HIGH-RATES OF FRAMESHIFT MUTATIONS WITHIN HOMOOLIGOMERIC RUNS DURING A SINGLE-CYCLE OF RETROVIRAL REPLICATION

Homo-oligomeric runs were inserted into a spleen necrosis virus-based retrovirus vector to determine the nature and rate of mutations within runs of 10 to 12 identical nucleotides during a single replication cy cle. Clones of helper cells containing integrated copies of retroviral vectors were used to produce virus for infection of target (nonhelper ) cells. Proviral sequences from target cell clones were compared with proviral sequences from helper cell clones to study mutations that oc curred during a single cycle of replication. In addition to the intern al region spanning the homo-oligomeric inserts, a naturally occurring run of 10 T's in the long terminal repeat (LTR) also was sequenced. Ra tes of mutation ranged from <0.01 to 0.38 frameshift mutations per run per cycle for different nucleotide runs. Frameshift mutations ranged from deletions of 2 bases to additions of 5 bases; the most common mut ations were +1 and -1. Frameshift mutation rates did not increase as t he run length increased from 10 to 12 bases. Rates of frameshift mutat ion for runs of T's and A's were significantly higher than rates for r uns of C's and G's, and rates for runs of pyrimidines were significant ly higher than those for runs of purines. Interestingly, the vast majo rity of frameshift mutations in the internal region (95%) were positiv e, suggesting that the primer strand tends to slip backward on the tem plate in this region. LTR runs had a significantly lower number of pos itive frameshift mutations than the internal runs. By analyzing the ty pes of frameshift mutations within runs and by comparing the patterns of frameshift mutations in the 5' and 3' LTRs of individual proviruses , we conclude that the majority of mutations observed in our system oc curred during minus-strand DNA synthesis of reverse transcription.