Rapid adaptation to human protein kinase R by a unique genomic rearrangement in rhesus cytomegalovirus

ABSTRACTCytomegaloviruses (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 fitness 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.AUTHOR SUMMARYRhesus macaque CMV (RhCMV) is an important model for human CMV (HCMV) pathogenesis and vaccine development. Therefore, it is important to understand the similarities and differences in infectivity and interaction of these viruses with their host species. In contrast to the strict species-specificity of HCMV, RhCMV is able to cross species barriers to replicate in human cells. We know from past work that a component of this broader host range is RhCMV’s ability to counteract both the rhesus and human versions of a key antiviral factor. Here we delve further into the mechanisms by which RhCMV can adapt to counteract human cellular defenses. We find that RhCMV appears to be poised to undergo a specific genomic rearrangement that facilitates increased replication efficiency in human cells. Besides providing insights into CMV species-specificity and host barriers to cross-species transmission, this work also provides more generalized clues about viral adaptative mechanisms.

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Rapid adaptation to human protein kinase R by a unique genomic rearrangement in rhesus cytomegalovirus

PLoS Pathogens ◽

10.1371/journal.ppat.1009088 ◽

2021 ◽

Vol 17 (1) ◽

pp. e1009088

Author(s):

Stephanie J. Child ◽

Alexander L. Greninger ◽

Adam P. Geballe

Keyword(s):

Protein Kinase ◽

World Monkey ◽

Human Fibroblasts ◽

Serial Passage ◽

Duplication Event ◽

Human Cells ◽

Rapid Adaptation ◽

Protein Kinase R ◽

Sequence Element ◽

Inverted Duplication

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.

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Antagonism of the Protein Kinase R Pathway in Human Cells by Rhesus Cytomegalovirus

Journal of Virology ◽

10.1128/jvi.01793-17 ◽

2017 ◽

Vol 92 (6) ◽

Cited By ~ 3

Author(s):

Stephanie J. Child ◽

Sarah E. Hickson ◽

Avraham Bayer ◽

Daniel Malouli ◽

Klaus Früh ◽

...

Keyword(s):

Protein Kinase ◽

Host Range ◽

Rhesus Macaque ◽

Host Species ◽

Vaccine Development ◽

Human Fibroblasts ◽

Human Cells ◽

Translation Initiation Factor ◽

Restriction Factor ◽

Protein Kinase R

ABSTRACTWhile cytomegalovirus (CMV) infections are often limited in host range by lengthy coevolution with a single host species, a few CMVs are known to deviate from this rule. For example, rhesus macaque CMV (RhCMV), a model for human CMV (HCMV) pathogenesis and vaccine development, can replicate in human cells, as well as in rhesus cells. Both HCMV and RhCMV encode species-specific antagonists of the broadly acting host cell restriction factor protein kinase R (PKR). Although the RhCMV antagonist of PKR, rTRS1, has very limited activity against human PKR, here, we show it is essential for RhCMV replication in human cells because it prevents human PKR from phosphorylating the translation initiation factor eIF2α, thereby allowing continued translation and viral replication. Although rTRS1 is necessary for RhCMV replication, it is not sufficient to rescue replication of HCMV lacking its own PKR antagonists in human fibroblasts. However, overexpression of rTRS1 in human fibroblasts enabled HCMV expressing rTRS1 to replicate, indicating that elevated levels or early expression of a weak antagonist can counteract a resistant restriction factor like human PKR. Exploring potential mechanisms that might allow RhCMV to replicate in human cells revealed that RhCMV makes no less double-stranded RNA than HCMV. Rather, in human cells, RhCMV expresses rTRS1 at levels 2 to 3 times higher than those of the HCMV-encoded PKR antagonists during HCMV infection. These data suggest that even a modest increase in expression of this weak PKR antagonist is sufficient to enable RhCMV replication in human cells.IMPORTANCERhesus macaque cytomegalovirus (RhCMV) offers a valuable model for studying congenital human cytomegalovirus (HCMV) pathogenesis and vaccine development. Therefore, it is critical to understand variations in how each virus infects and affects its host species to be able to apply insights gained from the RhCMV model to HCMV. While HCMV is capable only of infecting cells from humans and very closely related species, RhCMV displays a wider host range, including human as well as rhesus cells. RhCMV expresses an antagonist of a broadly acting antiviral factor present in all mammalian cells, and its ability to counter both the rhesus and human versions of this host factor is a key component of RhCMV's ability to cross species barriers. Here, we examine the molecular mechanisms that allow this RhCMV antagonist to function against a human restriction factor.

