Experimental Evolution Identifies Vaccinia Virus Mutations in A24R and A35R That Antagonize the Protein Kinase R Pathway and Accompany Collapse of an Extragenic Gene Amplification

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Abstract
Most new human infectious diseases emerge from cross-species pathogen transmissions; however, it's not clear how viruses adapt to productively infect new hosts. Host restriction factors represent one species-specific barrier that viruses may initially have little ability to inhibit in new hosts. For example, viral antagonists of protein kinase R (PKR) vary in their ability to block PKR-mediated inhibition of viral replication, in part due to PKR allelic variation between species. We previously reported that amplification of a weak PKR antagonist encoded by rhesus cytomegalovirus, rhtrs1, improved replication of a recombinant poxvirus (VVΔEΔK+RhTRS1) in several resistant primate cells. To test whether amplification increases the opportunity for mutations to evolve that improve virus replication with only a single copy of rhtrs1, we passaged rhtrs1-amplified viruses in semi-permissive primate cells. After passage, we isolated two viruses that contained only a single copy of rhtrs1 yet replicated as well as the amplified virus. Surprisingly, rhtrs1 was not mutated in these viruses; instead, we identified mutations in two VACV genes, A24R and A35R, either of which was sufficient to improve VVΔEΔK+RhTRS1 replication. Neither of these genes has previously been implicated in PKR antagonism. Furthermore, the mutation in A24R, but not A35R, increased resistance to the anti-poxviral drug isatin-β-thiosemicarbazone, suggesting that these mutations employ different mechanisms to evade PKR. This study supports our hypothesis that gene amplification may provide a “molecular foothold”, broadly improving replication to facilitate rapid adaptation while subsequent mutations maintain this efficient replication in the new host without requiring gene amplification.

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