Drosophila genome-wide RNAi screen identifies novel genes involved in Sindbis virus entry. Debasis Panda, Patrick Rose, Sheri Hanna, Beth Gold, Sara Cherry. Microbiology, University of Pennsylvania, Philadelphia, PA.

   Alphaviruses are a large class of insect-borne human pathogens which exhibit a broad host range in nature. Little is known about the host factor requirements for alphaviruses and thus we performed a genome-wide RNAi screen in Drosophila cells which validated 96 genes that impacted infection of Sindbis virus (SINV), the prototypical alphavirus. This led to the identification of Natural Resistance-Associated Macrophage Protein (NRAMP, Divalent Metal Transporter (DMT1)), as an entry receptor for SINV in insects and the mammalian homolog NRAMP2 as a receptor in vertebrates. NRAMP is the major iron transporter in cells, and thus NRAMP expression is tightly regulated: either iron deficiency (a major public health concern) or excess causes human disease. Further studies revealed that Endoplasmic Reticulum Associated Decay genes along with the proteasome promote viral infection in vitro and in vivo at the level of entry. Furthermore, we found that depletion of dSEC61A and dPSMD11 significantly reduced NRAMP protein levels, decreasing both SINV infectivity and reducing NRAMP-dependent iron transport, suggesting a role for these genes in iron metabolism. Altogether, our study reveals new genes involved in SINV infection and also sheds light onto novel modes of NRAMP regulation. The identification of genes and pathways involved in NRAMP stability are critical for our understanding of alphavirus pathogenesis as well as iron metabolism.