To investigate the accuracy of retroviral in vitro DNA replication we have examined with two fidelity assays the reverse transcriptases (RTs) from SIVagm, HIV-1, MoMLV as well for comparison the Klenow fragment from E. coli and DNA polymerase a from calf-thymus. These forward mutation assays measured the loss of bacteriophage M13 lacZa gene function by mutations. In the EnvlacZa assay frameshift mutations occurring during polymerisation of a 176 b long simian immunodeficiency virus (SIV) envelope (env) sequence were phenotypically detected by blue/white-plaque screening. To measure in addition substitutions, a 116 b long M13 lacZa gene DNA template was used as the mutational target (LacZa assay). With the SIVagm env gene DNA template, we observed similar levels of frameshift fidelity for all three RTs. Nevertheless, the SIVagm RT was slightly more accurate than the other RTs and nearly all frameshifts were observed at two homopolymeric runs of its homologous template. Measuring also substitution errors at the lacZa template the mutation frequency of the SIVagm RT increased 2.5 fold and that of the HIV-1 RT was enhanced by a factor of 3.

No abstract