Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe

Edited by Kevan M. Shokat, University of California, San Francisco, CA, and approved June 29, 2015 (received for review March 23, 2015)
July 27, 2015
112 (32) E4364-E4373

Significance

Many pathogens use molecular mimicry to subvert key cellular processes of the host. RNA-dependent protein kinase (PKR), a member of the eukaryotic translation initiation factor 2α (eIF2α) kinase family, is an important component of innate immunity in vertebrates and has often been subjected to inhibition by viral mimicry. In this study we show that the paradigm of host–virus mimicry extends to invertebrates where there is no discernable PKR homologue. We characterize an eIF2α kinase-mimic protein called “PK2,” encoded by baculoviruses, that inhibits a heme-regulated inhibitor kinase (HRI)-like eIF2α kinase, possibly through a lobe-swap mechanism. The inhibition of the HRI-like kinase confers a growth advantage to the baculovirus during infection of its insect host. These experiments suggest the independent emergence of eIF2α kinase antiviral defense mechanisms in vertebrates and invertebrates.

Abstract

Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by eIF2α family kinases is a conserved mechanism to limit protein synthesis under specific stress conditions. The baculovirus-encoded protein PK2 inhibits eIF2α family kinases in vivo, thereby increasing viral fitness. However, the precise mechanism by which PK2 inhibits eIF2α kinase function remains an enigma. Here, we probed the mechanism by which PK2 inhibits the model eIF2α kinase human RNA-dependent protein kinase (PKR) as well as native insect eIF2α kinases. Although PK2 structurally mimics the C-lobe of a protein kinase domain and possesses the required docking infrastructure to bind eIF2α, we show that PK2 directly binds the kinase domain of PKR (PKRKD) but not eIF2α. The PKRKD–PK2 interaction requires a 22-residue N-terminal extension preceding the globular PK2 body that we term the “eIF2α kinase C-lobe mimic” (EKCM) domain. The functional insufficiency of the N-terminal extension of PK2 implicates a role for the adjacent EKCM domain in binding and inhibiting PKR. Using a genetic screen in yeast, we isolated PK2-activating mutations that cluster to a surface of the EKCM domain that in bona fide protein kinases forms the catalytic cleft through sandwiching interactions with a kinase N-lobe. Interaction assays revealed that PK2 associates with the N- but not the C-lobe of PKRKD. We propose an inhibitory model whereby PK2 engages the N-lobe of an eIF2α kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism. Finally, we show that PK2 enhances baculovirus fitness in insect hosts by targeting the endogenous insect heme-regulated inhibitor (HRI)–like eIF2α kinase.

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Acknowledgments

We thank Keisuke Shoji for siRNA design, Dr. Harmit Malik for discussions and insights into the evolutionary analysis of PK2, Ines Drinnenberg for access to lepidopteran transcriptome assemblies, and Neroshan Thevakumaran and Dr. Daniel Mao for technical and advisory support. This work was supported in part by Canadian Institutes of Health Research Grant MOP-84370 (to F.S.), by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 19688004 and 25292196 (to S.K.), by NIH Grant GM090042 (to N.C.E.), by the Intramural Research Program of the NIH, Eunice Kennedy Shriver National Institute of Child Health and Human Development (T.E.D.), by NIH Grant P50 GM107632 (to J.M.Y.), and by an award from the Pew Charitable Trusts (to N.C.E.).

Supporting Information

Appendix (PDF)
Supporting Information

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Information & Authors

Information

Published in

The cover image for PNAS Vol.112; No.32
Proceedings of the National Academy of Sciences
Vol. 112 | No. 32
August 11, 2015
PubMed: 26216977

Classifications

Submission history

Published online: July 27, 2015
Published in issue: August 11, 2015

Keywords

  1. viral mimicry
  2. lobe-swap
  3. HRI
  4. eIF2α kinase inhibition
  5. PKR

Acknowledgments

We thank Keisuke Shoji for siRNA design, Dr. Harmit Malik for discussions and insights into the evolutionary analysis of PK2, Ines Drinnenberg for access to lepidopteran transcriptome assemblies, and Neroshan Thevakumaran and Dr. Daniel Mao for technical and advisory support. This work was supported in part by Canadian Institutes of Health Research Grant MOP-84370 (to F.S.), by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 19688004 and 25292196 (to S.K.), by NIH Grant GM090042 (to N.C.E.), by the Intramural Research Program of the NIH, Eunice Kennedy Shriver National Institute of Child Health and Human Development (T.E.D.), by NIH Grant P50 GM107632 (to J.M.Y.), and by an award from the Pew Charitable Trusts (to N.C.E.).

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

John J. Li
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada;
Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada;
Chune Cao
Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892;
Sarah M. Fixsen
Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112;
Janet M. Young
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109;
Chikako Ono
Laboratory of Applied Molecular Entomology, Division of Agrobiology, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan;
Present address: Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
Hisanori Bando
Laboratory of Applied Molecular Entomology, Division of Agrobiology, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan;
Nels C. Elde
Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112;
Susumu Katsuma2 [email protected]
Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan;
Thomas E. Dever2 [email protected]
Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892;
Frank Sicheri2 [email protected]
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada;
Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada;
Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Notes

2
To whom correspondence may be addressed. Email: [email protected], [email protected], or [email protected].
Author contributions: J.J.L., N.C.E., S.K., T.E.D., and F.S. designed research; J.J.L., C.C., S.M.F., J.M.Y., and S.K. performed research; C.O. and H.B. contributed new reagents/analytic tools; J.J.L., C.C., S.M.F., J.M.Y., N.C.E., S.K., T.E.D., and F.S. analyzed data; and J.J.L., S.M.F., J.M.Y., N.C.E., S.K., T.E.D., and F.S. wrote the paper.

Competing Interests

The authors declare no conflict of interest.

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    Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe
    Proceedings of the National Academy of Sciences
    • Vol. 112
    • No. 32
    • pp. 9787-E4509

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