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

Abstract

Most new human infectious diseases emerge from cross-species pathogen transmissions; however, it is 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 (VV⌬E⌬K؉RhTRS1) in several resistant primate cells. To test whether amplification increases the opportunity for mutations that improve virus replication with only a single copy of rhtrs1 to evolve, we passaged rhtrs1-amplified viruses in semipermissive 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 vaccinia virus (VACV) genes, A24R and A35R, either of which was sufficient to improve VV⌬E⌬K؉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 antipoxviral drug isatin-␤-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. IMPORTANCEUnderstanding how viruses adapt to a new host may help identify viruses poised to cross species barriers before an outbreak occurs. Amplification of rhtrs1, a weak viral antagonist of the host antiviral protein PKR, enabled a recombinant vaccinia virus to replicate in resistant cells from humans and other primates. After serial passage of rhtrs1-amplified viruses, there arose in two vaccinia virus genes mutations that improved viral replication without requiring rhtrs1 amplification. Neither of these genes has previously been associated with inhibition of the PKR pathway. These data suggest that gene amplification can improve viral replication in a resistant host species and facilitate the emergence of novel adaptations that maintain the foothold needed for continued replication and spread in the new host. Of the approximately 1,400 known human pathogens, over 60% are zoonotic (1). Furthermore, during the last 80 years, more than 70% of all new and emerging human infectious diseases were acquired from animal sources (2). These emerging zoonoses are often responsible for severe disease in humans, such as HIV-1 infection/AIDS (3) and the recent Ebola pandemic in West Africa (4). Similarly, pathogen transmission between both wild and domes...

Emergence of a Viral RNA Polymerase Variant during Gene Copy Number Amplification Promotes Rapid Evolution of Vaccinia Virus

“…Furthermore, another A24R mutation, resulting in a Lys452Asn amino acid substitution, was identified in a replicate population independently of the Leu18Phe variant. This result suggests that the A24R gene may be a common target for beneficial mutations in VACV, consistent with previous reports of other adaptive A24R mutations (20)(21)(22). Together, these high-frequency mutations suggest a common role for poxvirus genes encoding essential viral functions in adaptation to activated host innate immune responses and an altered cellular environment.…”

“…In this way, recombination-based CNV may enhance the viability of an expanded set of beneficial mutations that otherwise suffer from trade-offs (e.g., activation of PKR) and might otherwise be unable to sweep through populations. Given that copy number amplification events are likely to be transient (14), this foothold may be temporary, as suggested by previous work describing the accumulation of beneficial point mutations causing the collapse of CNV (20). In the current study, CNV of the K3L locus persisted through passage 10 ( Fig.…”

“…Vaccinia virus has proven to be a useful model for experimental evolution and continues to reveal the genetic basis of various poxvirus adaptations (14,20,25). We charted the rise of multiple point mutations over the course of 10 serial infections, which is consistent with adaptive evolution through several independent mechanisms.…”

“…These studies suggest that changes to transcript length, dictated by vRNAP, can influence the activation of innate immune dsRNA sensors in the cell. Indeed, another A24R mutation was recently identified that reduces the activation of the dsRNA sensor PKR (20). Without E3L, viruses are more vulnerable to PKR and OAS activities, and thus we hypothesized that the A24R substitutions we identified might act through a similar mechanism to reduce the activation of dsRNA sensors.…”

An Evolutionary View of the Arms Race between Protein Kinase R and Large DNA Viruses

“…A similar cycle of gene amplification, mutation, and collapse was observed upon serial passage of a VACV expressing RhCMV TRS1 as its sole PKR antagonist in cells containing a relatively resistant PKR allele (33). In this case, the RhCMV TRS1 locus amplified, but subsequent collapse of the locus appeared to be enabled by mutations in other loci affecting genes not previously implicated in PKR pathway antagonism (34).…”