Structural and functional characterisation of pUL21, an α-herpesvirus phosphatase adaptor protein

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.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2021

Author dc:creator

• Benedyk, Tomasz

Advisor dc:contributor.advisor

• Graham, Stephen

• hsv-1
• sphingolipids
• protein phosphatase 1
• ceramide transfer protein
• CERT
• PP1
• pUL21

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0001-6420-3665
0000-0003-4547-4034

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/337882