Reductive evolution enabled the adaption of a B1 deletion vaccinia virus by truncation and loss of function of the B12 pseudokinase.. Vaccinia virus WR strain:WR

Vaccinia virus has a double-stranded DNA genome and encodes for over 200 proteins. Among these are proteins essential for DNA replication in the cytoplasm of the cell. The vaccinia B1 kinase participates in a necessary role during DNA replication by restricting the antiviral function of a host protein that binds and condenses the viral DNA to inhibit replication. During the process of understanding the pathways regulated by the vaccinia B1 kinase, we discovered that the loss of the B12 pseudokinase rescues the attenuated phenotype of a B1 deletion virus. This system represents the first example of reductive evolution, explicitly shown by experimental evolution. Specifically, we came to these findings by serially passage of a B1 deletion vaccinia virus (ΔB1) in CV1 cells and quantifying the harvest titer at each passage until a rescue in titer was achieved. We sequenced the complete genomes of our lab ‘Wiebe’ WT, ΔB1, ΔB1-A1 and ΔB1-A3 viruses (raw sequence reads provided for each virus). The sequence analysis revealed a prominent insertion/deletion (indel) mutation within the B12 gene, predicted to cause a protein truncation. Further studies were completed to confirm that the indel mutation produces a truncated protein, the loss of B12 function rescues ΔB1 phenotype, and the reconstitution of B12 represses the ΔB1mutB12 virus growth phenotype. In summary, experimental evolution of ΔB1 established the first example of reduction evolution where the loss of a repressor, vaccinia B12, restores the growth phenotype of ΔB1 to near WT levels.Method: Virus stocks were purified by centrifugation through a sucrose gradient. The Nextera XT kit from Illumina was used to construct the libraries from purified viral DNA and the resultant multiplexed library was sequenced on the MiSeq V2 instrument. 150 base pair paired-end sequencing was performed. The 200kb genome sequences had between 1000 and 2000x coverage and was analyzed for gene duplication, point mutations and indel mutations for alignments of ΔB1, ΔB1mutB12-A1, and ΔB1mutB12-A3 to the WT_Wiebe sequenced genome or ΔB1mutB12-A1, and ΔB1mutB12-A3 compared to the ΔB1 sequence.