Viruses replicate their genomes at exceptionally high mutation rates. Their offspring evolve rapidly and therefore, are able to evade common immunological and chemical antiviral agents. In parallel, virus genomes cannot tolerate a further increase in mutation rate: Experimental evidence exists that even few additional mutations are sufficient for the extinction of a viral population. A future antiviral strategy might therefore aim at increasing the error-producing capacity of viral replication enzymes. We employed the principles of directed evolution and developed a scheme for the stringent positive selection of error-prone polymerase activity. A mutant T7 RNA polymerase with a nucleotide substitution error rate at least 20-fold greater than that of the wild-type was selected. This enzyme synthesized highly heterogeneous RNA products in vitro or in vivo and also decreased the replication efficiency of wild-type bacteriophage T7 during infection.

Viruses replicate their genomes at exceptionally high mutation rates. Their offspring evolve rapidly and therefore, are able to evade common immunological and chemical antiviral agents. In parallel, virus genomes cannot tolerate a further increase in mutation rate: Experimental evidence exists that even few additional mutations are sufficient for the extinction of a viral population. A future antiviral strategy might therefore aim at increasing the error-producing capacity of viral replication enzymes. We employed the principles of directed evolution and developed a scheme for the stringent positive selection of error-prone polymerase activity. A mutant T7 RNA polymerase with a nucleotide substitution error rate at least 20-fold greater than that of the wild-type was selected. This enzyme synthesized highly heterogeneous RNA products in vitro or in vivo and also decreased the replication efficiency of wild-type bacteriophage T7 during infection.

The cover picture shows a schematic representation of the positive genetic selection of an error-prone T7 RNA polymerase (T7 RNAP) mutant (shown in green). A system of two compatible plasmids (blue) carrying a T7 RNAP mutant library (bottom left) or an inactive mutant of the gene coding for tetracycline resistance under the exclusive control of a T7 promoter (top) rewarded inaccurate transcription with the survival of bacteria (symbolized by the smileys in the flask). Following a single round of selection, the survivors expressed a T7 RNAP variant with a nucleotide substitution error rate at least 20-fold greater than that of the wild-type enzyme. The heterogeneity of RNA products synthesized by this enzyme was demonstrated in vitro, andÐmost impressivelyÐin vivo by employing the transcription of Green Fluorescent Protein (GFP): In contrast to the uniform distribution of fluorescence that was observed after transcription by the wild-type T7 RNAP (left inset diagram), transcription by the mutant enzyme induced large deviations from the standard fluorescence behavior (right inset diagram), which were attributed to contributions of GFP mutants with altered excitation wavelengths. More about this successful application of the principle of directed evolution can be found in the paper by S. Brakmann and S. Grzeszik on p. 212 ff.

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