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Species Specificity of Protein Kinase R Antagonism by Cytomegalovirus TRS1 Genes

Journal of Virology ◽

10.1128/jvi.02155-12 ◽

2012 ◽

Vol 86 (22) ◽

pp. 12470-12470

Author(s):

S. J. Child ◽

G. Brennan ◽

J. E. Braggin ◽

A. P. Geballe

Keyword(s):

Protein Kinase ◽

Species Specificity ◽

Protein Kinase R

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Short Interfering RNA Inhibits Rift Valley Fever Virus Replication and Degradation of Protein Kinase R in Human Cells

Frontiers in Microbiology ◽

10.3389/fmicb.2016.01889 ◽

2016 ◽

Vol 7 ◽

Cited By ~ 5

Author(s):

Bonto Faburay ◽

Juergen A. Richt

Keyword(s):

Protein Kinase ◽

Virus Replication ◽

Rift Valley ◽

Rift Valley Fever ◽

Rift Valley Fever Virus ◽

Human Cells ◽

Short Interfering Rna ◽

Protein Kinase R ◽

Valley Fever ◽

Interfering Rna

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Species Specificity of Protein Kinase R Antagonism by Cytomegalovirus TRS1 Genes

Journal of Virology ◽

10.1128/jvi.06158-11 ◽

2012 ◽

Vol 86 (7) ◽

pp. 3880-3889 ◽

Cited By ~ 20

Author(s):

S. J. Child ◽

G. Brennan ◽

J. E. Braggin ◽

A. P. Geballe

Keyword(s):

Protein Kinase ◽

Species Specificity ◽

Protein Kinase R

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Highly Pathogenic New World Arenavirus Infection Activates the Pattern Recognition Receptor Protein Kinase R without Attenuating Virus Replication in Human Cells

Journal of Virology ◽

10.1128/jvi.01090-17 ◽

2017 ◽

Vol 91 (20) ◽

Cited By ~ 10

Author(s):

Cheng Huang ◽

Olga A. Kolokoltsova ◽

Elizabeth J. Mateer ◽

Takaaki Koma ◽

Slobodan Paessler

Keyword(s):

Protein Kinase ◽

Virus Replication ◽

New World ◽

Human Cells ◽

Host Protein ◽

Initiation Factor ◽

Protein Kinase R ◽

Highly Pathogenic ◽

Rna Sensors ◽

Infected Cells

ABSTRACT The arenavirus family consists of several highly pathogenic viruses, including the Old World (OW) arenavirus Lassa fever virus (LASV) and the New World (NW) Junin virus (JUNV) and Machupo virus (MACV). Host response to infection by these pathogenic arenaviruses is distinct in many aspects. JUNV and MACV infections readily induce an interferon (IFN) response in human cells, while LASV infection usually triggers an undetectable or weak IFN response. JUNV induces an IFN response through RIG-I, suggesting that the host non-self RNA sensor readily detects JUNV viral RNAs (vRNAs) during infection and activates IFN response. Double-stranded-RNA (dsRNA)-activated protein kinase R (PKR) is another host non-self RNA sensor classically known for its vRNA recognition activity. Here we report that infection with NW arenaviruses JUNV and MACV, but not OW LASV, activated PKR, concomitant with elevated phosphorylation of the translation initiation factor α subunit of eukaryotic initiation factor 2 (eIF2α). Host protein synthesis was substantially suppressed in MACV- and JUNV-infected cells but was only marginally reduced in LASV-infected cells. Despite the antiviral activity known for PKR against many other viruses, the replication of JUNV and MACV was not impaired but was slightly augmented in wild-type (wt) cells compared to that in PKR-deficient cells, suggesting that PKR or PKR activation did not negatively affect JUNV and MACV infection. Additionally, we found an enhanced IFN response in JUNV- or MACV-infected PKR-deficient cells, which was inversely correlated with virus replication. IMPORTANCE The detection of viral RNA by host non-self RNA sensors, including RIG-I and MDA5, is critical to the initiation of the innate immune response to RNA virus infection. Among pathogenic arenaviruses, the OW LASV usually does not elicit an interferon response. However, the NW arenaviruses JUNV and MACV readily trigger an IFN response in a RIG-I-dependent manner. Here, we demonstrate for the first time that pathogenic NW arenaviruses JUNV and MACV, but not the OW arenavirus LASV, activated the dsRNA-dependent PKR, another host non-self RNA sensor, during infection. Interestingly, the replication of JUNV and MACV was not restricted but was rather slightly augmented in the presence of PKR. Our data provide new evidence for a distinct interplay between host non-self RNA sensors and pathogenic arenaviruses, which also provides insights into the pathogenesis of arenaviruses and may facilitate the design of vaccines and treatments against arenavirus-caused diseases.

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