{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/337882"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/337882","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Structural and functional characterisation of pUL21, an α-herpesvirus phosphatase adaptor protein","abstract":"The current CoV-SARS-2 pandemic exemplifies the profound impact viruses can have upon our lives. Virus infection is a complex process where viral proteins cooperate to exploit host cell metabolism while evading immune responses. Herpes simplex virus 1 (HSV-1) is a very prevalent human herpesvirus that causes life-long infection and is known to dramatically remodel the cellular environment upon replication. This extensive manipulation is achieved using only a limited number of virus-encoded proteins, many of which possess numerous functions and exert their effects in multiple different subcellular compartments. HSV-1 pUL21 is an example of such multi-function protein: it is known to be important for assembly of new virus particles and viral cell-to-cell spread but its exact molecular functions remained unknown. Using a high throughput interactomics screen, previous members of the group identified potential cellular binding partners of pUL21: protein phosphatase 1 (PP1) and ceramide transfer protein (CERT). This thesis presents biophysical characterization of the direct interactions between pUL21 and these partners and describes the role of pUL21 as a novel viral phosphatase adaptor that recruits PP1 to multiple substrates, including CERT, to promote their dephosphorylation. Conservational and structural analyses led to the discovery of a non-canonical linear motif in pUL21, termed TROPPO, that is critical for PP1 binding and it is absolutely conserved across α-herpesviruses. In vitro evolution experiments using HSV-1 strains with mutated TROPPO motifs revealed that the phosphatase adaptor pUL21 antagonises the activity of the virus-encoded kinase pUS3. A correct balance of kinase and phosphatase activity is shown to be essential for correct subcellular localisation of the HSV-1 nuclear egress complex and for virus replication and dissemination. Using in vitro biochemical experiments, stable expression of pUL21 in cultured cells, and infection with wild-type HSV-1 or viruses expressing pUL21 with a mutated TROPPO motif, we confirmed that pUL21 stimulates PP1-dependent dephosphorylation of CERT and the viral nuclear egress complex component pUL31, plus additional as-yet unidentified proteins. The binding interface of the pUL21:CERT complex was determined using small-angle X-ray scattering, enabling the generation of pUL21 mutants where binding to CERT, but not to other substrates, was specifically disrupted. Generation of a CERT non-binding mutant facilitated a detailed characterization of the sphingolipid-modulatory role of pUL21 using ‘click chemistry’-based assays and revealed that pUL21-dependent upregulation of sphingomyelin turnover is required for the correct trafficking of maturating virions to the plasma membrane. In summary, this thesis presents structural and functional characterisation of HSV-1 pUL21, dissecting the multiple roles played by this protein during the replication of HSV-1. Furthermore, this study provides first insights into the modulation of sphingolipid homeostasis during virus infection, a critically understudied host:pathogen interaction.","abstract_html":"The current CoV-SARS-2 pandemic exemplifies the profound impact viruses can have upon our lives. Virus infection is a complex process where viral proteins cooperate to exploit host cell metabolism while evading immune responses. Herpes simplex virus 1 (HSV-1) is a very prevalent human herpesvirus that causes life-long infection and is known to dramatically remodel the cellular environment upon replication. This extensive manipulation is achieved using only a limited number of virus-encoded proteins, many of which possess numerous functions and exert their effects in multiple different subcellular compartments. HSV-1 pUL21 is an example of such multi-function protein: it is known to be important for assembly of new virus particles and viral cell-to-cell spread but its exact molecular functions remained unknown. Using a high throughput interactomics screen, previous members of the group identified potential cellular binding partners of pUL21: protein phosphatase 1 (PP1) and ceramide transfer protein (CERT). This thesis presents biophysical characterization of the direct interactions between pUL21 and these partners and describes the role of pUL21 as a novel viral phosphatase adaptor that recruits PP1 to multiple substrates, including CERT, to promote their dephosphorylation. Conservational and structural analyses led to the discovery of a non-canonical linear motif in