Cytomegaloviruses (CMVs) are a group of pathogens that replicate within many mammalian species, including humans and monkeys. After initial infection, these viruses evade the immune system and persist within a host for its lifetime, largely asymptomatically. However, CMVs cause life-threatening complications in immunocompromised individuals and congenital brain defects in newborns. The ability of CMVs to evade the host immune system relies on its success in an evolutionary ‘arms-race’ with host anti-viral immunity factors. For CMV this arms-race is extremely specific and contributes to the inability of human CMVs to cross species barriers. An important anti-viral mechanism is mediated by Protein Kinase R (PKR) that broadly inhibits translation initiation when activated by double-stranded RNA (dsRNA), which is a common by-product of viral replication but rare in uninfected mammalian cells. To bypass this mechanism CMVs encode host-specific PKR antagonists called TRS1 that inhibit kinase activity. Fred Hutch researchers in the Geballe Lab (Human Biology Division) are leveraging the species-specific co-evolution of PKR and TRS1 to understand human anti-viral immunity mechanisms. In a recent publication in PLoS Pathogens Kathryn Carpentier and colleagues identified a single amino acid in human PKR that is critical for inhibition by human CMV TRS1.
CMVs are unable to cross species barriers so little is known about how evolutionary changes in PKR has affected the evolution of TRS1. To deliver TRS1 homologs from monkeys to human cells researchers used recombinant vaccinia virus (VacV) in the VacV gene that blocks PKR is removed and replaced with the TRS1 gene from human (HCMV), African green monkey (AgmCMV), rhesus monkey (RhCMV) specific CMVs. All of these recombinant VacV strains could replicate in human HeLa cells lacking the PKR gene; however, when PKR was present the results were very different. HCMV TRS1 successfully inhibited PKR and allowed replication but both AgmCMV and RhCMV TRS1 genes failed to inhibit PKR and the viruses could not replicate.
To understand which region in PKR is important for the interaction with TRS1 researchers created chimeric PKR containing mostly Human PKR (HuPKR) with small regions replaced by the sequences from African green monkey PKR (AgmPKR). This approach revealed a 45 amino acid region at the C-terminus of HuPKR that was responsible for the species-specific response to TRS1. Within this region only six amino acids diverge between HuPKR and AgmPKR. Surprisingly, replacement of a single mutation in HuPKR with the corresponding AgmPKR sequence (F489S) within the alpha G helix prevented HCMV TRS1 from inhibiting HuPKR. This activity was verified in an infection context where cells carrying the mutant HuPKR F489S were resistant to infection with the virus containing HCMV TRS1.
Next researchers uncovered how this mutation confers HCMV TRS1 resistance. HuPKR stops viral replication by phosphorylating and inactivating translation initiation factors. This basic mechanism is the same for HuPKR F489S. This mutation does not affect HuPKR activity but rather prevents the physical association of HuPKR and HCMV TRS1. Although almost all other mutations at position 489 do not affect the ability of PKR to inactivate translation, most (75%) of them are resistant to inhibition by human CMV TRS1. Despite the fact that this position tolerates many mutations in these experiments, PKR in general is rarely mutated in humans, said Dr. Geballe, “In fact, PKR is not polymorphic in humans; in ~100,000 alleles we found in a database, there are almost no polymorphisms. In contrast, in even just a small number of other primates in which the gene has been sequenced there are multiple polymorphisms.”
The alpha G helix of PKR seems to be a critical site of interaction with viral factors. Consistent with this idea, poxviruses also inhibit PKR by targeting the alpha G helix with an antagonist. The F489S mutation in HuPKR actually conferred resistance not just to HCMV TRS1 but poxvirus K3L too. Thus it is important to think about PKR evolution in the context of many dsRNA viruses simultaneously pressuring on PKR to adapt. Dr. Geballe explained, “One plan we have is to take advantage of the F489S point mutant to better understand how the viral protein (TRS1) interacts with PKR. For example, we are trying to experimentally select a mutant virus in which HCMV TRS1 acquires the ability to bind to and inhibit the F489S human PKR allele. A surprise emerging from this work was our finding that one residue in the alpha G helix seems to be targeted by at least two unrelated viral antagonists. We are interested in exploring further how mutable the alpha G helix is and whether other viruses also target it. This seems to be a hot spot in the host-virus arms race between PKR and at least some of its antagonists.”
Carpentier KS, Esparo NM, Child SJ, Geballe AP. 2016. A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists. PLoS Pathog, 12(10), e1005966.
Funding for this research was provided by the National Institutes of Health
Basic Sciences Division
Human Biology Division
Maggie Burhans, Ph.D.
Public Health Sciences Division
Vaccine and Infectious Disease Division
Clinical Research Division
Julian Simon, Ph.D.
Clinical Research Division
and Human Biology Division
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