Computational protocol: Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection

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Protocol publication

[…] HFF cells stably expressing either FL IFI16-eGFP, PY-eGFP, HIN-eGFP, or eGFP alone were infected with RF HSV-1 (MOI of 10). Cells were harvested 6 hpi, and immunoaffinity isolations with a GFP antibody prepared in-house were conducted as described previously (24). Eluted proteins were digested with trypsin (Promega) and analyzed by nano-liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) with an ESI-LTQ Orbitrap Velos (Thermo Scientific). Raw MS/MS spectra were searched and extracted using Proteome Discoverer (v. 1.4), and assessed through SEQUEST HT (v. 1.4). Specificity filtering on weighted spectral counts was conducted using the SAINT (Significance Analysis of INTeractions) algorithm, and a value of 0.96 was used as a specificity threshold (see  in the supplemental material). Spectral counts were normalized by the length of the IFI16 bait (PY or HINAB) and log2 fold enrichment was assessed as PY/HIN. The specific proteins were submitted to the STRING database (), categorized by Gene Ontology, and visualized in Cytoscape (v. 3.4.0). […]

Pipeline specifications

Software tools Proteome Discoverer, Comet
Application MS-based untargeted proteomics
Organisms Homo sapiens, Human alphaherpesvirus 1, Human poliovirus 1 Mahoney, Viruses, Human betaherpesvirus 5
Diseases HIV Infections