Poxviruses are large DNA viruses that rapidly adapt to hosts despite a relatively low mutation rate. One mechanism for adaptive evolution is through transient copy number variation described as ‘gene accordions’. To understand the mechanisms by which these structural variants are generated, we performed serial infections with vaccina, the model poxvirus, under selective pressure from the host nucleic acid sensor Protein kinase R (PKR). Rapid fitness increases in the viral populations corresponded to both gene amplification of the vaccinia PKR inhibitor K3L, and high frequency single nucleotide changes in unrelated genes. These mutations positively influence the appearance of copy number variation and provide a greater understanding of viral adaptation through structural variation.