This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A large number of viruses, including many human pathogens (dengue, yellow fever, west nile virus, etc), are transmitted by insects. The long-term goal of this proposal is to examine the mechanism of the antiviral defense system in Drosophila melanogaster. RNA interference (RNAi) is an ancient and conserved nucleic acid-based immune system in insects. Accordingly, insect viruses evolved strategies to control this defense mechanism. For example, Cricket Paralysis Virus (a Dicistrovirus) encodes a protein that suppresses RNAi by interacting specifically with the endonuclease Ago2. In more general terms, our understanding of the interactions of RNA viruses with the invertebrate hosts is very limited nevertheless requires urgent attention. The outcome of virus infection is the result of multidimensional set interactions of virus and host factors. Indeed, many viral and host proteins function as a complex than acting as a single molecule to regulate a vast number of cellular processes. Chemical crosslinking mass spectrometry of purified complexes derived from virus infected cells is a useful technique for mapping the site of linkage between two interacting partners in a complex and provides valuable information on three dimensional functional architecture of the protein complex. In addition, post translational modification events (e.g. phosporylation) of cellular signaling pathways are critical to regulate the antiviral mechanisms and often takes place upon infection that leads to change in cellular protein expression. The present research plan is designed to address how RNA viruses particularly Dicistroviruses interacts with host machinery to their advantage using Drosophila as a model organism and crosslinking mass spectrometry and phospoproteomics approaches.