Multi-level proteomics profiling of virus restrictive and -permissive cellular environments reveal regulatory factors of virus infection

Virus proliferation inside host cells relies on a diverse range of host machineries and is also restricted by the host through antiviral factors. The configuration of virus-dependency and antiviral factors determine the permissiveness of host cells to virus infection, however, overall differences between highly permissive and restrictive cellular states remain largely unexplored. Here we employed integrated omics analysis combining RNA-seq, proteomics, and phosphoproteomics to study determinants of virus permissiveness on a model system comprising multiple cellular states: highly permissive cells (HEK293T), steady-state cells (HEK293), and restrictive cells (interferon alpha (IFN-a) stimulated HEK293) due to their similar genetic background and distinct permissiveness. Our in-depth proteomics map across cellular states revealed pathway-level depletion of innate immune response and enrichment of anabolic processes in HEK293T cell. RNA-seq and proteomics results depicted dynamic regulations of IFN-a response across early/late timepoints, highlighting a group of robustly upregulated antiviral factors. In addition, phosphoproteomics uncovered extensive alterations of phosphorylation in IFN-a response. Integrated analysis of multi-level omics results identified putative regulators of infection, and we experimentally validated their roles in virus infection. Overall design: This RNA-seq data is part of our integrated omics analysis that aim to identify determinants of virus permissiveness across distinct cellular states. RNA-seq was performed with steady-state cells (HEK293), and restrictive cells (IFN-a stimulated HEK293 cells), comparing gene expression changes between unstimulated, early timepoint (4 h) IFN-a stimulation, and late timepoint (20 h) IFN-a stimulation in HEK293 cell line. Integration of sequencing results and our proteomics analysis facilitated identification of virus regulating factors in human cells.