Biology subjects

Benedyk, T. H.

Publications and source records attributed to Benedyk, T. H..

3 recordsLinked to original sources

bioRxiv2022

Herpes simplex virus 1 protein pUL21 stimulates cellular ceramide transport by activating CERT

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 transport protein CERT. CERT mediates non- vesicular transport of ceramide from the ER to the trans-Golgi network, whereupon ceramide is converted to sphingomyelin and other sphingolipids that play important roles in cell proliferation, cell signalling and membrane trafficking. Using click chemistry to profile the kinetics of sphingolipid metabolism in cultured cells, we show that pUL21-mediated dephosphorylation activates CERT and increases the rate of ceramide to sphingomyelin conversion. Purified pUL21 and full-length CERT interact with sub-micromolar affinity and we map the domains responsible for the interaction. Solving the solution structure of the pUL21 C-terminal domain in complex with the CERT PH and START domains using small-angle X-ray scattering allows us to identify a single amino acid mutation on the surface of pUL21 that disrupts CERT binding in vitro and in cultured cells. Sphingolipid profiling demonstrates that ceramide to sphingomyelin 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 are not significantly altered when pUL21-mediated CERT dephosphorylation is abolished, highlighting that dephosphorylation of other cellular and/or viral targets underpins the important role of pUL21 in HSV-1 biology. SignificanceHerpes simplex virus (HSV)-1 causes a life-long dormant infection of neurons, sporadically reactivating to manifest as cold-sores or genital herpes. While the impact of HSV-1 upon the protein content of infected cells has been well studied, we know relatively little about its impact upon cellular lipids. Using bioorthogonal labelling in cultured cells we show that HSV-1 protein pUL21 activates the key cellular lipid transport protein CERT to accelerate the conversion of ceramide to sphingomyelin. HSV-1 infection dramatically alters the kinetics of ceramide metabolism, leading to ceramide accumulation. Mutation of HSV-1 pUL21 to prevent CERT activation further enhances ceramide accumulation but this does not alter the replication or spread of HSV-1, highlighting that other cellular and/or viral proteins represent the critical targets of pUL21-mediated dephosphorylation in cultured cells.

microbiology↗

bioRxiv2021

pUL21 is a viral phosphatase adaptor that promotes herpes simplex virus replication and spread

The herpes simplex virus (HSV)-1 protein pUL21 is essential for efficient virus replication and dissemination. While pUL21 has been shown to promote multiple steps of virus assembly and spread, the molecular basis of its function remained unclear. Here we identify that pUL21 is a virus-encoded adaptor of protein phosphatase 1 (PP1). pUL21 directs the dephosphorylation of cellular and virus proteins, including components of the viral nuclear egress complex, and we define a conserved non-canonical linear motif in pUL21 that is essential for PP1 recruitment. In vitro evolution experiments reveal that pUL21 directly antagonises the activity of the virus-encoded kinase pUS3, with growth and spread of pUL21 PP1-binding mutant viruses being restored when pUS3 activity is disrupted. This study shows that virus-directed phosphatase activity is essential for efficient herpesvirus assembly and spread, highlighting the fine balance between kinase and phosphatase activity required for optimal virus replication.

microbiology↗

bioRxiv2019

The lipid transfer protein Saposin B does not directly bind CD1d for lipid antigen loading

Lipid antigens are presented on the surface of cells by the CD1 family of glycoproteins, which have structural and functional similarity to MHC class I molecules. The hydrophobic lipid antigens are embedded in membranes and inaccessible to the lumenal lipid-binding domain of CD1 molecules. Therefore, CD1 molecules require lipid transfer proteins for lipid loading and editing. CD1d is loaded with lipids in late endocytic compartments, and lipid transfer proteins of the saposin family have been shown to play a crucial role in this process. However, the mechanism by which saposins facilitate lipid binding to CD1 molecules is not known and is thought to involve transient interactions between protein components to ensure CD1-lipid complexes can be efficiently trafficked to the plasma membrane for antigen presentation. Of the four saposin proteins, the importance of Saposin B (SapB) for loading of CD1d is the most well-characterised. However, a direct interaction between CD1d and SapB has yet to be described. In order to determine how SapB might load lipids onto CD1d, we used purified, recombinant CD1d and SapB and carried out a series of highly sensitive binding assays to monitor direct interactions. Using equilibrium binding analysis, chemical cross-linking and co-crystallisation experiments, under a range of different conditions, we could not demonstrate a direct interaction. This work establishes comprehensively that the role of SapB in lipid loading does not involve direct binding to CD1d. We discuss the implication of this for our understanding of lipid loading of CD1d and propose several factors that may influence this process.

biochemistry↗