Quantitative Proteomics Affinity Purification Reveals Novel Insights On Host-Virus Interactions During HCV Life Cycle

摘要

1. 3xFLAG-tagged Core, NS2, NS3/4A, NS4B, NS5A and NS5B were expressed in 293T cells, and immunoprecipitated samples were analyzed by nano-LC MS/MS following elution with NH4OH. 2. Triplicate LC-MS/MS analyses from all isolated viral protein complexes enabled the identication of 594 host proteins identied with at least two peptides that were reproducibly detected in multiple immunoanity protein extracts with a FDR below 1%. 3. 90% of the identied peptides showed a fold-change below 3 when compared to the their average intensity across the 7 IPs. 4. We identied 92 proteins were identied that interacted with the 6 viral proteins. Most of the interactors are unique to one of the 6 viral proteins and are recruited at the organelle membranes. 5. Interacting host proteins were conrmed by Western Blot following immunoprecipitation of viral proteins with anti-FLAG antibodies and 3xFLAG peptide elution. Using this technique, we conrmed previously reported interacting proteins DDX3 and C1QBP with Core, and FKBP8 with NS5a. Finally, the study identied 82 potential new binding partners of HCV. 6. sGene silencing screen shows that Interactors like FKBP5, C14orf166, C1QBP, TARDBP and DDX3 are required to HCV replication.