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Structural and functional characterisation of pUL21, an α-herpesvirus phosphatase adaptor protein 10.17863/CAM.85288 Benedyk, Tomasz 0000000164203665 Graham, Stephen 0000000345474034 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. University of Cambridge 2021-12-01 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/337882 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cab19c7d-d569-4ac0-9d47-e3332e43a547/download c2c69b4d1691214acea62f6182eb1c97 https://www.rioxx.net/licenses/all-rights-reserved/ hsv-1 sphingolipids protein phosphatase 1 ceramide transfer protein CERT PP1 pUL21