Dengue virus non-structural protein 1 (NS1) protein-protein interactions in mosquito cells reveal participation in gene expression regulation Abstract: The dengue virus (DENV) NS1 protein is a multifunctional protein essential for viral replication and also acts as an immunomodulator. Yet, increasing evidence indicates that some properties of NS1 differ between vertebrate and mosquito cells. In order to gain insight of DENV NS1 functions in mosquito cells, the protein-protein interactions (PPIs) of DENV NS1 in Aedes albopictus C6/36 cells was obtained using a proximity biotinylation system (BioID). This system is based on a plasmid that expresses the DENV NS1 protein fused to a promiscuous biotin-ligase (BirA) enzyme from E. coli, which will add biotin to proteins that potentially interacts with NS1. The biotinylated proteins were purified using streptavidin beads and identified by mass spectrometry. A total of 817 proteins possibly interacting with DENV NS1 were identified. Of these, near 10% matched with previously reported ontology groups for NS1 PPIs in vertebrate cells; including proteins of the oligosaccharide transferase complex (OST), the chaperonin containing TCP-1 (CCT), nuclear import and export, vesicle localization and ribosomal proteins. Interestingly, other protein pathways such as epigenetic regulation, RNA silencing and apoptosis, not previously reported in vertebrate cells, were also found as part of the NS1 PPIs in mosquito cells. The interactions between NS1 and 4 selected proteins (Dido1, RPL26, Sec61A and GRP78) were validated in DENV infected C6/36 cells by colocalization and by proximity ligation assays. Due to the strong and novel interaction observed between Dido1 (Death Inducer-Obliterator 1) and NS1, we further explore the role of Dido 1 in viral infection. Dido1 knockdown in C6/36 and Aag2 (Aedes aegypti) cells results in a significant (one log) reduction in DENV and ZIKV progeny, suggesting that Dido 1 is a host factor necessary for flavivirus replication in the mosquito vector. Transcription analysis of C6/36 cells where compared in conditions of normal or knockdown expression of Dido1 and infection with DENV2 or not. Results reveal variations in gene expression of multiple genes consistent with the molecular role of Dido1, which has been related to transcription and epigenetic regulation. Ontology of differentially expressed genes include pathways previously associated with DENV infection such as RNA surveillance, IMD and Toll pathways. These results could help to understand the heterogenicity of NS1 as a multifunctional protein essential for Flavivirus infection in mosquito cells. Autors: Caraballo-Hernández Gerson I.1, Rosales-Ramírez Romel1, Siyuan Ding2, Harry-B. Greenberg2; Ludert Juan E.1. 1 Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies (CINVESTAV-IPN), Mexico City, Mexico. Emails: email@example.com; firstname.lastname@example.org 2 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.