230 Utilizing in Vivo Proximity Labeling Combined with Affinity Purification-Mass Spectrometry to Investigate Interacting Partners of Vaccinia Dsrna-Binding Protein E3

Proximity labeling coupled with affinity purification-mass spectrometry is a newly developed method for studying protein-protein interaction in vivo. APEX is an ascorbate peroxidase that oxidizes biotin phenol to generate short-lived radicals, which react with side chains of amino acids in proximity. Streptavidin beads are then used to pull doen biotinylated proteins, which are subjected to mass spectrometry. Vaccinia virus is a large cytoplasmic DNA virus that belongs to the poxvirus family. The vaccinia E3L gene encodes a key virulence factor that is composed of Z-DNA-binding and dsRNA-binding domains. E3 targets multiple pathways including IFN production and stress responses. E3 is localized to the virosome, where viral DNA replication and intermediate and late gene transcription occur. To investigate E3-binding partners in the host, we generated two recombinant modified vaccinia virus Ankara (MVA) viruses- one with E3L tagged with FLAG-APEX2 and the other with FLAG-APEX2 replacing the E3L gene. In both cases, the expression of either E3L-FLAG-APEX2 or FLAG-APEX2 is under the control of endogenous E3L promoter. Imaging analyses of MVA-E3L-FLAG-APEX2 or MVAΔE3L-FLAG-APEX2 infected B16-F10 murine melanoma cells at 8 h post-infection revealed that E3-FLAG-APEX2 is located in the virosomes. Consistent with that, mass spectrometry of streptavidin-pulled down proteins showed that several viral proteins are enriched, including E3, major core proteins 4a and 4b, B1 kinase, H1 dual-specificity protein phosphatase. For host proteins, the majority of E3-interacting host proteins include nucleolysin TIA, RNA-binding protein 3, Caprin, RNA-binding protein 3, Caprin 1, and G3BP1. Imaging studies demonstrate that MVAΔE3L infection in B16-F10 cells induces the formation of stress granules, whereas MVA does not. These results provide strong evidence that E3 interacts with proteins involved in stress-granule formation in the virosomes.