MUTATIONS IN THE SIV ENV AND THE M13 LACZA GENE GENERATED IN-VITRO BYREVERSE TRANSCRIPTASES AND DNA-POLYMERASES

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 SIV E. coli and DNA polymerase an from calf-thymus. These forwar d mutation assays measured the loss of bacteriophage M13 lacZa gene fu nction by mutations. In the EnvlacZa assay frameshift mutations occurr ing during polymerisation of a 176 b long simian immunodeficiency viru s (SIV) envelope (env) sequence were phenotypically detected by blue/w hite-plaque screening. To measure in addition substitutions, a 116 b l ong M13 lacZa gene DNA template was used as the mutational target (Lac Za assay). With the SIVagm env gene DNA template, we observed similar levels of frameshift fidelity for all three RTs. Nevertheless, the SIV agm RT was slightly more accurate than the other RTs and nearly all fr ameshifts were observed at two homopolymeric runs of its homologous te mplate. Measuring also substitution errors at the lacZa template the m utation frequency of the SIVagm RT increased 2.5 fold and that of the HIV-1 RT was enhanced by a factor of 3.