide 5310, within the genomic region encodi ng polypeptide 3AB, which is involved in the initiation of RNA replica tion. Only this U --> C base substitution restores the wild-type pheno typic ability to form plaques at 39-degrees-C; the other two base subs titutions at this site are lethal. The observed frequency of this spec ific transition averaged 2 x 10(-5), and all revertant viruses forming plaques at 39-degrees-C contained the expected cytidine at nucleotide 5310. Incredibly, only 3 of 10 revertants exhibited this one specific U --> C transition whereas 7 of 10 exhibited this same transition plu s four additional base substitutions that precisely reverted temperatu re-sensitive 3AB-310/4 to wild-type poliovirus sequence (these latter four mutations had been introduced into 3AB-310/4 as silent third base mutations to provide new restriction sites in infectious cDNAs). No o ther mutations were detected in this polypeptide 3AB domain in either the single-base or the precise 5-base revertants. No intermediates wer e seen; all revertants exhibited either the single U --> C transition at nucleotide 5310 or the same transition plus four precise reversions to the wild-type sequence at sites 8, 11, 43, and 46 bases distant fr om nucleotide 5310. Similar results were obtained after transfection o f cDNA-derived transcripts. We discuss possible mechanisms for our dat a. These include (but may not be limited to) error-prone polymerase ac tivity, sequential RNA recombination events joining independent mutati ons, or some unusual RNA editing process.