Rapid Degradation Pathways of Host Proteins During HCMV Infection Revealed by Quantitative Proteomics.

Kai-Min Lin,Lior Soday,Katie Nightingale,Michael P Weekes,Robin Antrobus

doi:10.3389/fcimb.2020.578259

Kai-Min Lin, Lior Soday + Show 3 more

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https://doi.org/10.3389/fcimb.2020.578259

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Abstract

Human cytomegalovirus (HCMV) is an important pathogen in immunocompromised individuals and neonates, and a paradigm for viral immune evasion. We previously developed a quantitative proteomic approach that identified 133 proteins degraded during the early phase of HCMV infection, including known and novel antiviral factors. The majority were rescued from degradation by MG132, which is known to inhibit lysosomal cathepsins in addition to the proteasome. Global definition of the precise mechanisms of host protein degradation is important both to improve our understanding of viral biology, and to inform novel antiviral therapeutic strategies. We therefore developed and optimized a multiplexed comparative proteomic analysis using the selective proteasome inhibitor bortezomib in addition to MG132, to provide a global mechanistic view of protein degradation. Of proteins rescued from degradation by MG132, 34–47 proteins were also rescued by bortezomib, suggesting both that the predominant mechanism of protein degradation employed by HCMV is via the proteasome, and that alternative pathways for degradation are nevertheless important. Our approach and data will enable improved mechanistic understanding of HCMV and other viruses, and provide a shortlist of candidate restriction factors for further analysis.

Highlights

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that persistently infects the majority of the human population worldwide (Cannon et al, 2010)
To optimize conditions for proteomic analysis in HCMV-infected immortalized primary human foetal foreskin fibroblasts (HFFF-TERTs), a range of bortezomib concentrations were compared with 10 mM MG132, a concentration we previously showed to provide efficacious inhibition of protein degradation (Figures 1, S1A–B) (Nightingale et al, 2018)
Ratios of (HCMV with bortezomib)/HCMV and (HCMV with MG132)/HCMV were compared to quantify the relative efficacy of protein rescue

Summary

Introduction

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that persistently infects the majority of the human population worldwide (Cannon et al, 2010). Following primary infection under the control of a healthy immune system, a latent infection is established that persists lifelong (Reeves et al, 2005). Primary infection is mostly asymptomatic in healthy individuals, HCMV may lead to significant morbidity or mortality in immunocompromised patients, transplant recipients and AIDS patients (Griffiths et al, 2015). Vertical transmission of HCMV is a leading cause of congenital infection, resulting in deafness and intellectual disability in newborns (Manicklal et al, 2013). The identification and characterization of critical facets of host innate immunity that are targeted for degradation by HCMV proteins has important implications

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