In contrast to RNA viruses, double-stranded DNA viruses have low mutation rates yet must still adapt rapidly in response to changing host defenses. To determine mechanisms of adaptation, we subjected ...the model poxvirus vaccinia to serial propagation in human cells, where its antihost factor K3L is maladapted against the antiviral protein kinase R (PKR). Viruses rapidly acquired higher fitness via recurrent K3L gene amplifications, incurring up to 7%–10% increases in genome size. These transient gene expansions were necessary and sufficient to counteract human PKR and facilitated the gain of an adaptive amino acid substitution in K3L that also defeats PKR. Subsequent reductions in gene amplifications offset the costs associated with larger genome size while retaining adaptive substitutions. Our discovery of viral “gene-accordions” explains how poxviruses can rapidly adapt to defeat different host defenses despite low mutation rates and reveals how classical Red Queen conflicts can progress through unrecognized intermediates.
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► Poxviruses rapidly adapt against host defenses via highly specific gene amplification ► Gains in vaccinia fitness occur within only a few serial passages in human cells ► Gene expansions precede and can facilitate adaptation via point mutation ► Gene “accordions” reveal new mode of virus adaptation in double-stranded DNA viruses
Despite low mutation rates, poxviruses evolve rapidly via gene amplifications, which facilitate the emergence of better-adapted versions of viral factors by increasing the sampling potential for mutations. Gene amplifications are only transient, revealing a gene “accordion” strategy for virus adaptation.
Cytomegaloviruses (CMVs) are generally unable to cross species barriers, in part because prolonged coevolution with one host species limits their ability to evade restriction factors in other ...species. However, the limitation in host range is incomplete. For example, rhesus CMV (RhCMV) can replicate in human cells, albeit much less efficiently than in rhesus cells. Previously we reported that the protein kinase R (PKR) antagonist encoded by RhCMV, rTRS1, has limited activity against human PKR but is nonetheless necessary and sufficient to enable RhCMV replication in human fibroblasts (HF). We now show that knockout of PKR in human cells or treatment with the eIF2B agonist ISRIB, which overcomes the translational inhibition resulting from PKR activation, augments RhCMV replication in HF, indicating that human PKR contributes to the inefficiency of RhCMV replication in HF. Serial passage of RhCMV in HF reproducibly selected for viruses with improved ability to replicate in human cells. The evolved viruses contain an inverted duplication of the terminal 6.8 kb of the genome, including rTRS1. The duplication replaces ~11.8 kb just downstream of an internal sequence element, pac1-like, which is very similar to the pac1 cleavage and packaging signal found near the terminus of the genome. Plaque-purified evolved viruses produced at least twice as much rTRS1 as the parental RhCMV and blocked the PKR pathway more effectively in HF. Southern blots revealed that unlike the parental RhCMV, viruses with the inverted duplication isomerize in a manner similar to HCMV and other herpesviruses that have internal repeat sequences. The apparent ease with which this duplication event occurs raises the possibility that the pac1-like site, which is conserved in Old World monkey CMV genomes, may serve a function in facilitating rapid adaptation to evolutionary obstacles.
Decades of research on vaccinia virus (VACV) have provided a wealth of insights and tools that have proven to be invaluable in a broad range of studies of molecular virology and pathogenesis. Among ...the challenges that viruses face are intrinsic host cellular defenses, such as the protein kinase R pathway, which shuts off protein synthesis in response to the dsRNA that accumulates during replication of many viruses. Activation of PKR results in phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α), inhibition of protein synthesis, and limited viral replication. VACV encodes two well-characterized antagonists, E3L and K3L, that can block the PKR pathway and thus enable the virus to replicate efficiently. The use of VACV with a deletion of the dominant factor, E3L, enabled the initial identification of PKR antagonists encoded by human cytomegalovirus (HCMV), a prevalent and medically important virus. Understanding the molecular mechanisms of E3L and K3L function facilitated the dissection of the domains, species-specificity, and evolutionary potential of PKR antagonists encoded by human and nonhuman CMVs. While remaining cognizant of the substantial differences in the molecular virology and replication strategies of VACV and CMVs, this review illustrates how VACV can provide a valuable guide for the study of other experimentally less tractable viruses.
