Viral attenuation by engineered protein fragmentation

A possible but untested method of viral attenuation is protein fragmentation, engineering wild-type proteins as two or more peptides that self-assemble after translation. Here, the bacteriophage T7 was engineered to encode its essential RNA polymerase as two peptides. Initial fitness was profoundly suppressed. Subjecting the engineered virus to over 100 generations of adaptation by serial transfer resulted in a large fitness increase, still remaining below that of evolved wild-type. The fitness increase was accompanied by three substitutions in the fragmented peptides as well as six mutations in other parts of the genome, but the fragmentation was retained. This study thereby demonstrates the feasibility of using gene fragmentation as a possibly permanent method of attenuation, but the initial fitness of the engineered genome may be a poor measure of its fitness on extended adaptation.