pUL21, termed TROPPO, that is critical for PP1 binding and it is absolutely conserved across α-herpesviruses. In vitro evolution experiments using HSV-1 strains with mutated TROPPO motifs revealed that the phosphatase adaptor pUL21 antagonises the activity of the virus-encoded kinase pUS3. A correct balance of kinase and phosphatase activity is shown to be essential for correct subcellular localisation of the HSV-1 nuclear egress complex and for virus replication and dissemination. Using in vitro biochemical experiments, stable expression of pUL21 in cultured cells, and infection with wild-type HSV-1 or viruses expressing pUL21 with a mutated TROPPO motif, we confirmed that pUL21 stimulates PP1-dependent dephosphorylation of CERT and the viral nuclear egress complex component pUL31, plus additional as-yet unidentified proteins. The binding interface of the pUL21:CERT complex was determined using small-angle X-ray scattering, enabling the generation of pUL21 mutants where binding to CERT, but not to other substrates, was specifically disrupted. Generation of a CERT non-binding mutant facilitated a detailed characterization of the sphingolipid-modulatory role of pUL21 using ‘click chemistry’-based assays and revealed that pUL21-dependent upregulation of sphingomyelin turnover is required for the correct trafficking of maturating virions to the plasma membrane. In summary, this thesis presents structural and functional characterisation of HSV-1 pUL21, dissecting the multiple roles played by this protein during the replication of HSV-1. Furthermore, this study provides first insights into the modulation of sphingolipid homeostasis during virus infection, a critically understudied host:pathogen interaction.","abstract_has_math":false,"creators":["Benedyk, Tomasz"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Graham, Stephen"],"committee_chairs":[],"committee_members":[],"year":2021,"date_issued":"2021-12-01","date_published":"2021-12-01","updated_at":"2026-07-22T22:24:00Z","subjects":["hsv-1","sphingolipids","protein phosphatase 1","ceramide transfer protein","CERT","PP1","pUL21"],"languages":["eng"],"rights":[],"rights_urls":["https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000164203665","0000000345474034"],"render_values":[{"text":"0000-0001-6420-3665","href":"https://orcid.org/0000-0001-6420-3665","code":true},{"text":"0000-0003-4547-4034","href":"https://orcid.org/0000-0003-4547-4034","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.85288","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Graham, Stephen"]},{"key":"dc:creator","label":"Author","values":["Benedyk, Tomasz"]},{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000164203665","0000000345474034"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2021-12-01"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/337882"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["hsv-1","sphingolipids","protein phosphatase 1","ceramide transfer protein","CERT","PP1","pUL21"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.85288"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cab19c7d-d569-4ac0-9d47-e3332e43a547/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The current CoV-SARS-2 pandemic exemplifies the profound impact viruses can have upon our lives. Virus infection is a complex process where viral proteins cooperate to exploit host cell metabolism while evading immune responses. Herpes simplex virus 1 (HSV-1) is a very prevalent human herpesvirus that causes life-long infection and is known to dramatically remodel the cellular environment upon replication. This extensive manipulation is achieved using only a limited number of virus-encoded proteins, many of which possess numerous functions and exert their effects in multiple different subcellular compartments. HSV-1 pUL21 is an example of such multi-function protein: it is known to be important for assembly of new virus particles and viral cell-to-cell spread but its exact molecular functions remained unknown. Using a high throughput interactomics screen, previous members of the group identified potential cellular binding partners of pUL21: protein phosphatase 1 (PP1) and ceramide transfer protein (CERT). This thesis presents biophysical characterization of the direct interactions between pUL21 and these partners and describes the role of pUL21 as a novel viral phosphatase adaptor that recruits PP1 to multiple substrates, including CERT, to promote their dephosphorylation. Conservational and structural analyses led to the discovery of a non-canonical linear motif in pUL21, termed TROPPO, that is critical for PP1 binding and it is absolutely conserved across α-herpesviruses. In vitro evolution experiments using HSV-1 strains with mutated TROPPO motifs revealed that the phosphatase adaptor