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Human cytomegalovirus degrades DMXL1 to inhibit autophagy, lysosomal acidification and viral assembly

ABSTRACT: Human cytomegalovirus (HCMV) is an important human pathogen and a master regulator of host intrinsic, innate, and adaptive immunity. HCMV hijacks host intracellular compartments to assemble new virions, and lysosomes are essential for this process. We combined a comprehensive proteomic analysis of host proteins targeted for degradation by HCMV with a database of proteins involved in vacuolar acidification. Dmx-like protein 1 (DMXL1) was the only protein that acidifies vacuoles yet is degraded by HCMV. Systematic comparison of viral deletion mutants revealed that the uncharacterised 7kDa US33A protein is necessary and sufficient for DMXL1 degradation. Functional experiments using cells stably expressing US33A, or infected with adenovirus vectors expressing US33A, or infected with recombinant HCMV deleted for US33A, demonstrated that HCMV degrades DMXL1 to inhibit lysosomal acidification and autophagic cargo degradation. Formation of the viral assembly compartment, which is known to require lysosomes, occurred significantly later in cells infected with US33A-expressing virus, with reduced viral replication. These data thus identify an entirely new viral strategy for cellular remodelling. US33A recruits the E3 ubiquitin ligase Kip1 ubiquitination-promoting complex (KPC) and acts akin to a proteolysis-targeting chimera (PROTAC) to degrade DMXL1. The potential thus exists to employ US33A in novel therapies for viral infection or rheumatic conditions, in which inhibition of lysosome acidification can attenuate disease.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Cytomegalovirus Homo Sapiens (human)

TISSUE(S): Cell Culture, Fibroblast

DISEASE(S): Human Cytomegalovirus Infection

SUBMITTER: Michael Weekes

LAB HEAD: Professor Michael Weekes

PROVIDER: PXD044273 | Pride | 2024-03-13

REPOSITORIES: Pride

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Project description:Infection or reactivation with human cytomegalovirus (HCMV) is still a major cause of morbidity and mortality in patients undergoing solid organ transplantation or allogeneic stem cell transplantation (alloSCT). Recent studies have shown that protection against latent HCMV infection relies on a much broader antigen spectrum than previously expected. The identification of novel immunogenic HCMV-derived peptides that facilitate HCMV control is therefore essential to improve immune monitoring of HCMV-specific T cells and HCMV vaccine development. In particular, only a few HCMV-derived peptides have been described for less common HLA alleles, such as HLA-A*03:01 and HLA-B*15:01, limiting the implementation of these techniques for a substantial number of patients. Here, we identified novel HCMV-derived HLA-A*03:01- and HLA-B*15:01-restricted immunogenic peptides by an innovative combined in vitro and in silico approach. We utilized the computational tools Peptide-PRISM (1) and PRICE (2) to analyze mass spectrometric (MS) and ribosome sequencing (ribo-seq) datasets, respectively. To refine our in silico approach, we utilized machine learning to rate the identified peptide candidates based on their translational activity, binding affinity and positioning within the small open reading frames (sORFs), with the goal of assessing their likelihood to be presented on HLA-A*03:01 and HLA-B*15:01 molecules. After identifying the highest-scoring 49 canonical and 49 cryptic potentially immunogenic peptides for each HLA allele, we assessed their immunogenicity by screening HCMV-seropositive and -seronegative healthy donors, as well as alloSCT patients for their peptide-specific T-cell responses. We used a stimulation procedure in two consecutive steps: peripheral blood cells (PBMCs) were stimulated starting with peptide pools and subsequently with potentially reactive single peptides. In vitro T-cell stimulation resulted in the direct identification of three canonical and one cryptic HLA-A*03-restricted immunogenic peptides as well as five canonical and one cryptic HLA-B*15-restricted immunogenic peptide, with a specific IFNγ+/CD8+ T cell response of ≥ 0.02%. Highest T-cell responses were identified against two HLA-A*03-restricted and three HLA-B*15-restricted canonical peptides with up to 3.42% of IFNγ+/CD8+ T cells in patients 180 days post alloSCT. In conclusion, we identified specific T-cell responses against eight canonical and two cryptic HLA-A*03:01- and HLA-B*15:01-restricted peptides in healthy individuals and post-transplanted patients and which were classified as immunogenic. Of these, six peptides are novel HCMV-derived, immunogenic peptides according to the UniProt database, broadening the spectrum of immunogenic HCMV-derived peptides for personalized immune monitoring and vaccine development.

			Action	DRS
	DUS29_34A_HCMV_F1.raw	Raw
	DUS29_34A_HCMV_F10.raw	Raw
	DUS29_34A_HCMV_F11.raw	Raw
	DUS29_34A_HCMV_F12.raw	Raw
	DUS29_34A_HCMV_F2.raw	Raw

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Human cytomegalovirus degrades DMXL1 to inhibit autophagy, lysosomal acidification and viral assembly

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