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Evolutionary Potential of an RNA Virus

Eugene V. Makeyev and Dennis H. Bamford^*

Institute of Biotechnology and Department of Biosciences, University of Helsinki, Finland

Received 2 June 2003/ Accepted 28 October 2003

RNA viruses are remarkably adaptable to changing environments. This is medically important because it enables pathogenic viruses to escape the immune response and chemotherapy and is of considerable theoretical interest since it allows the investigation of evolutionary processes within convenient time scales. A number of earlier studies have addressed the dynamics of adapting RNA virus populations. However, it has been difficult to monitor the trajectory of molecular changes in RNA genomes in response to selective pressures. To address the problem, we developed a novel in vitro evolution system based on a recombinant double-stranded RNA bacteriophage, 6, containing a ß-lactamase (bla) gene marker. Carrier-state bacterial cells are resistant to ampicillin, and after several passages, they become resistant to high concentrations of another ß-lactam antibiotic, cefotaxime, due to mutations in the virus-borne bla gene. We monitored the changes in bla cDNAs induced by cefotaxime selection and observed an initial explosion in sequence variants with multiple mutations throughout the gene. After four passages, a stable, homogeneous population of bla sequences containing three specific nonsynonymous mutations was established. Of these, two mutations (E104K and G238S) have been previously reported for ß-lactamases from cefotaxime-resistant bacterial isolates. These results extend our understanding of the molecular mechanisms of viral adaptation and also demonstrate the possibility of using an RNA virus as a vehicle for directed evolution of heterologous proteins.

Present address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138.

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