Evasion of the African green monkey protein kinase R pathway by mutation of vaccinia virus RNA polymerase

Abstract

Abstract One of the earliest barriers to cross-species transmission is mediated by host restriction factors, a group of antiviral proteins that have evolved to detect and then inhibit viral replication. To productively infect a new species, viruses must rapidly adapt to overcome host-specific orthologs of these restriction factors. To model this process we created a recombinant vaccinia virus (VACV), replacing its antagonists of the host restriction factor PKR with the rhesus cytomegalovirus PKR antagonist RhTRS1 (VACVΔEΔK+RhTRS1). Relative to wildtype virus, VACVΔEΔK+RhTRS1 replication was reduced 100-fold in primary African green monkey (AGM) fibroblasts. This replication defect was mediated entirely by PKR. After several rounds of serial passage, we found that genetic amplification of rhtrs1 rescued VACVΔEΔK+RhTRS1 replication in AGM cells. Further serial passage identified mutations in two VACV genes that were individually sufficient to rescue VACVΔEΔK+RhTRS1 replication in AGM cells. Fixation of these mutations coincided with collapse of the rhtrs1 amplification. One of these mutations occurred in the catalytic subunit of the viral RNA polymerase, A24R, and provided a replication benefit independent of rhtrs1. Different mutations in A24R have been shown to prevent activation of RNase L by reducing dsRNA production during viral infection. However, the A24R mutation that evolved during this serial passage does not prevent RNase L activation in AGM cells. Furthermore, this mutation does not decrease the total concentration of dsRNA produced during infection. This work identifies new PKR evasion genes, and suggests that the viral RNA polymerase is an important modulator of multiple host innate immune responses during infection.