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Herpes simplex virus 1 protein pUL21 alters ceramide metabolism by activating the interorganelle transport protein CERT.

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Benedyk, Tomasz H 
Connor, Viv 
Caroe, Eve R 
Shamin, Maria 
Svergun, Dmitri I 


Herpes simplex virus (HSV)-1 dramatically alters the architecture and protein composition of cellular membranes during infection, but its effects upon membrane lipid composition remain unclear. HSV-1 pUL21 is a virus-encoded protein phosphatase adaptor that promotes dephosphorylation of multiple cellular and virus proteins, including the cellular ceramide (Cer) transport protein CERT. CERT mediates nonvesicular Cer transport from the endoplasmic reticulum to the trans-Golgi network, whereupon Cer is converted to sphingomyelin (SM) and other sphingolipids that play important roles in cellular proliferation, signaling, and membrane trafficking. Here, we use click chemistry to profile the kinetics of sphingolipid metabolism, showing that pUL21-mediated dephosphorylation activates CERT and accelerates Cer-to-SM conversion. Purified pUL21 and full-length CERT interact with submicromolar affinity, and we solve the solution structure of the pUL21 C-terminal domain in complex with the CERT Pleckstrin homology and steroidogenic acute regulatory-related lipid transfer domains using small-angle X-ray scattering. We identify a single amino acid mutation on the surface of pUL21 that disrupts CERT binding in vitro and in cultured cells. This residue is highly conserved across the genus Simplexvirus. In addition, we identify a pUL21 residue essential for binding to HSV-1 pUL16. Sphingolipid profiling demonstrates that Cer-to-SM conversion is severely diminished in the context of HSV-1 infection, a defect that is compounded when infecting with a virus encoding the mutated form of pUL21 that lacks the ability to activate CERT. However, virus replication and spread in cultured keratinocytes or epithelial cells is not significantly altered when pUL21-mediated CERT dephosphorylation is abolished. Collectively, we demonstrate that HSV-1 modifies sphingolipid metabolism via specific protein-protein interactions.



ceramide transport, herpesvirus, lipid metabolism, protein phosphatases, virus–host interactions, Herpesvirus 1, Human, Carrier Proteins, Protein Serine-Threonine Kinases, Ceramides, Sphingomyelins, Sphingolipids, Biological Transport, Viral Proteins, Golgi Apparatus

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J Biol Chem

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Elsevier BV
Wellcome Trust (098406/Z/12/B)
Biotechnology and Biological Sciences Research Council (BB/M021424/1)
MRC (MR/T016493/1)
Wellcome Trust (219447/Z/19/Z)
Wellcome Trust (098406/Z/12/Z)
This work was funded by a Royal Society University Research Fellowship (UF100371), a Royal Society Enhancement Award (RGF\EA\180151) and a Wellcome Trust Senior Research Fellowship (219447/Z/19/Z) to JED, a Biotechnology and Biological Sciences Research Council (BBSRC) Research Grant (BB/M021424/1) and a Medical Research Council Research Grant (MR/T016493/1) to CMC, and by a Sir Henry Dale Fellowship, jointly funded by the Wellcome Trust and the Royal Society (098406/Z/12/B) to SCG. DIS and CMJ acknowledge the support of iNEXT-Discovery (project number 871037) funded by the Horizon 2020 programme of the European Commission. A Titan V graphics card used for this research was donated by the NVIDIA Corporation.
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