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

Benedyk, Tomasz H.; Muenzner, Julia; Connor, Viv; Han, Yue; Brown, Katherine A.; Wijesinghe, Kaveesha J.; Zhuang, Yunhui; Colaco, Susanna; Stoll, Guido A.; Tutt, Owen S.; Svobodova, Stanislava; Svergun, Dmitri I.; Bryant, Neil; Deane, Janet E.; Firth, Andrew E.; Jeffries, Cy M.; Crump, Colin M.; Graham, Stephen C.

doi:10.1371/journal.ppat.1009824

Cited by 24 publications

(82 citation statements)

References 107 publications

(162 reference statements)

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“…Consistent with this hypothesis, the abundance alkyne-SM as a fraction of total alkyne-Cer plus alkyne-SM was higher in HaCaT21 versus HaCaT cells at early time points (0-30 min) during the chase (Fig pUL21 has multiple functions during infection, promoting the PP1-mediated dephosphorylation of not just CERT but of multiple different cellular and viral proteins [18]. It was therefore essential to identify a point mutant of pUL21 with impaired binding to CERT, but not to other cellular or viral partners, to dissect the functional significance of increased CERT activity during infection.…”

Section: Resultssupporting

confidence: 57%

“…It was therefore essential to identify a point mutant of pUL21 with impaired binding to CERT, but not to other cellular or viral partners, to dissect the functional significance of increased CERT activity during infection. Defining the interaction surfaces of transient protein complexes presents a considerable challenge [45], made more difficult in the case of pUL21 and CERT by their containing multiple domains and regions of intrinsic disorder [18].…”

Section: Resultsmentioning

confidence: 99%

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

Benedyk

Connor

Caroe

et al. 2022

Preprint

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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. The accumulation of ceramide in the pUL21 mutant virus infection is correlated with accelerated virus replication, illuminating how HSV-1 manipulates lipid metabolism via altering protein phosphorylation to fine-tune its replication.

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Section: Resultssupporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

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

Benedyk

Connor

Caroe

et al. 2022

Preprint

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“…An alternative explanation for the hyperphosphorylation of pUs3, pUL31 and pUL34 in ΔUL21 infected cells is that pUL21 may promote their dephosphorylation ( Fig 9B ). This idea is strongly supported by a recent study from Graham and co-workers that demonstrated HSV-1 pUL21 is an adaptor protein for protein phosphatase 1 (PP1) and furthermore that pUL21 has a penchant for delivery of PP1 to pUs3 substrates to mediate their dephosphorlyation [ 36 ]. The fact that pUL21 and pUs3 are only found in members of the Alphaherpesvirinae raises the intriguing possibility that pUL21 exists, in part, to specifically counter the activities of pUs3.…”

Section: Discussionmentioning

confidence: 91%

“…HSV-2 pUL21 regulates the phosphorylation status of the NEC dephosphorlyation [36]. The fact that pUL21 and pUs3 are only found in members of the Alphaherpesvirinae raises the intriguing possibility that pUL21 exists, in part, to specifically counter the activities of pUs3.…”

Section: Plos Pathogensmentioning

confidence: 99%

pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex

et al. 2021

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It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.

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“…These genes are essential for the viral life cycle in vivo, and many of them play roles in viral assembly and egress. Some of these conserved assembly proteins participate in self-assembly complexes, such as major and minor capsid proteins; while others, including the viral nuclear egress complex, the virus protein kinases pUS3 and pUL13, pUL21, pUL36, VP26, pUS9, and gE interact with host proteins to mediate other aspects of viral egress (5)(6)(7)(8)(9)(10)(11)(12)(13). Targeting these viral-host interactions for therapeutics may reduce development of drug resistance since the host partner is not subject to selective pressure (14).…”

Section: Introductionmentioning

confidence: 99%

HSV Tegument Protein pUL51 Interacts with Host Dynactin for Viral Spread

White

Tran

Roller

2022

Preprint

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Dynein motors are microtubule associated protein complexes that mediate multiple essential cellular processes, such as long-distance cargo trafficking and stabilization of the microtubule organization center. Most of these functions and their regulations depend on the dynein motor subunit dynactin. By using an infection-inducible system, we disrupted dynein motor function after HSV entry by overexpressing a dominant-negative inhibitor of dynein, resulting in a 5-fold growth defect in Vero cells and 1000-fold growth defect in CAD neuronal cells. Also, we found that in infected CAD cells, the dynein complex was recruited to viral assembly sites regardless of microtubule polymerization. Based on these observations, we then identified a novel interaction between conserved HSV-1 tegument protein pUL51 and p150Glued. pUL51 is a palmitoylated Golgi membrane-associated protein that is required for efficient virus assembly and spread. Overexpression of pUL51 alone was sufficient to recruit p150Glued to Golgi membranes. Sequences that are important and sufficient for pUL51-p150Glued interaction were mapped to residues 90 to 124 in pUL51 and residues 548 to 911 in p150Glued. Deletion of a.a 90-124 in pUL51 resulted in a moderate viral growth defect, a profound spread defect, and failure to accumulate both dynactin and the viral spread factor glycoprotein E (gE) at cell-cell junctions. A synthetic peptide that contains pUL51 a.a 90-125 could also inhibit viral growth and spread in pUL51-dependent manner. Taken together, our results suggest that the proper function of pUL51 in efficient viral assembly and spread depends on its interaction with p150Glued.

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

Cited by 24 publications

References 107 publications

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

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

pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex

HSV Tegument Protein pUL51 Interacts with Host Dynactin for Viral Spread

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