University of Cambridge
Functional Characterisation of Protein Degradation during Human Cytomegalovirus Infection
Abstract
dc:description.abstractHuman cytomegalovirus (HCMV) is a ubiquitous and clinically significant herpesvirus, causing substantial morbidity and mortality in immunocompromised individuals and congenital infection in up to 1/100 pregnancies. HCMV has evolved a myriad of strategies to evade the host immune response, including targeting host proteins for degradation. Insights into this viral antagonism can facilitate the development of novel antiviral therapies by restoring the activity of endogenous proteins important for antiviral restriction and immunity. A previous multiplexed proteomic approach identified 35 ‘high-confidence’ and 133 ‘medium-confidence’ host proteins that are likely degraded during early stages of HCMV infection. It was hypothesised that protein degradation plays an active role in the establishment of infection and that these targeted proteins are of significance for HCMV. It has already been shown that a subset of these proteins are functionally important in innate and adaptive immunity. However, roles for the remaining proteins during HCMV infection remain uncharacterised. The overall aim of this thesis was to characterise viral mechanisms of protein degradation and to investigate the functional role that this plays during infection, focusing on select cellular proteins. All three results chapters include their own separate introduction and discussion sections. Chapter three and five characterised two cellular proteins lectin mannose-binding like-2 protein (LMAN2L) and neurabin-II, respectively, that are degraded during early stages of HCMV infection. The use of HCMV deletion mutants and gene overexpression/knockdown systems identified US2 as the viral protein responsible for LMAN2L degradation in concert with the cellular E3 ligase TRC8 (translocation in renal carcinoma, chromosome 8 gene). Several potential viral interactors of neurabin-II were identified by proximity-labelling mass spectrometry, however all candidates appeared to be dispensable for neurabin-II downregulation and could not elucidate the viral mechanism of degradation. Chapter four utilised the neddylation inhibitor MLN4924 which impairs cullin-RING ubiquitin ligase (CRL) activation and proteomic analysis identified several HCMV targets that are likely degraded in a CRL-dependent manner, including the positive control HLTF and antiviral restriction factor Sp100. As inhibition of CRL-mediated degradation restricts HCMV infection, we speculated that these proteins are of particular functional importance and offers mechanistically tractable candidates for future studies. While neither LMAN2L nor neurabin-II have previously been implicated in viral infection, we hypothesised that their degradation could alter cellular processes to favour the establishment of infection. The function of LMAN2L is largely uncharacterised but is believed to be involved in the trafficking of glycoproteins out of the endoplasmic reticulum. Plasma membrane profiling experiments identified several proteins that are downregulated on the surface of LMAN2L deficient cells, including US2 target integrin alpha 6 (ITGA6), which proposed an alternative mechanism by which US2 prevents the expression of key proteins at the plasma membrane. Neurabin-II, on the other hand, is a multifunctional cellular protein with defined roles in diverse cellular processes, such as the regulation of protein phosphatase 1 and organisation of the actin cytoskeleton. Several assays were employed to characterise the impact of neurabin-II downregulation on viral dissemination, protein phosphorylation, cell migration and NK cell degranulation. These results show that neurabin-II expression restricts HCMV spread and alters protein phosphorylation, whereas no consistent cell migration or immunostimulatory phenotype was observed in the latter assays. Overall, this thesis demonstrates how studying HCMV-mediated protein degradation can provide insights into the function of both viral and cellular proteins, virus-host interactions, and highlight biologically significant pathways for infection.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2023
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Hunter, Leah
- Advisor dc:contributor.advisor
-
- Weekes, Michael
Subjects
dc:subject × 6Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.108565
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/368253