Cut, flip, paste: unique genomic rearrangement enables improved human PKR antagonism

From the Geballe Lab, Human Biology Division

As the 2020 SARS-CoV-2 pandemic has brought into intense focus, viruses can switch from one host to another, sometimes leading to severe disease in the new host. Introduction of a novel virus can lead to a rapid spread in a population without pre-existing immunity. The infection in the natural host could look very different, often causing little disease yet replicating at high levels and infecting a large portion of the population. The lack of pathogenicity in the natural host might reflect an adaptation where the host-virus interaction has achieved a well-balanced relationship dependent on viral and host adaptations that developed over evolutionary time.

Cytomegaloviruses (CMVs), a type of herpesvirus, represent a prime example of host-virus co-evolution as CMVs have been co-speciating with their host lineages for approximately 60 to 80 million years. Not surprisingly, this long-standing relationship has resulted in a fairly strict species-specificity. In other words, a species-specific CMV will only be capable of infecting cells from a natural host and, at best, replicate poorly in a new host.

Researchers in the Geballe lab are interested in understanding the mechanisms and evolution underlying the interactions between large DNA viruses such as CMV and their host cells. Viruses like CMV are large and complex, and there are significant gaps in understanding how they have evolved to be successful pathogens. In a new study published in the journal PLoS Pathogens, the group describes a genome rearrangement event in rhesus CMV as a mechanism for rapid adaptation to overcome evolutionary obstacles. 

Schematic showing the deletion and inverted duplication events that yielded the EV genomes.
Schematic showing the deletion and inverted duplication events that yielded the EV genomes. Image provided by Dr. Stephanie Child

The Geballe lab has previously shown that one of the factors limiting CMV cross-species transmission is the host restriction factor protein kinase R (PKR). HCMV encodes two PKR antagonists IRS1 and TRS1, which potently block human PKR. Interestingly, the RhCMV genome encodes for rTRS1, a potent rhesus PKR antagonist with weak human PKR antagonism. However, this level of antagonism is sufficient to allow limited RhCMV replication in human cells. The investigators thus asked: Can RhCMV adapt to fully overcome human PKR restriction? And, if so, what would be the genetic basis of this gain-of-function?

To answer this question, the investigators designed and executed an in vitro evolution experiment where RhCMV was serially passaged in human fibroblasts. Each passage yielded viruses with increased replication efficiency relative to the parental virus. Deep sequencing of the pooled evolved viruses revealed a large deletion (>11kb) and an inverted duplication inserted in the corresponding deleted region. PCR analysis and sequencing of plaque-isolated evolved viruses showed that the duplication event includes a segment containing rTRS1, which resulted in its overexpression.

Dr. Stephanie Child, senior staff scientist and first author in the publication, explained the functional implications of the new genomic rearrangement in the evolved viruses: "Human cytomegalovirus and several other herpesviruses possess complex genome structures with two connected regions, one long and one short, that are each flanked by inverted repeats. These repeats enable the segments to flip independently of each other during DNA replication, yielding an equal mixture of four genomic isomers. However, Rhesus cytomegalovirus is one of those herpesviruses that have a simple genome structure, with just one unique region flanked by direct terminal repeats, so only a single genomic arrangement is produced.”

Surprisingly, Southern blot analyses showed that the inverted duplication present in serially passaged RhCMV generates a complex genome structure that enables genome isomerization. Dr. Child added, “When we began analyzing the RhCMV viruses that had been evolved in human cells, it took us awhile to decipher the data and solve the puzzle that the inverted duplication driven by adaptation of Rhesus cytomegalovirus to human PKR created a genomic structure that can now isomerize. We were quite surprised by this, but excited by the evolutionary clues that this type of adaptation might suggest." 

Dr. Adam Geballe, principal investigator in the study, highlighted the implications of the study: "Our studies uncovered a small genomic element common among Old World monkey CMVs that seems poised to facilitate adaptation when the viruses encounter an unfamiliar host or cellular changes in their natural host, pressures that may have played a part in shaping some of the variation in genomic structure found in different herpesviruses."

Child, S. J., Greninger, A. L., & Geballe, A. P. (2021). Rapid adaptation to human protein kinase R by a unique genomic rearrangement in rhesus cytomegalovirus. PLoS pathogens17(1), e1009088.

Fred Hutch/UW Cancer Consortium member Adam P. Geballe contributed to this study.

This work was supported by grants from the National Institute of Allergy and Infectious Diseases and by the Genomic Core Shared Resource of the Fred Hutch/University of Washington Cancer Consortium