The specific aims of this proposal are part of a long term goal to understand completely the infection course of a non-enveloped arthropod-borne virus, Bluetongue virus (BTV), a member of the Reoviridae family that include viruses infecting a wide range of hosts, including human, animals, plants, fungi, and bacteria. As such BTV shares a family relationship with several other scientifically and medically important viruses (e.g. Rotaviruses). BTV has been studied extensively as an agent of economically important disease and also, increasingly, as a model system for related viruses. Substantial progress has been achieved in the molecular, biochemical and structural fields; however, certain areas of BTV and related viruses' biology and structure remain unclear due to the unavailability of appropriate tools and assay systems. BTV and other members (rotavirus and reoviruses) are complex, multi-layered capsid viruses with 10-12 segmented double-stranded RNA genomes but the details of how virus genomes are packaged into their protective capsids or the mechanism by which a copy of each segment is incorporated for the virus to be viable remain elusive. In this proposal we aim to maintain and extend the leading position of BTV towards our long term goal of understanding, in totality, the infection course of the virus. In particular, this application wll utilize a groundbreaking technological advance (i.e., the first in vitro packaging system for a complex dsRNA virus) recently pioneered by us (through our current NIH funding), to understand the genome packaging and replication events of this complex capsid virus and by inference for all multi-segmented RNA viruses. In specific aim 1 we will determine the packaging order of ten ssRNAs and the signals involved in assembly of BTV particles. In specific aim 2 we will focus on identification of the protein and protein complexes essential for ssRNA packaging while in the specific aim 3, the replication competence of mutant constructs will be assessed in cell culture using our established reverse genetics system. Current data suggest that ssRNAs drive assembly of the BTV transcription complex (TC), leading to capsid (core) assembly and that packaged genome precursor RNA molecules are positioned at the 12 vertices of the assembling icosahedral capsid, precisely priming them for subsequent transcription. In specific aim 4 we will seek direct structural evidence of the TC organization at the vertices of the packaged particles, using both native and mutant particles to determine the consequences of mutation, using the state-of-the art single particle cryo-electron microscopy and cryo-tomography. Completion of these goals will pave the way for the rational design of inhibitors to block virus replication and provide lead compounds for related viruses of pathogenic significance to man and animals.