An error-prone T7 RNA polymerase mutant generated by directed evolution

Viruses replicate their genomes at exceptionally high mutation rates. Their offspring evolve rapidly and therefore, are able to evade common immunolog ical and chemical antiviral agents. In parallel, virus genomes cannot toler ate 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 t he error-producing capacity of viral replication enzymes. We employed the i principles of directed evolution and developed a scheme for the stringent positive selection of error-prone polymerase activity. A mutant T7 RNA poly merase with a nucleotide substitution error rate at least 20-fold greater t han that of the wild-type was selected. This enzyme synthesized highly hete rogeneous RNA products in vitro or in vivo and also decreased the replicati on efficiency of wildtype bacteriophage T7 during infection.