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Characterization of the Golgi c10orf76-PI4KB complex, and its necessity for Golgi PI4P levels and enterovirus replication

J.A. McPhail, H.R. Lyoo, J.G. Pemberton, View ORCID ProfileR.M. Hoffmann, W. van Elst, View ORCID ProfileJ.R.P.M. Strating, M.L. Jenkins, J.T.B. Stariha, F.J.M. van Kuppeveld, View ORCID ProfileT. Balla, View ORCID ProfileJ.E. Burke
doi: https://doi.org/10.1101/634592
J.A. McPhail
1Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada
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H.R. Lyoo
2Department of Infectious Diseases & Immunology, Virology Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
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J.G. Pemberton
3Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
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R.M. Hoffmann
1Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada
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  • ORCID record for R.M. Hoffmann
W. van Elst
2Department of Infectious Diseases & Immunology, Virology Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
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J.R.P.M. Strating
2Department of Infectious Diseases & Immunology, Virology Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
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  • ORCID record for J.R.P.M. Strating
M.L. Jenkins
1Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada
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J.T.B. Stariha
1Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada
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F.J.M. van Kuppeveld
2Department of Infectious Diseases & Immunology, Virology Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
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  • For correspondence: jeburke@uvic.ca ballat@mail.nih.gov F.J.M.vanKuppeveld@uu.nl
T. Balla
3Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
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  • ORCID record for T. Balla
  • For correspondence: jeburke@uvic.ca ballat@mail.nih.gov F.J.M.vanKuppeveld@uu.nl
J.E. Burke
1Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada
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  • ORCID record for J.E. Burke
  • For correspondence: jeburke@uvic.ca ballat@mail.nih.gov F.J.M.vanKuppeveld@uu.nl
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Summary

The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi, and is essential for the viral replication of specific enteroviruses. We used hydrogen deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 was also required for proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.

Highlights

  • c10orf76 forms a direct complex with PI4KB, with the interface formed by a disorder-to-order transition in the kinase linker of PI4KB

  • The c10orf76 binding site of PI4KB can be phosphorylated by PKA, with phosphorylation leading to decreased affinity for c10orf76

  • Complex-disrupting mutants of PI4KB and c10orf76 reveal that PI4KB recruits c10orf76 to the Golgi/TGN

  • Depletion of c10orf76 leads to decreases in both active Arf1 and Golgi PI4P levels

  • Enteroviruses that rely on c10orf76 for replication depend on formation of the c10orf76-PI4KB complex

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted May 10, 2019.
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Characterization of the Golgi c10orf76-PI4KB complex, and its necessity for Golgi PI4P levels and enterovirus replication
J.A. McPhail, H.R. Lyoo, J.G. Pemberton, R.M. Hoffmann, W. van Elst, J.R.P.M. Strating, M.L. Jenkins, J.T.B. Stariha, F.J.M. van Kuppeveld, T. Balla, J.E. Burke
bioRxiv 634592; doi: https://doi.org/10.1101/634592
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Characterization of the Golgi c10orf76-PI4KB complex, and its necessity for Golgi PI4P levels and enterovirus replication
J.A. McPhail, H.R. Lyoo, J.G. Pemberton, R.M. Hoffmann, W. van Elst, J.R.P.M. Strating, M.L. Jenkins, J.T.B. Stariha, F.J.M. van Kuppeveld, T. Balla, J.E. Burke
bioRxiv 634592; doi: https://doi.org/10.1101/634592

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