Chikungunya virus is a human pathogen transmitted by mosquitos. After a genetic adaptation of the virus that allows increased spreading by mosquitos, the past decade, Chikungunya virus has spread across the globe. In a significant number of patients, Chikungunya virus causes a chronic, debilitating, arthritic joint pain.

Chikungunya virus replicates in cells of both its vertebrate host and insect vector. To identify cellular pathways that the virus engages to allow optimal replication in these evolutionary distinct organisms we performed AP-MS to identify interaction partners of the viral non-structural proteins (nsPs) in both human and mosquito cells. Mass spectrometric analysis of on-bead-digestions of affinity purifications coupled to MiST analysis allowed sensitive and reproducible identification of a significant number of cellular protein interaction partners of nsP1, -3 and -4. The retrieval of well-established nsP3 interactors, G3BP and Bin1, in both human and mosquito cells validated our approach. Separate nsPs were associated with both shared and unique interaction partners, the latter belonging to different cellular pathways. Comparison of high-confidence interactors of nsP3 in human and mosquito cells identified 25 proteins that associate with nsP3 in both organisms. Functional classification of these shared nsP3 interactors using GO annotation showed engagement of cell-cell adhesion-, Hippo signaling-, ribosomal function- and innate immune signaling pathways by nsP3 in both human and mosquito cells. Interaction of members of each functional group with nsP3 were validated in AP-WB experiments. Functional roles in viral replication of several of these interactors are evaluated using knockouts.

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