pUL21 antagonises the activity of the virus-encoded kinase pUS3. A correct balance of kinase and phosphatase activity is shown to be essential for correct subcellular localisation of the HSV-1 nuclear egress complex and for virus replication and dissemination. Using in vitro biochemical experiments, stable expression of pUL21 in cultured cells, and infection with wild-type HSV-1 or viruses expressing pUL21 with a mutated TROPPO motif, we confirmed that pUL21 stimulates PP1-dependent dephosphorylation of CERT and the viral nuclear egress complex component pUL31, plus additional as-yet unidentified proteins. The binding interface of the pUL21:CERT complex was determined using small-angle X-ray scattering, enabling the generation of pUL21 mutants where binding to CERT, but not to other substrates, was specifically disrupted. Generation of a CERT non-binding mutant facilitated a detailed characterization of the sphingolipid-modulatory role of pUL21 using ‘click chemistry’-based assays and revealed that pUL21-dependent upregulation of sphingomyelin turnover is required for the correct trafficking of maturating virions to the plasma membrane. In summary, this thesis presents structural and functional characterisation of HSV-1 pUL21, dissecting the multiple roles played by this protein during the replication of HSV-1. Furthermore, this study provides first insights into the modulation of sphingolipid homeostasis during virus infection, a critically understudied host:pathogen interaction."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["c2c69b4d1691214acea62f6182eb1c97"]},{"key":"dc:title","label":"Title","values":["Structural and functional characterisation of pUL21, an α-herpesvirus phosphatase adaptor protein"]}]}],"canonical_facts":{"dc:contributor.advisor":["Graham, Stephen"],"dc:creator":["Benedyk, Tomasz"],"dc:creator.authoridentifier":["0000000164203665","0000000345474034"],"dc:date.issued":["2021-12-01"],"dc:description.abstract":["The current CoV-SARS-2 pandemic exemplifies the profound impact viruses can have upon our lives. 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This thesis presents biophysical characterization of the direct interactions between pUL21 and these partners and describes the role of pUL21 as a novel viral phosphatase adaptor that recruits PP1 to multiple substrates, including CERT, to promote their dephosphorylation. Conservational and structural analyses led to the discovery of a non-canonical linear motif in pUL21, termed TROPPO, that is critical for PP1 binding and it is absolutely conserved across α-herpesviruses. In vitro evolution experiments using HSV-1 strains with mutated TROPPO motifs revealed that the phosphatase adaptor pUL21 antagonises the activity of the virus-encoded kinase pUS3. A correct balance of kinase and phosphatase activity is shown to be essential for correct subcellular localisation of the HSV-1 nuclear egress complex and for virus replication and dissemination. Using in vitro biochemical experiments, stable expression of pUL21 in cultured cells, and infection with wild-type HSV-1 or viruses expressing pUL21 with a mutated TROPPO motif, we confirmed that pUL21 stimulates PP1-dependent dephosphorylation of CERT and the viral nuclear egress complex component pUL31, plus additional as-yet unidentified proteins. The binding interface of the pUL21:CERT complex was determined using small-angle X-ray scattering, enabling the generation of pUL21 mutants where binding to CERT, but not to other substrates, was specifically disrupted. Generation of a CERT non-binding mutant facilitated a detailed characterization of the sphingolipid-modulatory role of pUL21 using ‘click chemistry’-based assays and revealed that pUL21-dependent upregulation of sphingomyelin turnover is required for the correct trafficking of maturating virions to the plasma membrane. In summary, this thesis presents structural and functional characterisation of HSV-1 pUL21, dissecting the multiple roles played by this protein during the replication of HSV-1. Furthermore, this study provides first insights into the modulation of sphingolipid homeostasis during virus infection, a critically understudied host:pathogen interaction."],"dc:format.checksum.md5":["c2c69b4d1691214acea62f6182eb1c97"],"dc:identifier.doi":["10.17863/CAM.85288"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cab19c7d-d569-4ac0-9d47-e3332e43a547/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/337882"],"dc:rights":["https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["hsv-1","sphingolipids","protein phosphatase 1","ceramide transfer protein","CERT","PP1","pUL21"],"dc:title":["Structural and functional characterisation of pUL21, an α-herpesvirus phosphatase adaptor protein"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:00Z"}