During millions of years of coevolution with their hosts, cytomegaloviruses (CMVs) have succeeded in adapting to overcome host-specific immune defenses, including the protein kinase R (PKR) pathway. ...Consequently, these adaptations may also contribute to the inability of CMVs to cross species barriers. Here, we provide evidence that the evolutionary arms race between the antiviral factor PKR and its CMV antagonist TRS1 has led to extensive differences in the species-specificity of primate CMV TRS1 proteins. Moreover, we identify a single residue in human PKR that when mutated to the amino acid present in African green monkey (Agm) PKR (F489S) is sufficient to confer resistance to HCMVTRS1. Notably, this precise molecular determinant of PKR resistance has evolved under strong positive selection among primate PKR alleles and is positioned within the αG helix, which mediates the direct interaction of PKR with its substrate eIF2α. Remarkably, this same residue also impacts sensitivity to K3L, a poxvirus-encoded pseudosubstrate that structurally mimics eIF2α. Unlike K3L, TRS1 has no homology to eIF2α, suggesting that unrelated viral genes have convergently evolved to target this critical region of PKR. Despite its functional importance, the αG helix exhibits extraordinary plasticity, enabling adaptations that allow PKR to evade diverse viral antagonists while still maintaining its critical interaction with eIF2α.
Detection of intracellular DNA triggers activation of the STING-dependent interferon-stimulatory DNA (ISD) pathway, which is essential for antiviral responses. Multiple DNA sensors have been proposed ...to activate this pathway, including AIM2-like receptors (ALRs). Whether the ALRs are essential for activation of this pathway remains unknown. To rigorously explore the function of ALRs, we generated mice lacking all 13 ALR genes. We found that ALRs are dispensable for the type I interferon (IFN) response to transfected DNA ligands, DNA virus infection, and lentivirus infection. We also found that ALRs do not contribute to autoimmune disease in the Trex1−/− mouse model of Aicardi-Goutières Syndrome. Finally, CRISPR-mediated disruption of the human AIM2-like receptor IFI16 in primary fibroblasts revealed that IFI16 is not essential for the IFN response to human cytomegalovirus infection. Our findings indicate that ALRs are dispensable for the ISD response and suggest that alternative functions for these receptors should be explored.
•Generated mice lacking all 13 mouse ALR genes to explore the function of ALRs•ALRs are not required for the ISD response or autoimmune disease in Trex1−/− mice•The human ALR IFI16 is not essential for the IFN response to HCMV infection.•ALR gene expression is altered in Aim2–/– (B6.129) cells due to 129-derived SNPs
Detection of intracellular DNA triggers an antiviral type I interferon response. Multiple DNA sensors have been proposed to trigger this response, including AIM2-like receptors (ALRs). Gray and colleagues generated mice lacking all 13 mouse ALR genes and show that ALRs are dispensable for the interferon response to intracellular DNA.
Herpesviruses present a major global disease burden. Understanding the host cell mechanisms that block viral infections, as well as how viruses can evolve to counteract these host defenses, is ...critically important for understanding viral disease pathogenesis. This study reveals that the major human variant of the antiviral protein myxovirus resistance protein B (MxB) inhibits the human pathogen herpes simplex virus (HSV-1), whereas a minor human variant and orthologous MxB genes from even closely related primates do not. Thus, in contrast to the many antagonistic virus-host interactions in which the virus is successful in thwarting the host's defense systems, here the human gene appears to be at least temporarily winning at this interface of the primate-herpesvirus evolutionary arms race. Our findings further show that a polymorphism at amino acid 83 in a small fraction of the human population is sufficient to abrogate MxB's ability to inhibit HSV-1, which could have important implications for human susceptibility to HSV-1 pathogenesis.
Distinguishing self from non-self is a fundamental biological challenge. Many pathogens exploit the challenge of self discrimination by employing mimicry to subvert key cellular processes including ...the cell cycle, apoptosis and cytoskeletal dynamics. Other mimics interfere with immunity. Poxviruses encode K3L, a mimic of eIF2 , which is the substrate of protein kinase R (PKR), an important component of innate immunity in vertebrates. The PKR-K3L interaction exemplifies the conundrum imposed by viral mimicry. To be effective, PKR must recognize a conserved substrate (eIF2 ) while avoiding rapidly evolving substrate mimics such as K3L. Using the PKR-K3L system and a combination of phylogenetic and functional analyses, we uncover evolutionary strategies by which host proteins can overcome mimicry. We find that PKR has evolved under intense episodes of positive selection in primates. The ability of PKR to evade viral mimics is partly due to positive selection at sites most intimately involved in eIF2 recognition. We also find that adaptive changes on multiple surfaces of PKR produce combinations of substitutions that increase the odds of defeating mimicry. Thus, although it can seem that pathogens gain insurmountable advantages by mimicking cellular components, host factors such as PKR can compete in molecular 'arms races' with mimics because of evolutionary flexibility at protein interaction interfaces challenged by mimicry.
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