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High definition analysis of protein stability during cytomegalovirus infection informs on cellular restriction

ABSTRACT: Human cytomegalovirus (HCMV) is an important pathogen and a paradigm of viral immune evasion. Here, we describe a multiplexed approach to discover proteins with novel innate immune functions on the basis of their active proteasomal or lysosomal degradation during the early phase of HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, we identified 35 proteins with high confidence that were enriched in known antiviral restriction factors. A final screen facilitated global analysis of the mechanism of viral immune-modulation by predicting which viral genes target >250 human proteins. Helicase-like Transcription Factor (HLTF), a DNA helicase important in error-free post-replication DNA repair, was rapidly degraded during infection and potently inhibited early viral gene expression. HCMV UL145, a protein of previously unknown function, recruits the Cullin 4 E3 ligase complex to degrade HLTF. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Cytomegalovirus Homo Sapiens (human)

TISSUE(S): Fibroblast

SUBMITTER: Michael Weekes

LAB HEAD: Michael Weekes

PROVIDER: PXD009945 | Pride | 2018-08-16

REPOSITORIES: Pride

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			Action	DRS
	Block_1_HpRP1.raw	Raw
	Block_1_HpRP10.raw	Raw
	Block_1_HpRP11.raw	Raw
	Block_1_HpRP12.raw	Raw
	Block_1_HpRP2.raw	Raw

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Rapid degradation pathways of host proteins during HCMV infection revealed by quantitative proteomics

Project description:Human cytomegalovirus 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 optimised 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, 62-85% 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.

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