Characterisation of Novel Autophagy and Necroptosis Antagonists Encoded by Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes significant morbidity and mortality in immunocompromised individuals. To support its intracellular survival, HCMV has evolved diverse strategies to manipulate key host pathways and evade intrinsic immune defences. In this thesis, I investigated novel HCMV-encoded inhibitors of two critical host responses—autophagy and necroptosis—by combining comprehensive proteomic screening with conventional biochemical assays. The initial focus was on lysosomal acidification, a process essential for autophagic cargo degradation. By overlapping quantitative proteomic data from HCMV-infected cells with a database of vacuolar acidification proteins, Dmx-like protein-1 (DMXL1) was identified as the only factor that regulates vacuolar acidification and is also targeted for degradation by HCMV. Systematic analysis of viral deletion mutants revealed that the uncharacterised 7 kDa protein US33A is both necessary and sufficient for DMXL1 degradation, acting via the E3 ubiquitin ligase Kip1 ubiquitination-promoting complex (KPC). This US33A-mediated DMXL1 degradation impairs lysosomal acidification and autophagic flux, thereby delaying the formation of the virion assembly compartment and reducing viral replication. Necroptosis, another key intrinsic antiviral defence, eliminates infected cells by inducing lytic cell death and thereby preventing viral dissemination. Only two HCMV-encoded necroptosis inhibitors have been reported to date: UL36, which targets mixed lineage kinase-like protein (MLKL) for degradation, and IE1, which enhances the ubiquitination of receptor-interacting protein kinase 3 (RIPK3). A systematic screen of HCMV recombinants lacking blocks of accessory genes dispensable for viral replication indicated that deleting either the US18–US22 or UL13–UL20 gene blocks significantly increases necroptosis in infected cells. Further investigation in fibroblasts stably expressing individual genes from these blocks identified four additional necroptosis inhibitors, most notably US22, which represses RIPK3 transcription and prevents MLKL phosphorylation. A multi-step viral growth assay showed that the absence of US22 markedly attenuates viral replication, highlighting the importance of necroptosis inhibition for efficient HCMV propagation. Current therapies for HCMV are often limited by toxicity and the emergence of drug resistance. The identification of viral factors such as US33A and US22, which remodel key host intrinsic immune responses, may provide promising avenues for the development of novel therapeutic strategies. Further elucidation of their functional mechanisms could enable innovative approaches to controlling HCMV infection.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Li, Hanqi

Advisor dc:contributor.advisor

• Weekes, Michael

• Autophagy
• HCMV
• Necroptosis
• Virology

Licence

• https://www.repository.cam.ac.uk/bitstreams/2f11082f-bdbc-4ff7-95f3-5189645b1485/download
• http://purl.org/NET/rdflicense/allrightsreserved

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.118824

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/385027