Accelerated selection of a viral RNA polymerase variant during gene copy number amplification promotes rapid evolution of vaccinia virus

Abstract

Viruses are under relentless selective pressure from host immune defenses. To study how poxviruses adapt to innate immune detection pathways, we performed serial infections of vaccinia virus in primary human cells. Independent courses of experimental evolution with a recombinant strain lacking E3L revealed several high frequency point mutations in conserved poxvirus genes, suggesting important roles for essential poxvirus proteins in innate immune subversion. Two distinct mutations were identified in the viral RNA polymerase gene A24R, which seem to act through different mechanisms to increase virus replication. Specifically, a Leu18Phe substitution in A24R conferred fitness tradeoffs, including increased activation of the antiviral factor Protein kinase R (PKR). Intriguingly, this A24R variant underwent a drastic selective sweep during passaging, despite enhanced PKR activity. We show that the sweep of this variant can be accelerated by the presence of copy number variation (CNV) at the K3L locus, which with multiple copies strongly reduces PKR activation. Therefore, adaptive cases of CNV can also facilitate the accumulation of point mutations separate from the expanded locus. This study reveals how rapid bouts of gene copy number amplification during accrual of distant point mutations can potently facilitate poxvirus adaptation to host defenses.\n\nImportanceViruses can quickly evolve to defeat host immune functions. For poxviruses, little is known about how multiple adaptive mutations might concurrently emerge in populations. In this study, we uncovered a means of vaccinia virus adaptation involving the accumulation of distinct genetic variants within a single population. We identified adaptive point mutations in the viral RNA polymerase gene A24R, and show that these mutations can affect the activation of host nucleic acid sensing pathways through different mechanisms. We also found that structural variants within viral genomes in the form of gene copy number variation (CNV) provide dual benefits to evolving populations, including evidence that CNV facilitates the accumulation of a point mutation distant from the expanded locus. Our data suggest that transient CNV can accommodate the accumulation of mutations conferring modest benefits, or even fitness tradeoffs, and highlight how structural variation might aid poxvirus adaptation through both direct and indirect action.