MCA77G, Serotec), rabbit anti-C6 (1:1000, described in (Unterholzner et?al., 2011), mouse anti-D8 (1:1000, explained in (Parkinson and Smith, 1994)). within proteins shown in (A), compared to all quantified proteins. mmc3.xlsx (52K) GUID:?FEA2C782-6C58-41D7-9142-F1F7B5B96D05 Table S3. Candidate Immunoreceptors, Related to Figures 2 and S2 mmc4.xlsx (12K) GUID:?47DC012D-5401-4EB7-A6F4-5C199F97FF97 Table S4. Protein Regulation by VACV Difloxacin HCl and HCMV, Related to Physique?3 (A) All proteins quantified in both this study and a previous quantitative temporal analysis of HCMV contamination (Weekes et?al., 2014). (B) All proteins downregulated >2-fold by both VACV and HCMV, at 1 time point during the course of Difloxacin HCl both infections. (C) Enrichment of functional pathways within proteins shown in (B), compared to all quantified proteins shown in (A). mmc5.xlsx (317K) GUID:?6203D9D9-E2EE-4658-A645-083F44060612 Table S5. VACV Protein and Transcriptional Classes, Related to Physique?4 Transcriptional classes and functional category information were derived from Yang et?al., 2010, Yang et?al., 2015). A comparison to a prior analysis (Croft et?al., 2015) is included in this table. Croft et?al. analyzed VACV contamination in two impartial time courses, with time course 1 from 0.5C9.5?h of contamination, sampled at 3?h intervals and time course 2 from 0.5C8.5?h of contamination sampled at 2?h intervals. Each generated four temporal classes of viral proteins. As some of these data are discordant, a further column is included in the table indicating concordant temporal classes for 47 viral proteins, to which our data were compared (Physique?S4B). mmc6.xlsx (20K) GUID:?A93460E4-3E92-454A-801E-C2851458B1FA Table S6. Systematic Analysis of Proteasomal Degradation, Related to Physique?5 (A) Human proteins downregulated >2-fold at 12?h of VACV contamination compared to mock. Rescue ratios are shown as defined in Physique?5A. (B) Data for all those 173 viral proteins quantified in this experiment. mmc7.xlsx (37K) GUID:?6699F270-B0D0-4313-9F1D-9928F3F86B29 Table S7. Confirmation of Genetic Knockout and Details of TMT Labeling, Related to Physique?7 and STAR Methods Rabbit polyclonal to LRRC15 (A) Confirmation of genetic knockout in indie HeLa and HEK293T HDAC5?/? clones. Sequencing of the genomic region targeted by the gRNAs confirmed frameshift mutations had been launched into each allele. Primers and gRNA sequences Difloxacin HCl employed are also shown. (B) Details of TMT labeling. mmc8.xlsx (13K) GUID:?4EB66DD8-7CD1-4541-BB83-3CE237C7B17A Document S2. Article plus Supplemental Information mmc9.pdf (5.9M) GUID:?08E7D5A8-993C-48B9-A235-56D429B861E1 Summary Vaccinia virus (VACV) has numerous immune evasion?strategies, including multiple mechanisms of inhibition of interferon regulatory factor 3 (IRF-3), nuclear factor B (NF-B), and type I interferon (IFN) signaling. Here, we use highly multiplexed proteomics to quantify 9,000 cellular proteins and 80% of viral proteins at seven time points throughout VACV contamination. A total of 265 cellular proteins are downregulated >2-fold by VACV, including putative natural killer cell ligands and IFN-stimulated genes. Two-thirds of these viral targets, including class II histone deacetylase 5 (HDAC5), are degraded proteolytically during contamination. In follow-up analysis, we demonstrate that HDAC5 restricts replication of both VACV and herpes simplex virus type 1. By generating a protein-based temporal classification of VACV gene expression, we identify protein C6, a multifunctional IFN antagonist, as being necessary and sufficient for proteasomal degradation of?HDAC5. Our approach thus identifies both a host?antiviral factor and a viral mechanism of innate immune evasion. of the 5 guanosine. Direct inhibition of RNA viral translation has also been reported (Daffis et?al., 2010, Pichlmair et?al., 2011). Our observation that both VACV and HCMV downregulate IFITs (Physique?3C; Table S4C) suggests that this whole class of proteins may have an as yet unrecognized mechanism of restricting DNA viruses in addition to RNA viruses. We Difloxacin HCl quantified 29 tripartite motif containing proteins (TRIMs), of which TRIM 5, 13, 25, 26, and 56 were downregulated during contamination. TRIM5 was also targeted by HCMV (Physique?3C; Furniture S2A and S2B). TRIM5 can restrict retroviruses, and TRIM56 inhibits diverse RNA viruses including influenza, dengue, and yellow fever computer virus (Liu et?al., 2014, Liu.