Immunoprecipitation of the human cytomegalovirus (HCMV) gHgLgO glycoprotein complex
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human) Human Cytomegalovirus (hhv-5) (human Herpesvirus 5)
SUBMITTER: 
Thomas Fröhlich
LAB HEAD: Thomas Fröhlich
PROVIDER: PXD058164 | Pride | 2025-05-13
REPOSITORIES: Pride
Dataset's files
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|---|---|---|---|---|
| Cell_control_R1.raw | Raw | |||
| Cell_control_R2.raw | Raw | |||
| Cell_control_R3.raw | Raw | |||
| WT_R1.raw | Raw | |||
| WT_R2.raw | Raw |
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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.
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Project description:Background: Intrinsic defence mechanisms are pivotal host strategies to restrict viruses already at early stages of their infection. Here we addressed the question how the autophagy receptor sequestome 1 (SQSTM1/p62, herein termed p62) interfered with human cytomegalovirus (HCMV) infection. (2) Methods: CRISPR/Cas9- mediated genome editing, mass spectrometry and expression of p62 phosphovariants from recombinant HCMVs were used to address the role of p62 during infection. (3) Results: Knockout of p62 led to enhanced HCMV progeny release. Mass spectrometry revealed an interaction of p62 with cellular proteins required for nucleo-cytoplasmic transport. Phosphoproteomics further revealed that p62 is hyperphosphorylated at position S272 in HCMV-infected cells. Phosphorylated p62 showed enhanced nuclear retention, concordant with enhanced interaction with viral proteins relevant for genome replication and nuclear capsid egress. This modification led to reduced HCMV progeny release, compared to a unphosphorylated version of p62. (4) Conclusions: p62 is a restriction factor for HCMV replication The activity of the receptor appears to be regulated by phosphorylation at position S272, leading to enhanced nuclear localization, viral protein degradation and impairment of progeny production.
2025-05-13 | PXD055196 | Pride